U.S. patent application number 15/650568 was filed with the patent office on 2018-01-18 for compounds and methods for treatment and prevention of flavivirus infection.
The applicant listed for this patent is FLORIDA STATE UNIVERSITY RESEARCH FOUNDATION, INC., JOHNS HOPKINS UNIVERSITY, The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to YICHEN CHENG, RUILI HUANG, WENWEI HUANG, EMILY M. LEE, GUO-LI MING, HONGJUN SONG, HENGLI TANG, ZHEXING WEN, MENGHANG XIA, MIAO XU, WEI ZHENG, YI ZHOU.
Application Number | 20180015153 15/650568 |
Document ID | / |
Family ID | 60941627 |
Filed Date | 2018-01-18 |
United States Patent
Application |
20180015153 |
Kind Code |
A1 |
TANG; HENGLI ; et
al. |
January 18, 2018 |
COMPOUNDS AND METHODS FOR TREATMENT AND PREVENTION OF FLAVIVIRUS
INFECTION
Abstract
The present invention concerns the use of compounds for the
treatment or prevention of Flavivirus infections, such as Zika
virus infections. Aspects of the invention include methods for
treating or preventing Flavivirus virus infection, such as Zika
virus infection, by administering a compound or class of compound
disclosed herein, such as a niclosamide compound, an emricasan
compound, a cyclin-dependent kinase inhibitor, a proteasome
inhibitor, or a combination of two or more of the foregoing, to a
subject in need thereof; methods for inhibiting Flavivirus
infections such as Zika virus infections in a cell in vitro or in
vivo; pharmaceutical compositions; packaged dosage formulations;
and kits for treating or preventing Flavivirus infections, such
Zika virus infections.
Inventors: |
TANG; HENGLI; (TALLAHASSEE,
FL) ; LEE; EMILY M.; (TALLAHASSEE, FL) ;
CHENG; YICHEN; (TALLAHASSEE, FL) ; ZHOU; YI;
(TALLAHASSEE, FL) ; ZHENG; WEI; (ROCKVILLE,
MD) ; HUANG; RUILI; (ROCKVILLE, MD) ; XU;
MIAO; (ROCKVILLE, MD) ; HUANG; WENWEI;
(ROCKVILLE, MD) ; XIA; MENGHANG; (ROCKVILLE,
MD) ; SONG; HONGJUN; (BALTIMORE, MD) ; MING;
GUO-LI; (BALTIMORE, MD) ; WEN; ZHEXING;
(ATLANTA, GA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FLORIDA STATE UNIVERSITY RESEARCH FOUNDATION, INC.
The United States of America, as represented by the Secretary,
Department of Health and Human Serv
JOHNS HOPKINS UNIVERSITY |
TALLAHASSEE
Bethesda
BALTIMORE |
FL
MD
MD |
US
US
US |
|
|
Family ID: |
60941627 |
Appl. No.: |
15/650568 |
Filed: |
July 14, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62430107 |
Dec 5, 2016 |
|
|
|
62380778 |
Aug 29, 2016 |
|
|
|
62374467 |
Aug 12, 2016 |
|
|
|
62366855 |
Jul 26, 2016 |
|
|
|
62363238 |
Jul 16, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/55 20130101;
A61K 38/00 20130101; A61K 31/24 20130101; A61K 31/609 20130101;
A61K 38/43 20130101; Y02A 50/391 20180101; Y02A 50/30 20180101;
C07C 239/08 20130101; A61K 45/06 20130101; C07C 239/02 20130101;
A61K 31/609 20130101; A61K 2300/00 20130101; A61K 31/24 20130101;
A61K 2300/00 20130101 |
International
Class: |
A61K 38/55 20060101
A61K038/55; A61K 31/24 20060101 A61K031/24; A61K 38/43 20060101
A61K038/43; C07C 239/08 20060101 C07C239/08; C07C 239/02 20060101
C07C239/02 |
Claims
1. A method for treating or preventing Flavivirus infection in a
human or non-human animal subject, said method comprising
administering an effective amount of at least one compound to a
subject in need thereof, wherein the at least one compound inhibits
Flavirus infection or suppresses Flavivirus-induced caspase-3
activity, and wherein the at least one compound comprises: (a) a
niclosamide compound, or (b) an emricasan compound, or (c) a
cyclin-dependent kinase (CDK) inhibitor, or (d) a proteasome
inhibitor, or (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
2. The method of claim 1, wherein the at least one compound
comprises a combination of two or more of the compounds.
3. The method of claim 1, wherein the Flavivirus infection is Zika
virus infection.
4. The method of claim 1, wherein the at least one compound
comprises a compound in Table 7, or a prodrug, metabolite, or
derivative thereof, or a pharmaceutically acceptable salt of any of
the foregoing.
5. The method of claim 1, wherein the at least one compound
comprises the niclosamide compound, and the niclosamide compound
comprises: (a) niclosamide, (b) a niclosamide derivative, (c) a
prodrug or metabolite of (a) or (b), or (d) a pharmaceutically
acceptable salt of (a), (b), or (c).
6. The method of claim 5, wherein the niclosamide or niclosamide
derivative has the structure of Formula 1: ##STR00064## or a
pharmaceutically acceptable salt thereof, wherein R.sub.1, R.sub.2,
R.sub.3, R.sub.4, R.sub.5, R.sub.6 are independently selected from
the group consisting of a H; F; Cl; Br; I; OH; ketone (.dbd.O);
(C.sub.1-6)alkyl; (C.sub.2-6)alkenyl; (C.sub.2-6)alkynyl; ether
[--OR, where R can include (C.sub.1-6)alkyl; aralkyl; alkaryl;
halogenated alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl;
cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl; amino;
alkylamino; arylamino; dialkylamino; alkylarylamino; diarylamino;
acylamino; hydroxyl; alkoxy; alkoxyalkyl; aryloxy; arylalkoxy;
acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy; benzyloxy;
alkaryl; arylalkyl; carbamate; amino; alkylamino; arylamino;
dialkylamino; alkylarylamino; diarylamino; heteroalkyl;
alkyltriphenylphosphonium; heterocyclyl; acyl halide [--COX, where
X is selected from F, Cl, Br, and I]; carbonyl [--COR, where R
selected from (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl]; aldehyde
(--CHO); ester [--OC(.dbd.O)R, --ROC(.dbd.O)R', RC(.dbd.O)OR',
--C(.dbd.O)OR', where R and R' is selected from
(C.sub.1-14)alkyland (C.sub.2-14)alkenyl; carbonate ester [--OCOOR,
where R is selected from (C.sub.1-6)alkyland (C.sub.1-6)alkenyl;
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' is selected
from hydrogen; alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl;
amines [--NR'R'', where R' and R'' is selected from hydrogen;
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl; cyanate (--OCN); isocynate
(--NCO); nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC);
nitroso (--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; borono
and boronate [--B(OR')(R''), where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; borinate [--B(R')(OR''),
where R' and R'' is selected from H; (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; phosophino [--PR.sub.2, where R is selected
from H; (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphate
[--OP(.dbd.O)(OR).sub.2, where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphono
[--RP(.dbd.O)(OH), where R is selected from (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; thiol (--SH); thioalkyl; alkylthio; sulfide
[--SR, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; disulfide [--SSR, where R is selected from
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl, sulfonamide; sulfinyl
[--S(.dbd.O)R, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; sulfino (--SO.sub.2H); sulfo (--SO.sub.3H);
thiocyanate; isothiocyanate; carbonothioyl [--C(.dbd.S)R where R is
selected from (C.sub.1-6)alkyland (C.sub.2-6)alkenyl; wherein
R.sub.2 and R.sub.4 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.4 and R.sub.6 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; and wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring.
7. The method of claim 5, wherein the niclosamide or niclosamide
derivative has the structure of Formula 2: ##STR00065## or a
pharmaceutically acceptable salt thereof, where R.sub.7, R.sub.8,
R.sub.9, R.sub.10, R.sub.11, R.sub.12 are independently selected
from the group consisting of a H; F; Cl; Br; I; OH; ketone
(.dbd.O); (C.sub.1-6)alkyl; (C.sub.2-6)alkenyl; (C.sub.2-6)alkynyl;
ether [--OR, where R can include (C.sub.1-6)alkyl; aralkyl;
alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl;
cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl;
amino; alkylamino; arylamino; dialkylamino; alkylarylamino;
diarylamino; acylamino; hydroxyl; alkoxy; alkoxyalkyl; aryloxy;
arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy;
benzyloxy; alkaryl; arylalkyl; carbamate; amino; alkylamino;
arylamino; dialkylamino; alkylarylamino; diarylamino; heteroalkyl;
alkyltriphenylphosphonium; heterocyclyl; acyl halide [--COX, where
X is selected from F, Cl, Br, and I]; carbonyl [--COR, where R
selected from (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl]; aldehyde
(--CHO); ester [--OC(.dbd.O)R, --ROC(.dbd.O)R', RC(.dbd.O)OR',
--C(.dbd.O)OR', where R and R' is selected from
(C.sub.1-14)alkyland (C.sub.2-14)alkenyl; carbonate ester [--OCOOR,
where R is selected from (C.sub.1-6)alkyland (C.sub.1-6)alkenyl;
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' is selected
from hydrogen; alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl;
amines [--NR'R'', where R' and R'' is selected from hydrogen;
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl; cyanate (--OCN); isocynate
(--NCO); nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC);
nitroso (--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; borono
and boronate [--B(OR')(R''), where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; borinate [--B(R')(OR''),
where R' and R'' is selected from H; (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; phosophino [--PR.sub.2, where R is selected
from H; (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphate
[--OP(.dbd.O)(OR).sub.2, where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphono
[--RP(.dbd.O)(OH), where R is selected from (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; thiol (--SH); thioalkyl; alkylthio; sulfide
[--SR, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; disulfide [--SSR, where R is selected from
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl, sulfonamide; sulfinyl
[--S(.dbd.O)R, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; sulfino (--SO.sub.2H); sulfo (--SO.sub.3H);
thiocyanate; isothiocyanate; carbonothioyl [--C(.dbd.S)R where R is
selected from (C.sub.1-6)alkyland (C.sub.2-6)alkenyl; wherein
R.sub.7 and R.sub.10 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-s)alkene ring; and wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.8 and R.sub.10 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-s)alkene ring; wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.10 and R.sub.11 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; and wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring.
8. The method of claim 5, wherein the niclosamide or niclosamide
derivative has the structure of Formula 3: ##STR00066## or a
pharmaceutically acceptable salt thereof, where R.sub.13 can
include (C.sub.1-14)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-14)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers.
9. The method of claim 5, wherein the niclosamide or niclosamide
derivative has the structure of Formula 4: ##STR00067## or a
pharmaceutically acceptable salt thereof.
10. The method of claim 1, wherein the at least one compound
comprises an emricasan compound, and wherein the emricasan compound
comprises: (a) emricasan, (b) an emricasan derivative, (c) a
prodrug or metabolite of (a) or (b), or (d) a pharmaceutically
acceptable salt of (a), (b), or (c).
11. The method of claim 10, wherein the emricasan or emricasan
derivative has the structure of Formula 5: ##STR00068## or a
pharmaceutically acceptable salt thereof, where R.sub.23, R.sub.24,
R.sub.25, R.sub.26, R.sub.27, R.sub.28, R.sub.29, R.sub.30,
R.sub.31, R.sub.32, R.sub.33, and R.sub.34 are independently
selected from the group consisting of a H; F; Cl; Br; I; OH; ketone
(.dbd.O); (C.sub.1-6)alkyl; (C.sub.2-6)alkenyl; (C.sub.2-6)alkynyl;
ether [--OR, where R can include (C.sub.1-6)alkyl; aralkyl;
alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl;
cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl;
amino; alkylamino; arylamino; dialkylamino; alkylarylamino;
diarylamino; acylamino; hydroxyl; alkoxy; alkoxyalkyl; aryloxy;
arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy;
benzyloxy; alkaryl; arylalkyl; carbamate; amino; alkylamino;
arylamino; dialkylamino; alkylarylamino; diarylamino; heteroalkyl;
alkyltriphenylphosphonium; heterocyclyl; acyl halide [--COX, where
X is selected from F, Cl, Br, and I]; carbonyl [--COR, where R
selected from (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl]; aldehyde
(--CHO); ester [--OC(.dbd.O)R, --ROC(.dbd.O)R', RC(.dbd.O)OR',
--C(.dbd.O)OR', where R and R' is selected from
(C.sub.1-14)alkyland (C.sub.2-14)alkenyl; carbonate ester [--OCOOR,
where R is selected from (C.sub.1-6)alkyland (C.sub.1-6)alkenyl;
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' is selected
from hydrogen; alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl;
amines [--NR'R'', where R' and R'' is selected from hydrogen;
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl; cyanate (--OCN); isocynate
(--NCO); nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC);
nitroso (--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; borono
and boronate [--B(OR')(R''), where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; borinate [--B(R')(OR''),
where R' and R'' is selected from H; (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; phosophino [--PR.sub.2, where R is selected
from H; (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphate
[--OP(.dbd.O)(OR).sub.2, where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphono
[--RP(.dbd.O)(OH), where R is selected from (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; thiol (--SH); thioalkyl; alkylthio; sulfide
[--SR, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; disulfide [--SSR, where R is selected from
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl, sulfonamide; sulfinyl
[--S(.dbd.O)R, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; sulfino (--SO.sub.2H); sulfo (--SO.sub.3H);
thiocyanate; isothiocyanate; carbonothioyl [--C(.dbd.S)R where R is
selected from (C.sub.1-6)alkyland (C.sub.2-6)alkenyl; wherein
R.sub.23 and R.sub.24 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.23 and R.sub.25 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene ring; where
R.sub.24 and R.sub.26 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.27 and R.sub.30 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene; wherein R.sub.28
and R.sub.29 can be bonded together to form an (C.sub.2-8)alkane
ring and/or (C.sub.2-8)alkene ring; and wherein R.sub.27 and
R.sub.30 can be bonded together to form an (C.sub.2-8)alkane ring
and/or (C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring; and wherein R.sub.29 and R.sub.30 can be bonded
together to form an (C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene
ring.
12. The method of claim 1, wherein the at least one compound
comprises a combination of the niclosamide compound and the
emricasan compound, and wherein the niclosamide compound and the
emricasan compound are administered simultaneously, together within
the same composition or in separate compositions.
13. The method of claim 1, wherein the at least one compound
comprises a combination of the niclosamide compound and the
emricasan compound, and wherein the niclosamide compound and the
emricasan compound are administered consecutively in any order.
14. The method of claim 1, wherein the at least one compound
comprises the CDK inhibitor, and the CDK inhibitor has a structure
shown in FIG. 11 (PHA-690509 or a derivative thereof), FIG. 12
(Alvocidib or a derivative thereof), FIG. 13 (PHA-793887 or a
derivative thereof), FIG. 14 (Dinaciclib or a derivative thereof),
FIG. 15 (Seliciclib or a derivative thereof), or a pharmaceutically
acceptable salt of any of the foregoing.
15. The method of claim 1, wherein the at least one compound
comprises the CDK inhibitor, and the CDK inhibitor has a structure
shown in FIG. 16, or a pharmaceutically acceptable salt
thereof.
16. The method of claim 1, wherein the at least one compound
comprises a CDK inhibitor, and the CDK inhibitor is selected from
among Alvocidib, Kenpaullone, Olomoucine, Purvalanol A, Purvalanol
B, Seliciclib, NU-6027, Indirubin, Flavopiridol, AT7519,
PD-0332991, SNS-032, PHA-793887, PHA-690509, RGB-286147, BS-194,
BS-181, AZD-5438, R-547, Dinaciclib, Milciclib, BMS-265246,
7-Hydroxystaurosporine, CGP-60474, CDK9 inhibitor, NU-6102,
Fascaplysin, Cdk4/6 Inhibitor IV, or a prodrug, metabolite,
derivative, or pharmaceutically acceptable salt of any of the
foregoing.
17. The method of claim 1, wherein the at least one compound
comprises a combination of the CDK inhibitor and the emricasan
compound, and wherein the CDK inhibitor and the emricasan compound
are administered simultaneously, together within the same
composition or in separate compositions.
18. The method of claim 1, wherein the at least one compound
comprises a combination of the CDK inhibitor and an emricasan
compound, and wherein the CDK inhibitor and the emricasan compound
are administered consecutively in any order.
19. The method of claim 1, wherein the subject has the Flavivirus
infection at the time of said administering, and the at least one
compound is administered as therapy.
20. The method of claim 19, further comprising, prior to said
administering, identifying the subject as having the Flavivirus
infection.
21. The method of claim 20, wherein said identifying comprises
assaying a biological sample obtained from the subject for the
presence of Flavivirus nucleic acids or Flavivirus proteins.
22. The method of claim 21, wherein said assaying comprises use of
reverse transcriptase-polymerase chain reaction (RT-PCR),
immunological assay, or Plaque-reduction neutralization testing
(PRNT).
23. The method of claim 1, wherein the subject does not have the
Flavivirus infection at the time of said administering, and the at
least one compound is administered as prophylaxis.
24. The method of claim 1, wherein the at least one compound is
administered orally, nasally, rectally, parenterally,
subcutaneously, intramuscularly, or intravascularly.
25. The method of claim 1, further comprising administering an
additional agent for treating or preventing Flavivirus infection,
or a symptom thereof, in the same formulation as at least one
compound, or in a separate formulation before, during, or after
administration of the at least one compound.
26. The method of claim 1, wherein said administering comprises
administering a composition to the subject, wherein the composition
comprises the at least one compound and a pharmaceutically
acceptable buffer, carrier, or diluent.
27. A method for inhibiting Flavivirus infection in human or
non-human animal cells in vitro or in vivo, said method comprising
contacting an effective amount of at least one compound to a human
or non-human animal cell in vitro or in vivo before or after
exposure of the cell to Flavivirus, wherein the at least one
compound inhibits Flavirus infection or suppresses
Flavivirus-induced caspase-3 activity, and wherein the at least one
compound comprises: (a) a niclosamide compound, or (b) an emricasan
compound, or (c) a cyclin-dependent kinase (CDK) inhibitor, or (d)
a proteasome inhibitor, or (e) a compound selected from among
Teriflunomide, Hydroxocobalamin, Ensulizole, Tenonitrozole,
Isoliquiritigenin, Nitazoxanide, Febuxostat, Leflunomide,
Vidofludimus, SB-366791, Emodin, Diphenyl isophthalate, Benzoylpas,
Fenobam, Indobufen, 2-(2H-Benzotriazol-2-yl)-4-methylphenol,
Tiaprofenic acid, Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
28. The method of claim 27, wherein the Flavivirus is Zika
virus.
29. A composition comprising a combination of two or more of the
following: (a) a niclosamide compound, or (b) an emricasan
compound, or (c) a cyclin-dependent kinase (CDK) inhibitor, or (d)
a proteasome inhibitor, or (e) a compound selected from among
Teriflunomide, Hydroxocobalamin, Ensulizole, Tenonitrozole,
Isoliquiritigenin, Nitazoxanide, Febuxostat, Leflunomide,
Vidofludimus, SB-366791, Emodin, Diphenyl isophthalate, Benzoylpas,
Fenobam, Indobufen, 2-(2H-Benzotriazol-2-yl)-4-methylphenol,
Tiaprofenic acid, Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
30. A composition comprising: (a) a niclosamide compound, or (b) an
emricasan compound, or (c) a cyclin-dependent kinase (CDK)
inhibitor, or (d) a proteasome inhibitor, or (e) a compound
selected from among Teriflunomide, Hydroxocobalamin, Ensulizole,
Tenonitrozole, Isoliquiritigenin, Nitazoxanide, Febuxostat,
Leflunomide, Vidofludimus, SB-366791, Emodin, Diphenyl
isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing, and an additional agent
effective for the treatment of one or more symptoms of Flavivirus
infection.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of U.S.
Provisional Application Ser. No. 62/430,107, filed Dec. 5, 2016;
62/380,778, filed Aug. 29, 2016; 62/374,467, filed Aug. 12, 2016;
62/366,855, filed Jul. 26, 2016; and 62/363,238, filed Jul. 16,
2016, each of which is hereby incorporated by reference herein in
its entirety, including any figures, tables, nucleic acid
sequences, amino acid sequences, or drawings.
BACKGROUND OF THE INVENTION
[0002] Zika virus (ZIKV), a mosquito-borne flavivirus, has
re-emerged and spread across the Western Hemisphere in the past
year. First isolated in 1947 from a sentinel rhesus macaque in the
Ziika Forest region of Uganda [1], ZIKV had remained in relative
obscurity for many years until outbreaks in the Pacific islands and
then the Americas in the past decade [2-4]. A large outbreak
started in Brazil in late 2014 and is a growing public health
concern [5]. Currently, active transmission has been reported in 58
countries and territories globally. About 20% of ZIKV infected
individuals develop symptoms, which mostly resemble symptoms caused
by other arboviruses, such as dengue viruses or chikungunya virus.
Unlike these viruses, however, ZIKV causes congenital defects,
including microcephaly [6,7], and is also associated with
Guillain-Barre syndrome in infected adults [8,9].
BRIEF SUMMARY OF THE INVENTION
[0003] The present invention concerns the use of compounds for the
treatment or prevention of Flavivirus infections, such as Zika
virus infections. Aspects of the invention include methods for
treating or preventing Flavivirus virus infection, such as Zika
virus infection, by administering a niclosamide compound, an
emricasan compound, a cyclin-dependent kinase inhibitor, a
proteasome inhibitor, another compound or class of compounds
disclosed herein, or a combination of two or more of the foregoing,
to a subject in need thereof; methods for inhibiting Flavivirus
infections such as Zika virus infections in a cell in vitro or in
vivo; pharmaceutical compositions; packaged dosage formulations;
and kits for treating or preventing Flavivirus infections, such
Zika virus infections.
[0004] The inventors have identified a novel use for certain
compounds in the treatment and prevention of Zika virus (ZIKV)
infections and other Flavivirus infections. In some embodiments,
the compounds are selected from among:
[0005] (a) a niclosamide compound, or
[0006] (b) an emricasan compound, or
[0007] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0008] (d) a proteasome inhibitor, or
[0009] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or
a pharmaceutically acceptable salt of any of the foregoing.
[0010] In some embodiments, the compounds are selected from those
in Tables 7-10 or FIGS. 9-22, or a prodrug, metabolite, or
derivative thereof.
[0011] The inventors identified Niclosamide from a library of
compounds used in a large scale compound screen measuring
cell-death inhibition in ZIKV infected cells. However, the compound
screen failed to identify Niclosamide as being highly effective
against ZIKV-induced cell death. In spite of the initial negative
results, the inventors chose to focus on Niclosamide due to its
status as a FDA approved drug and category B pregnancy rating. By
directly testing Niclosamide in human cell culture against ZIKV
infection, the inventors have found strong evidence that
Niclosamide is highly effective against ZIKV infection.
[0012] The inventors have also identified a novel use for CDK
inhibitors, such as PHA690509, in having antiviral properties in
Zika virus (ZIKV) infection. The inventors identified PHA690509
from a library of compounds used in a large scale compound screen
(in collaboration with NIH) measuring cell-death inhibition in ZIKV
infected cells. The compound screen identified PHA690509 as being
highly effective against ZIKV-induced cell death. The inventors
have also tested PHA690509 in several human cell culture systems
(human neural progenitor cells, iPSC-derived astrocytes, organoids,
and glioblastoma cells) against ZIKV infection, and have found
strong evidence that PHA690509 is highly effective against ZIKV
infection.
[0013] The inventors have also identified Emricasan, a pan-caspase
inhibitor, as a compound that potently inhibits ZIKV-induced
increase in caspase-3 activity and reduces cell death. The
inventors confirmed its efficacy to protect human cortical neural
progenitors in both monolayer and organoid cultures. PHA-690509, a
cyclin-dependent kinase (CDK) inhibitor, and four structurally
unrelated CDK inhibitors, inhibit ZIKV replication in human cells
in a dose-dependent manner. In addition, Niclosamide, an FDA
approved category B anthelmintic drug, inhibits ZIKV replication at
sub-micromolar concentrations. Both CDK inhibitors and Niclosamide
are effective in reducing ZIKV replication when added either before
or after ZIKV exposure. Furthermore, a combination of two classes
of compounds showed a synergistic effect in protecting human neural
cells from ZIKV-induced cell death. The inventors' results
demonstrate the effectiveness of this compound screening strategy
and provide lead compounds for drug development against ZIKV and
other Flavivirus.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Patent
and Trademark Office upon request and payment of the necessary
fee.
[0015] FIGS. 1A-1, 1A-2, 1A-3, 1B-1, 1B-2, 1C-1, and 1C-2.
Emricasan suppresses cell death of ZIKV-infected human astrocytes
and hNPCs in 2D monolayer cultures and 3D brain organoids.
Emricasan (FIG. 1A-1) inhibits caspase-3 activity and improves cell
viability in human astrocytes infected with three different strains
of ZIKV in a concentration dependent manner. Value represent
mean.+-.s.d. (n=3). The curves (FIGS. 1A-2 and 1A-3) represent best
fit. (FIG. 1B) Sample images of immunostaining (FIG. 1B-1) of
forebrain-specific hNPCs for ZIKV envelop protein (ZIKVE; green)
and cleaved-caspase 3 (Cas3; red) and DAPI (gray) 72 hours after
ZIKV FSS-13025 infection (MOI=0.04-0.08; Scale bar: 20 .mu.m), and
quantification (FIG. 1B-2). Values represent mean.+-.s.e.m. (n=3;
**P<0.01; Student's t-test). Sample images (FIG. 1C-1) of
immunostaining of day 28 human forebrain-specific organoids for
ZIKVE (green) and Cas3 (red) and DAPI (gray) 10 days after ZIKV
FSS-13025 infection (Scale bar: 100 .mu.m), and quantification
(FIG. 1C-2). Values represent mean.+-.s.e.m. (n=6; ***P<0.001;
Student's t-test).
[0016] FIGS. 2A-1, 2A-2, 2B-1, 2B-2, 2C-1, 2C-2, 2D-1, 2D-2, 2D-3,
2E-1, 2E-2, and 2F. Identification of PHA-690509 and Niclosamide as
antiviral compounds. Screening of select compounds for antiviral
activity against ZIKV infection was carried out. Glioblastoma
SNB-19 cells were treated with indicated compound at 10.times. AC50
(determined in the primary caspase screen) for 1 hour prior to
inoculation with FSS-13025 (at MOI=1). Cells were harvested 24
hours post infection and analyzed by Western blot for ZIKV-NSI1 or
GAPDH (FIGS. 2A-1 and 2A-2). Titration of PHA-690509 on
glioblastoma cells for anti-ZIKV activity. Three strains of virus
(FSS-13025, PRVABC59, and MR766; MOI=1) were tested. Shown are
sample western blot images (FIG. 2B-1) and ImageJ quantification
(FIG. 2B-2). Values represent mean.+-.s.d. (n=3; ***P<0.001;
One-way ANOVA). Titration of Emricasan on SNB-19 cells for
anti-ZIKV activity was conducted similarly (FIGS. 2C-1 and 2C-2).
Values represent mean.+-.s.d. (n=3; no significant differences were
found; One-way ANOVA). Titration of Niclosamide (FIG. 2D-1) on
SNB-19 cells for anti-ZIKV activity. Sample western blot (FIG.
2D-2) and ImageJ quantification (FIG. 2D-3) are shown for infection
with the PRVABC59 strain (MOI=1). Results for additional strains
are shown in FIGS. 6A-F. Values represent mean.+-.s.d. (n=2;
**P<0.01; ***P<0.001; One-way ANOVA). Secretion of ZIKV NS1
protein into the medium of infected cells was inhibited by
anti-ZIKV compounds in a dosage-dependent manner. The concentration
of NS1 in the medium of infected SNB-19 cells collected 24 hours
post-infection was quantified by a ZIKV-NS1 ELISA kit (FIGS. 2E-2
and 2E-2). Values represent mean.+-.s.d. (n=3; ***P<0.001;
One-way ANOVA). FIG. 2F shows a summary of titration of niclosamide
and PHA-690509 on ZIKV production. SNB-19 cells were treated with
each indicated compound at increasing concentrations for 1 hour
before infection with PRVABC59 at a MOI=0.5 (n=3 cultures). Cell
culture supernatant was collected 24 hours after infection, and
infectious virions were quantified for focus-forming units (FFU)
using Vero cells. Data represent mean.+-.s.d. Curves represent best
fits for calculating IC50 values, and the insets report the
calculated IC50 value for each compound.
[0017] FIGS. 3A, 3B, 3C-1, 3C-2, 3C-3, 3C-4, 3C-5, 3D-1, 3D-2, and
3E. Time-of-addition compound treatments and blockade of ZIKV
infection by additional CDK inhibitors. FIG. 3A shows a schematic
illustration of time-of-addition experiment for Niclosamide and
PHA-690509. Niclosamide and PHA-690509 inhibit ZIKV infection at a
post-entry step. SNB-19 cells were treated with 10 .mu.g/ml
anti-AXL antibody, 2 .mu.M Niclosamide, or 92 .mu.M PHA-690509 for
1 hour prior or 4 hours post infection with PRVABC59 (MOI=1). Cells
were harvested 24 hours post infection and analyzed by western blot
for ZIKV-NS1 or GAPDH (shown in FIG. 3B). Structures of PHA-690509
and four structurally unrelated CDK inhibitors (Alvocidib,
PHA-793887, Dinaciclib, and Seliciclib) are shown in FIGS. 3C-5,
3C-1, 3C-2, 3C-3, and 3C-4, respectively. Inhibition of ZIKV
infection in CDK inhibitor-treated cells. SNB-19 cells were treated
with the indicated compound at 92 .mu.M for one hour prior to
infection with FSS13025 or PRVABC59 (MOI=1). Cells were harvested
24 hours post infection and analyzed by western blot for ZIKV-NS1
or GAPDH. Shown are sample images and quantification (FIGS. 3D-1
and 3D-2). Values represent mean.+-.s.d. (n=2; ***P<0.01;
One-way ANOVA). Quantification of NS1 protein band intensities,
relative to those for GAPDH (n=3 cultures), in FIG. 3B, are shown
in FIG. 3E. Data were normalized to those obtained for DMSO-treated
cells. Values represent mean.+-.s.d. *P<0.05; **P<0.01;
***P<0.001; by one-way ANOVA, as compared to DMSO-treated
cells.
[0018] FIGS. 4A-1, 4A-2, 4B-1, 4B-2, 4B-3, 4C-1, 4C-2, 4D-1, and
4D-2. Niclosamide and PHA-690509 inhibit ZIKV infection in human
astrocytes and forebrain-specific hNPCs. Sample immunostaining
images of astrocytes treated with 2 .mu.M Niclosamide, 92 .mu.M
PHA-690509, 9 .mu.M Emricasan, or a combination of 92 .mu.M
PHA-690509 and 9 .mu.M Emricasan for 1 hour prior to infection with
PRVABC59 (MOI=0.5) are shown in FIG. 4A-1. Cells were fixed 24
hours post infection and stained for ZIKVE (green) and DAPI (blue).
Scale bar: 50 .mu.m. Also shown is the quantification of
ZIKV-infected astrocytes with multiple compound concentrations
(FIG. 4A-2). Values represent mean.+-.s.d. (n=6; ***P<0.001;
One-way ANOVA). Shown in FIG. 4B-3 is virus production from
compound-treated iPSC-derived human astrocytes (as in FIG. 2F) (n=3
cultures). Data represent mean.+-.s.d. Niclosamide and PHA-690509
inhibit ZIKV infection in hNPCs. hNPCs were infected at a MOI of
0.1 and analyzed by Western blot 48 hours post infection for
ZIKV-NS1 or GAPDH. Shown are sample images and quantification
(FIGS. 4B-1 and 4B-2). Values represent mean.+-.s.d. (n=2;
***P<0.01; One-way ANOVA). Synergistic effect of
Emricasan/PHA-690509 combination on improving cell viability in
ZIKV infected astrocytes. Note that Emricasan and PHA-690509
inhibit the increased caspase-3 activity in a
concentration-dependent manner in human astrocytes infected with
FSS13025-ZIKV or MR766 ZIKV, and improve cell viability as measured
by ATP production (FIGS. 4C-1, 4C-2, 4D01, and 4D-2). Values
represent mean.+-.s.d. (n=3; **P<0.01; One-way ANOVA).
[0019] FIGS. 5A-5F. Two high-throughput assays that measure
increased caspase-3 activity and reduced cell viability of ZIKV
infected cells. (FIG. 5A) Flowchart of caspase-3 activity and cell
viability assays for compound screening. (FIG. 5B) Increased
caspase-3 activity in the ZIKV infected hNPCs, SNB-19 cells, and
human astrocytes. Values represent mean.+-.s.d. (n=3; **P<0.01;
One-way ANOVA). (FIG. 5C) Decreased cell viability after ZIKV
infection for three days. Values represent mean.+-.s.d. (n=3;
*P<0.01; One-way ANOVA). (FIG. 5D) Schematic diagram of compound
screening and hit follow-up process. (FIG. 5E) Summary of numbers
of confirmed compounds in the caspase 3/7 activity assay.
Thirty-five compounds were confirmed in all three cell types. (FIG.
5F) Summary of confirmed compounds in the cell viability assay that
improved cell viability in cells infected with ZIKV.
[0020] FIGS. 6A-6F. Niclosamide and PHA-690509 inhibit ZIKV
infection in a dose-dependent manner. FIG. 6A shows representative
Western blot images of SNB-19 cells treated with compounds at
10.times.IC50 concentration (determined in primary caspase-3
screen) for 1 hour prior to infection with ZIKV-FSS13025 and
harvested 24 hours later for NS1 detection. FIG. 6B shows
representative Western blot images of SNB-19 cells treated with 2,
10, or 20 .mu.M Niclosamide 1 hour prior to infection with
indicated ZIKV strains and harvested 24 hours later and analyzed as
in FIG. 6A. Dose-dependence of Niclosamide on ZIKV-NS1 protein
levels is shown in FIG. 6C. Cells were treated with 0.02-2 .mu.M
Niclosamide, infected with PRVABC59, and analyzed as in FIG. 6B.
FIGS. 6D and 6E show dose-dependence of Niclosamide and PHA-690509
on intracellular ZIKV-RNA levels. Cells were treated with compounds
at indicted concentrations for one hour then infected with
ZIKV-FSS13025, and harvested 48 hours later for RNA purification.
ZIKV RNA levels were measured using qRT-PCR and normalized to that
with the DMSO treatment. Values represent mean+s.d. (n=3 cultures;
**P<0.01; ***P<0.001; One-way ANOVA for comparison with the
DMSO treatment). Niclosamide inhibits a post-entry step in the ZIKV
infection lifecycle. SNB-19 cells were treated with 2 .mu.M
Niclosamide or DMSO for 1 hour prior to infection. Cells were
incubated with viral inoculum at MOI=1 for 2 hours on ice, then at
37.degree. C. (denoted as 0 hr time point). Cells were washed with
PBS and total RNA was collected and purified at each time point for
qRT-PCR analysis of ZIKV RNA. Relative ZIKV RNA levels after
normalizing to GAPDH RNA in the same sample are plotted in FIG. 6F.
Values represent mean+s.d. (n=3 cultures; **P<0.01;
***P<0.001; One-way ANOVA for comparison with the DMSO
treatment).
[0021] FIGS. 7A, 7B-1, 7B-2, and 7B-3. Additional cyclic-dependent
kinase inhibitors (CDKis) and non-CDK kinase inhibitors that were
tested for antiviral activity. Chemical structures of nine
additional CDKis that inhibited ZIKV infection are shown in FIG.
7A. Representative Western blot images of SNB-19 cells treated with
1 .mu.M of each indicated compound for 1 hour prior to infection
with ZIKV-FSS13025 and harvested 24 hours post infection, shown in
FIG. 7B-1. FIG. 7B-2 shows chemical structures of four non-CDK
kinase inhibitors tested on ZIKV infection. FIG. 7B-3 shows
representative Western blot images of SNB-19 cells treated with 1
.mu.M of each indicated non-CDK kinase inhibitor 1 hour prior to
infection with PRVABC59 and harvested 24 hours post infection.
[0022] FIGS. 8A-8E. Additional benefit of combinatorial treatment
with two classes of compounds. Human astrocytes were treated with
DMSO, PHA-690509, Emricasan, or PHA-690509 and Emricasan at
indicated concentrations for 1 hour prior to infection with
ZIKV-FSS13025. Cell lysates were harvested 24 hours post infection
and analyzed for ZIKV-NS1 and GAPDH protein levels by western blot.
Shown are representative Western blot images (FIG. 8A) and
quantification (FIG. 8B). Data were normalized to that of the DMSO
treatment. Values represent mean+s.d. (n=3 cultures; *P<0.001;
One-way ANOVA for comparison with the DMSO treatment). Secreted NS1
levels from DMSO-, Niclosamide-, PHA-690509-, or Emricasan-treated
astrocytes. Astrocytes were infected as in FIG. 8A and supernatants
were collected 24 hours post infection for analysis by NS1 ELISA.
Results are shown in FIG. 8C. Values represent mean+s.d. (n=3
cultures; *P<0.001; One-way ANOVA for comparison with the DMSO
treatment). Combined treatment of hNPCs with Emricasan (Em) and
Niclosamide (Nic). hNPCs were treated with 15 .mu.M Emricasan 1
hour prior to addition of PRVABC59 (MOI=0.08). At 72 hours post
infection, 1 .mu.M Niclosamide or DMSO was added to hNPCs and cells
were cultured for an additional 48 hours prior to staining with
anti-ZIKVE. Shown in FIG. 8D are representative images of
immunostaining for ZIKVE (green) and DAPI (gray, left; Scale bar:
20 .mu.m) and quantification (FIG. 8E). Values represent
mean+s.e.m. (n=3 cultures; ***P<0.001; One-way ANOVA).
[0023] FIGS. 9A-9D. Formulas encompassing Niclosamide and
derivatives of Niclosamide (Formulas 1-4, respectively).
[0024] FIG. 10. Formula encompassing Emricasan and derivatives of
Emricasan, (Formula 5).
[0025] FIG. 11. Formula encompassing the CDK inhibitor PHA-690509
and derivatives of PHA-690509 (Formula 6).
[0026] FIG. 12. Formula encompassing the CDK inhibitor Alvocidib
and derivatives of Alvocidib (Formula 7).
[0027] FIG. 13. Formula encompassing the CDK inhibitor PHA-793887
and derivatives of PHA-793887 (Formula 8).
[0028] FIG. 14. Formula encompassing the CDK inhibitor Dinaciclib
and derivatives of Dinaciclib (Formula 9).
[0029] FIG. 15. Formula encompassing the CDK inhibitor Selicicib
and derivatives of Selicicib (Formula 10).
[0030] FIG. 16. Table of embodiments of CDK inhibitory compounds of
the invention, with corresponding chemical structures, including
Kenpaullone, Olomoucine, Purvalanol A, Purvalanol B, Seliciclib,
NU-6027, Indirubin, Flavopiridol, AT7519, PD-0332991, SNS-032,
PHA-793887, PHA-690509, RGB-286147, BS-194, BS-181, AZD-5438,
R-547, Dinaciclib, Milciclib, BMS-265246, 7-Hydroxystaurosporine,
CGP-60474, CDK9 inhibitor, NU-6102, Fascaplysin, and Cdk4/6
Inhibitor IV.
[0031] FIGS. 17A-17C. Analysis of potential toxicity of CDKis on
human astrocyte viability and hNPC proliferation. Human
iPSC-derived astrocytes (BJ line) were treated with each indicated
compound (cpd) for 25 hours prior to cell viability analysis as
measured in relative luciferase units for ATP production. Results
are shown in FIG. 17A. Values represent mean+s.d. (n=3 cultures;
***P<0.001; **P<0.01; One-way ANOVA for comparison with the
DMSO treatment). RLU: relative luminescence units. hNPCs were
treated with saline, PHA-690509 (PHA, 1 .mu.M) or Seliciclib (5
.mu.M), 1 hour prior to ZIKV PRVABC59 infection (MOI=0.08). At 72
hours post infection, EdU (10 .mu.M) was added to hNPCs and cells
were cultured for additional 4 hours prior to fixation and staining
for EdU and DAPI. Results are shown in FIG. 17B. Values represent
mean+s.e.m. (n=3 cultures; ***P<0.001; One-way ANOVA for
comparison with the mock treatment). Day 20 forebrain-specific
brain organoids were treated with PHA-690509 (1 .mu.M) or
Seliciclib (5 .mu.M) for 3 days and hNPC proliferation was
evaluated by phospho-Histone 3 (PH3) expression within the
ventricular zone. Results are shown in FIG. 17C. Values represent
mean+s.e.m. (n=8 organoids; P>0.05; One-way ANOVA for comparison
with the mock treatment).
[0032] FIGS. 18A, 18B, 18C-1, 18C-2, 18D, 18E, 18F, 18G-1, and
18G-2. Antiviral activity of emetine against ZIKV infection. ZIKV
production from human glioblastoma SNB-19 cells treated with
increasing concentration of emetine. 24 hours post infection,
supernatants from SNB-19 cells were titrated onto naive vero cells
plated in a monolayer in 96-well plates for infectious
focus-forming unit assay as previously described and focus forming
units quantified [47]. Results are shown in FIG. 18A. The chemical
structure of emetine is shown in FIG. 18B. Emetine reduces ZIKV
infection in a dosage dependent manner. Immunofluorescence (IF)
staining (FIG. 18C-1) and quantification (FIG. 18C-2) of ZIKV
positive SNB-19 cells following infection with one of three ZIKV
strains and treatment with emetine at 0.01, 0.1, or 1 .mu.M.
Emetine is effective against all three ZIKV strains tested. FIG.
18D shows Western blot analysis of intracellular ZIKV NS1 levels in
SNB-19 cells. FIG. 18E shows that infectious ZIKV production is
reduced for all three strains of ZIKV tested in SNB-19 cells. IC50
for each strain is .ltoreq..about.10 nM. Emetine is effective
against ZIKV infection when added both before or after infection.
FIG. 18F shows Western blot analysis of ZIKV NS1 protein levels in
SNB-19 cells after a 24-hour infection. Drug was added at 1 .mu.M
either 1-hour prior to infection with PRVABC59-ZIKV, or 4-hours
post infection with PRVABC59-ZIKV.
[0033] FIG. 18G-1 shows IF staining of ZIKV-ENV vero cells after a
24-hour infection. Drug was added at 1 .mu.M either 1-hour prior to
infection with PRVABC59-ZIKV, or 4-hours post infection with
PRVABC59-ZIKV. Percentage of ZIKV-ENV+ cells are shown in FIG.
18G-2.
[0034] FIGS. 19A, 19B-1, 19B-2, 19C, 19D, 19E-1, and 19E-2.
Antiviral activity of proteasome inhibitors against ZIKV and DENV
infection. FIG. 19A shows Western blot analysis of ZIKV NS1 protein
levels in SNB-19 cells after a 24-hour infection (strain PRVABC59,
MOI=1). Cells were treated for 1 hour prior to addition of 1 .mu.M
indicated compound. FIG. 19B-1 shows immunofluorescence staining
and quantification (FIG. 19B-2) of ZIKV-ENV positive SNB-19 cells
after a 24-hour infection (strain PRVABC59, MOI=1). Cells were
treated for 1 hour prior to addition of 1 .mu.M indicated compound.
ZIKV production from human glioblastoma SNB-19 cells treated with
increasing concentration of indicated compound. 24-hours post
infection, supernatants from SNB-19 cells were titrated onto naive
vero cells plated in a monolayer in 96-well plates for infectious
focus-forming unit assay as previously described and focus forming
units quantified [47], as shown in FIG. 19C. IC50s were calculated
using graphpad PRISM software and listed next to the graph. FIG.
19D shows the Western blot analysis of DENV NS3 protein levels in
SNB-19 cells after a 24-hour infection (DENV serotype II, MOI=0.2)
Cells were treated for 1 hour prior to addition of 1 .mu.M
indicated compound.
[0035] FIG. 19E-1 shows immunofluorescence staining and
quantification (FIG. 19E-2) of DENV-ENV positive SNB-19 cells after
a 24-hour infection (DENV serotype II, MOI=0.2). Cells were treated
for 1 hour prior to addition of 1 .mu.M indicated compound.
[0036] FIG. 20. Synthesis scheme for the niclosamide derivative
p-niclosamide. Reagent and conditions: (a) (EtO).sub.2POH, DIPEA,
CCl.sub.4, DMAP, MeCN, -10.degree. C.; (b) (CH.sub.3).sub.3SiBr,
CHCl.sub.3, r.t.; (c) CH.sub.3OH, r.t.
[0037] FIG. 21. Antiviral activity of p-niclosamide against ZIKV
infection. Infectious PRVABC59-ZIKV production from human
glioblastoma SNB-19 cells treated with increasing concentration of
indicated compound. 24-hours post infection, supernatants from
SNB-19 cells were titrated onto naive vero cells plated in a
monolayer in 96-well plates for infectious focus-forming unit assay
as previously described and focus forming units quantified [47].
P-niclosamide IC50 in SNB-19 cell culture production of
ZIKV=0.000289M. IC50's calculated using graphpad PRISM
software.
[0038] FIG. 22. Antiviral activity of DK-520 against ZIKV
infection. Infectious PRVABC59-ZIKV production from human
glioblastoma SNB-19 cells treated with increasing concentration of
indicated compound. 24-hours post infection, supernatants from
SNB-19 cells were titrated onto naive vero cells plated in a
monolayer in 96-well plates and stained for ZIKV-ENV protein and
DAB substrate. Shown are phase-contrast images of ZIKV-ENV stained
cells (in black). Results indicate IC50 of DK520 against ZIKV
infection in human glioblastoma cell culture is <3 .mu.M.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The inventors have identified compounds useful for the
treatment and prevention of Zika virus (ZIKV) infections and other
Flavivirus infections based at least on their ability to inhibit
Flavivirus infection in vitro or in vivo, or their ability to
suppress Flaviviurs-induced capsase-3 activity (e.g., in one or
more host neural cell types).
[0040] One aspect of the invention concerns a method for treating
or preventing Flavivirus infection in a human or non-human animal
subject, the method comprising administering an effective amount of
at least one compound to a subject in need thereof, wherein the at
least one compound inhibits Flavirus infection or suppresses
Flavivirus-induced caspase-3 activity, and wherein the at least one
compound comprises:
[0041] (a) a niclosamide compound, or
[0042] (b) an emricasan compound, or
[0043] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0044] (d) a proteasome inhibitor, or
[0045] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
[0046] In some embodiments, a combination of two or more of the
foregoing compounds from (a), (b), (c), (d), or (e) are
administered.
[0047] In some embodiments, one or more compounds from Tables 7-10
or FIGS. 9-22, or a pharmaceutically acceptable salt thereof, are
administered. In some embodiments, a prodrug, metabolite, or
derivative of a compound of Tables 7-10, or FIGS. 9-22, is
administered to the subject. In some embodiments, the compound is
one that reduces virally-induced caspase activation and apoptosis
by either directly preventing Zika virus-induced cell death or
suppressing Zika replication in a subject.
[0048] Another aspect of the invention concerns a method for
inhibiting Flavivirus infection in human or non-human animal cells
in vitro or in vivo, said method comprising contacting an effective
amount of at least one compound to a human or non-human animal cell
in vitro or in vivo before or after exposure of the cell to
Flavivirus, wherein the at least one compound inhibits Flavirus
infection or suppresses Flavivirus-induced caspase-3 activity, and
wherein the at least one compound comprises:
[0049] (a) a niclosamide compound, or
[0050] (b) an emricasan compound, or
[0051] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0052] (d) a proteasome inhibitor, or
[0053] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing. In some embodiments, the
cells are contacted with a combination of two or more of the
foregoing compounds from (a), (b), (c), (d), or (e). In some
embodiments of the method for inhibiting Flavivirus infection in
cells, the cell is a neural cell, such as a neural progenitor cell
or astrocyte. Contacting of cells in vitro or in vivo with one or
more of the compounds of the invention may be carried out before
Flavivirus infection (pre-infection) or after Flavivirus infection
(post-infection). When carried out in vivo, the one or more
compounds are administered to a human or non-human animal subject.
Optionally, when carried out in vivo, the method for inhibiting
Flavivirus infection in cells may be treat or prevent Flavivirus
infection in accordance with the method of treatment herein.
[0054] In some embodiments, the cells are contacted with one or
more compounds from Tables 7-10, or FIGS. 9-22, or a
pharmaceutically acceptable salt thereof. In some embodiments, the
cells are contacted with a prodrug, metabolite, or derivative of a
compound of Tables 7-10, or FIGS. 9-22, or a pharmaceutically
acceptable salt thereof.
[0055] In some embodiments, the compound is one that reduces
virally-induced caspase activation and apoptosis by either directly
preventing Zika virus-induced cell death or suppressing Zika
replication in a subject.
[0056] In some embodiments of the methods, compositions, kits, and
packaged dosage formulations of the invention, the Flavivirus
infection is Zika virus, West Nile virus, dengue virus (e.g., type
1, 2, 3, or 4), tick-borne encephalitis virus, Japanese
encephalitis virus, St. Louis encephalitis virus, or yellow fever
virus. Other members of the genus can be found in Kuno G et al.,
Journal of Virology, 1998, "Phylogeny of the Genus Flavivirus,"
72(1):73-83, which is incorporated herein by reference. In some
embodiments, the Flavivirus is Zika virus. The Zika virus may be
any origin or lineage (e.g., African, Asian, American, Brazilian).
Examples of Zika virus strains include but are not limited to MR766
(1947 Uganda strain), FSS13025 (2010 Cambodian strain), PRVABC59
(2015 Puerto Rican strain), GZ01/2016 (2016 Chinese strain (ex
Venezuela)), H/PF/2013 (2013 French Polynesian strain), IBH30656
(1968 Nigerian strain), Paraiba 2015 (2015 Brazilian strain),
PLCal_ZV (2013 Canadian strain (ex Thailand)), SMGC-1 (2016 Chinese
strain), SPH 2015 (2015 Brazilian strain), and SZO1 (2016 Chinese
strain).
[0057] In the various methods, compositions, kits and packaged
dosage formulations of the invention, the compound or compounds
used may comprise:
[0058] (a) a niclosamide compound, or
[0059] (b) an emricasan compound, or
[0060] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0061] (d) a proteasome inhibitor, or
[0062] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
[0063] Derivatives of any of the compounds can be synthesized by
chemical transformations of the compounds' functional groups using
standard chemical reactions. For example, these standard chemical
reactions can include, but are not limited to: polar reactions
under basic conditions, polar reactions under acidic conditions,
pericyclic reactions, and free radical reactions. In another
example, these standard chemical reactions can include, but are not
limited to: addition reactions, substitution reactions, oxidation
reactions, reduction reactions, elimination reactions, hydrolysis,
acylation, amidations, etherification, and esterification. Alkane
functional group transformations can include, but are not limited
to: free radical chlorination (hv, Cl.sub.2), free radical
bromination (hv, Br.sub.2), and allylic bromination (NBS). Alkene
functional group transformations can include, but are not limited
to: addition of HCl, addition of HBr, addition of HI, addition of
H.sub.3O(+), chlorination (Cl.sub.2) bromination (Br.sub.2),
iodination (I.sub.2), chlorohydrin formation (Cl.sub.2/H.sub.2O),
bromohydrin formation (Br.sub.2/H.sub.2O), ether formation
(H+/ROH), oxymercuration (Hg(OAc).sub.2/H.sub.2O), oxymercuration,
(Hg(OAc).sub.2/ROH), hydroboration, epoxidation (RCO.sub.3H),
dihydroxylation (OsO.sub.4), dihydroxylation (KMnO.sub.4),
cyclopropanation, dichlorocyclopropanation, ozonolysis (reductive
workup), ozonolysis (oxidative workup), oxidative cleavage
(KMnO.sub.4), hydrogenation, rearrangements (H shift),
rearrangements (alkyl shift), free radical addition of HBr, and
Sharpless epoxidation. Alkyne functional group transformations can
include, but are not limited to: deprotonation (acetylide
formation), S.sub.N2 with alkyl halides, partial reduction
(Lindlar), partial, reduction (Na/NH.sub.3), hydroboration,
oxymercuration, addition of HCl, HBr, or HI, addition of HCl, HBr,
or HI, hydrogenation, ozonolysis, oxidative cleavage (KMnO.sub.4),
and halogenation (Cl.sub.2, Br.sub.2, I.sub.2). The substitution
reaction can include, but is not limited to: alcohol formation,
nitrile formation, thiol formation, ether formation, thioether
formation, azides, ester formation, acetylide addition, alkanes
(Gilman reagents), ammonium salt formation, alkyl chloride
formation, alkyl bromide formation, alkyl iodide formation, alkyl
shift, and hydride shift. Elimination reactions can include, but
are not limited to: alkenes from alkyl halides, alkenes from
alcohols (strong acid), alkenes from alcohols (POCl.sub.3), alkenes
from alkyl halides, E1 with rearrangement (alkyl shift), Hoffmann
elimination, and alkyne formation via elimination E1 with
rearrangement (hydride shift). Organometallic reactions can
include, but are not limited to: Grignard formation (alkyl
halides), Grignard formation (alkenyl halides), reaction of
Grignards with acids, addition of Grignards to aldehydes, addition
of Grignards to ketones, addition of Grignards to esters, reaction
of Grignards with CO.sub.2, addition of Grignards to nitriles,
formation of organolithium reagents, formation of Gilman reagents,
S.sub.N2 with Gilman reagents, addition of Gilman reagents to
enones, addition of Gilman to acyl halides, Heck reaction, Suzuki
reaction, and Stille reaction. Reactions of epoxides can include,
but are not limited to: epoxide opening (basic conditions), epoxide
opening (acidic conditions), epoxide opening (diol formation),
epoxide formation (from halohydrins), epoxide formation (from
alkenes), and Sharpless epoxidation of alkenes. Reactions of
alcohols and thiols can include, but are not limited to:
deprotonation (alkoxide formation), protonation (onium ion
formation), conversion to tosylates/mesylates, conversion to alkyl
chlorides (SOCl.sub.2), conversion to alkyl bromides (PBr.sub.3),
oxidation to aldehydes (PCC), oxidation to ketones (PCC+others),
oxidation to carboxylic acid, (H.sub.2CrO.sub.4+others), protection
as silyl ethers, thiol formation (S.sub.N2), and thiol oxidation to
disulfides. Reactions of dienes can include, but are not limited
to: Diels-alder reaction, polymerization of dienes, reactions of
aromatics (arenes), nitration (HNO.sub.3/H.sub.2SO.sub.4),
chlorination (Cl.sub.2 plus catalyst), bromination (Br.sub.2 plus
catalyst), sulfonylation (SO.sub.3/H.sub.2SO.sub.4), Friedel Crafts
alkylation (R-X plus catalyst), Friedel Crafts acylation (RCOX plus
catalyst), iodination (I.sub.2/catalyst), Side chain oxidation
(KMnO.sub.4), reduction of nitro groups, reduction of aromatic
ketones, Side chain bromination, nucleophilic aromatic substitution
(S.sub.NAr), and aryne formation (S.sub.NAr via arynes). Reactions
of aldehydes and ketones can include, but are not limited to:
hydrate formation (H.sub.2O), cyanohydrin formation (CN), reduction
of aldehydes (NaBH.sub.4), reduction of aldehydes (LiAlH.sub.4),
reduction of ketones (NaBH.sub.4), reduction of ketones
(LiAlH.sub.4), Grignard addition to aldehydes, Grignard addition to
ketones, acetal formation (ROH/H.sup.+), acetal hydrolysis
(H.sub.3O.sup.+), imine, formation (RNH.sub.2), Enamine formation
(R.sub.2NH), Wolff-Kishner: reduction to alkanes, Clemmensen,
reduction to alkanes, oxidation to carboxylic acid
(H.sub.2CrO.sub.4 or KMnO.sub.4), keto-enol tautomerism, enolate
formation, aldol addition reaction, alkylation of enolates, Wittig
reaction (alkene formation), thioacetal formation, imine
hydrolysis, oxidation to carboxylic acids (Tollens), haloform
reaction, Baeyer-Villiger reaction, aldol condensation, Cannizarro
reaction. Reactions of carboxylic acids can include, but are not
limited to: deprotonation (carboxylate formation), formation via
Grignard and CO.sub.2, conversion to acid chloride (SOCl.sub.2),
reduction (LiAlH.sub.4), Fischer esterification, and
decarboxylation (of .beta.-keto acids). Reactions of esters can
include, but are not limited to: reduction to aldehydes (DIBAL-H),
reduction to alcohols (LiAlH.sub.4), hydrolysis to carboxylic acid
(acidic), hydrolysis to carboxylic acid (basic), addition of
Grignard reagents to esters, Claisen condensation, and
transesterification (basic conditions). Reactions of acyl halides
can include, but are not limited to: conversion to esters (ROH),
conversion to carboxylic acids (H.sub.2O), conversion to anhydrides
(RCO.sub.2), conversion to amides (RNH.sub.2), conversion to
ketones (Gilman reagents), and conversion to aldehydes
(LiAlH(OtBu).sub.3). Reactions of .alpha.,.beta.-unsaturated
ketones (enones) can include, but are not limited to: Michael
reaction (conjugate addition of enolates), conjugate addition of
Gilman reagents, conjugate addition of other nucleophiles.
Reactions of amines and amides can include, but are not limited to:
dehydration of amides to nitriles (P.sub.2O.sub.5), Hofmann
rearrangement, Gabriel synthesis of amines, reductive amination,
formation of diazonium salts, reactions of diazonium salts, amide
formation using DCC, amide formation from acid halides, and Curtius
rearrangement. Reactions of nitriles can include, but are not
limited to: addition of Grignard reagents to nitriles, reduction to
amines (LiAlH.sub.4), hydrolysis to carboxylic acids. Optionally,
potential derivatives of compounds disclosed herein can be tested
for caspase activity and/or the ability to inhibit virally-induced
apoptosis and/or suppress viral replication using methods disclosed
herein (e.g., caspase 3/7 activity assay, ATP cell viability assay)
or using other methods known in the art.
[0064] The niclosamide compound may be: (a) niclosamide, (b) a
niclosamide derivative, (c) a metabolite or prodrug of (a) or (b),
or (d) a pharmaceutically acceptable salt of (a), (b), or (c). In
some embodiments, the niclosamide or niclosamide derivative has a
structure of FIG. 9A, FIG. 9B, FIG. 9C, or FIG. 9D (Formula 1,
Formula 2, Formula 3, or Formula 4, respectively).
[0065] In some embodiments, the niclosamide or niclosamide
derivative has a structure of FIG. 9A (Formula 1):
##STR00001##
or a pharmaceutically acceptable salt thereof, where R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 can be independently
selected from the group consisting of a H; F; Cl; Br; I; OH; ketone
(.dbd.O); (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers;
(C.sub.2-6)alkynyl, where the triple bond can be located at any
position in the alkenyl carbon chain, including any alkynyl
conformational isomers; ether [--OR, where R can include
(C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; and (C.sub.2-6)alkenyl,
such as ethenyl, propenyl, butenyl, where the double bond can be
located at any position in the alkenyl carbon chain, including any
alkenyl conformational isomers]; aralkyl; alkaryl; halogenated
alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl;
cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino;
arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino;
hydroxyl; alkoxy; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro;
carbamoyl; trifluoromethyl; phenoxy; benzyloxy; alkaryl; arylalkyl;
carbamate; amino; alkylamino; arylamino; dialkylamino;
alkylarylamino; diarylamino; heteroalkyl;
alkyltriphenylphosphonium; heterocyclyl; acyl halide [--COX, where
X can include F, Cl, Br, and I]; carbonyl [--COR, where R can
include (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; aldehyde
(--CHO); ester [--OC(.dbd.O)R, --ROC(.dbd.O)R', RC(.dbd.O)OR',
--C(.dbd.O)OR', where R and R' can include (C.sub.1-14)alkyl, such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; and (C.sub.2-14)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, including any alkenyl
conformational isomers]; carbonate ester [--OCOOR, where R can
include (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.1-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; carboxyl
(--COOH); amide [--CONR'R'', where R' and R'' can be independently
selected from hydrogen; alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; amines
[--NR'R'', where R' and R'' can be independently selected from
hydrogen; (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; cyanate
(--OCN); isocynate (--NCO); nitrate (--ONO.sub.2); nitrile (--CN);
isonitrile (--NC); nitroso (--NO); oxime (--CH.dbd.NOH); borono
--B(OH).sub.2; borono and boronate [--B(OR')(R''), where R can
include H; (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; borinate
[--B(R')(OR''), where R' and R'' can be independently selected from
hydrogen; (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; phosophino
[--PR.sub.2, where R can include hydrogen; (C.sub.1-6)alkyl, such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; and (C.sub.2-6)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, including any alkenyl
conformational isomers]; phosphate [--OP(.dbd.O)(OR).sub.2, where R
can include H; (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers; phosphono
[--RP(.dbd.O)(OH), where R can include (C.sub.1-6)alkyl, such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; and (C.sub.2-6)alkenyl, such as ethenyl, propenyl,
butenyl, where the double bond can be located at any position in
the alkenyl carbon chain, including any alkenyl conformational
isomers]; thiol (--SH); thioalkyl; alkylthio; sulfide [--SR, where
R can include (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; disulfide
[--SSR, where R can include (C.sub.1-6)alkyl, such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; and (C.sub.2-6)alkenyl, such as ethenyl, propenyl,
butenyl, where the double bond can be located at any position in
the alkenyl carbon chain, including any alkenyl conformational
isomers], sulfonamide; sulfinyl [--S(.dbd.O)R, where R can include
(C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; and (C.sub.2-6)alkenyl,
such as ethenyl, propenyl, butenyl, where the double bond can be
located at any position in the alkenyl carbon chain, including any
alkenyl conformational isomers]; sulfino (--SO.sub.2H); sulfo
(--SO.sub.3H); thiocyanate; isothiocyanate; carbonothioyl
[--C(.dbd.S)R where R can include (C.sub.1-6)alkyl, such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; and (C.sub.2-6)alkenyl, such as ethenyl, propenyl,
butenyl, where the double bond can be located at any position in
the alkenyl carbon chain, including any alkenyl conformational
isomers]; where R.sub.2 and R.sub.4 can be bonded together to form
an (C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring, such as a
five-membered ring or a six-membered ring; and where R.sub.4 and
R.sub.5 can be bonded together to form an (C.sub.1-8)alkane ring
and/or (C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring; and where R.sub.4 and R.sub.6 can be bonded
together to form an (C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene
ring, such as a five-membered ring or a six-membered ring; and
where R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring, such as a
five-membered ring or a six-membered ring.
[0066] In some embodiments, the niclosamide or niclosamide
derivative has the structure of FIG. 9B (Formula 2):
##STR00002##
or a pharmaceutically acceptable salt thereof, where R.sub.7,
R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12 can be independently
selected from the group consisting of a H; F; Cl; Br; I; OH; ketone
(.dbd.O); (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers;
(C.sub.2-6)alkynyl, where the triple bond can be located at any
position in the alkenyl carbon chain, including any alkynyl
conformational isomers; ether [--OR, where R can include
(C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; and (C.sub.2-6)alkenyl,
such as ethenyl, propenyl, butenyl, where the double bond can be
located at any position in the alkenyl carbon chain, including any
alkenyl conformational isomers]; aralkyl; alkaryl; halogenated
alkyl; heteroalkyl; aryl; heterocyclyl; cycloalkyl; cycloalkenyl;
cycloalkynyl; hydroxyalkyl; aminoalkyl; amino; alkylamino;
arylamino; dialkylamino; alkylarylamino; diarylamino; acylamino;
hydroxyl; alkoxy; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro;
carbamoyl; trifluoromethyl; phenoxy; benzyloxy; alkaryl; arylalkyl;
carbamate; amino; alkylamino; arylamino; dialkylamino;
alkylarylamino; diarylamino; heteroalkyl;
alkyltriphenylphosphonium; heterocyclyl; acyl halide [--COX, where
X can include F, Cl, Br, and I]; carbonyl [--COR, where R can
include (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; aldehyde
(--CHO); ester [--OC(.dbd.O)R, --ROC(.dbd.O)R', RC(.dbd.O)OR',
--C(.dbd.O)OR', where R and R' can include (C.sub.1-14)alkyl, such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; and (C.sub.2-14)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, including any alkenyl
conformational isomers]; carbonate ester [--OCOOR, where R can
include (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.1-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; carboxyl
(--COOH); amide [--CONR'R'', where R' and R'' can be independently
selected from hydrogen; alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; amines
[--NR'R'', where R' and R'' can be independently selected from
hydrogen; (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; cyanate
(--OCN); isocynate (--NCO); nitrate (--ONO.sub.2); nitrile (--CN);
isonitrile (--NC); nitroso (--NO); oxime (--CH.dbd.NOH); borono
--B(OH).sub.2; borono and boronate [--B(OR')(R''), where R can
include H; (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; borinate
[--B(R')(OR''), where R' and R'' can be independently selected from
hydrogen; (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; phosophino
[--PR.sub.2, where R can include hydrogen; (C.sub.1-6)alkyl, such
as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; and (C.sub.2-6)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, including any alkenyl
conformational isomers]; phosphate [--OP(.dbd.O)(OR).sub.2, where R
can include H; (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers; phosphono
[--RP(.dbd.O)(OH), where R can include (C.sub.1-6)alkyl, such as
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; and (C.sub.2-6)alkenyl, such as ethenyl, propenyl,
butenyl, where the double bond can be located at any position in
the alkenyl carbon chain, including any alkenyl conformational
isomers]; thiol (--SH); thioalkyl; alkylthio; sulfide [--SR, where
R can include (C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-6)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers]; disulfide
[--SSR, where R can include (C.sub.1-6)alkyl, such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; and (C.sub.2-6)alkenyl, such as ethenyl, propenyl,
butenyl, where the double bond can be located at any position in
the alkenyl carbon chain, including any alkenyl conformational
isomers], sulfonamide; sulfinyl [--S(.dbd.O)R, where R can include
(C.sub.1-6)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; and (C.sub.2-6)alkenyl,
such as ethenyl, propenyl, butenyl, where the double bond can be
located at any position in the alkenyl carbon chain, including any
alkenyl conformational isomers]; sulfino (--SO.sub.2H); sulfo
(--SO.sub.3H); thiocyanate; isothiocyanate; carbonothioyl
[--C(.dbd.S)R where R can include (C.sub.1-6)alkyl, such as methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; and (C.sub.2-6)alkenyl, such as ethenyl, propenyl,
butenyl, where the double bond can be located at any position in
the alkenyl carbon chain, including any alkenyl conformational
isomers]; where R.sub.7 and R.sub.10 can be bonded together to form
an (C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring, such as a
five-membered ring or a six-membered ring; and where R.sub.4 and
R.sub.5 can be bonded together to form an (C.sub.1-8)alkane ring
and/or (C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring; where R.sub.8 and R.sub.10 can be bonded
together to form an (C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene
ring, such as a five-membered ring or a six-membered ring; and
where R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring, such as a
five-membered ring or a six-membered ring; and where R.sub.10 and
R.sub.11 can be bonded together to form an (C.sub.1-8)alkane ring
and/or (C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring; and where R.sub.4 and R.sub.5 can be bonded
together to form an (C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene
ring, such as a five-membered ring or a six-membered ring.
[0067] In some embodiments, the niclosamide or niclosamide
derivative has the structure of FIG. 9C (Formula 3):
##STR00003##
or a pharmaceutically acceptable salt thereof, where R.sub.13 can
include (C.sub.1-14)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-14)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers.
[0068] In some embodiments, the niclosamide or niclosamide
derivative has the structure of FIG. 9D (Formula 4):
##STR00004##
or a pharmaceutically acceptable salt thereof.
[0069] In some embodiments, the compound is a derivative of
niclosamide such as p-niclosamide [48], which is incorporated
herein by reference in its entirety, or an acyl derivative of
niclosamide, such as DK-520 [49], which is incorporated herein by
reference in its entirety, or a pharmaceutically acceptable salt
thereof.
[0070] The emricasan compound may comprise: (a) emricasan, (b) an
emricasan derivative, (c) a metabolite or prodrug of (a) or (b), or
(d) a pharmaceutically acceptable salt of (a), (b), or (c). In some
embodiments, the emricasan or emricasan derivative has the
structure of FIG. 10 (Formula 5):
##STR00005##
or a pharmaceutically acceptable salt thereof, where R.sub.23,
R.sub.24, R.sub.25, R.sub.26, R.sub.27, R.sub.28, R.sub.29,
R.sub.30, R.sub.31, R.sub.32, R.sub.33, and R.sub.34, can be
independently selected from the group consisting of: H; F; Cl; Br;
I; OH; ketone (.dbd.O); ether [--OR, where R can include methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
acyl halide (--COX); carbonyl [--COR, where R can include methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
aldehyde (--CHO); carbonate ester [--OCOOR, where R can include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' can be
independently selected from hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; amines
[--NR'R'', where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; cyanate (--OCN); isocynate (--NCO);
nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC); nitroso
(--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; boronare
[--B(OR')(R''), where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; borinate [--B(R')(OR''), where R' and R''
can be independently selected from hydrogen, methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
phosophino [--PR.sub.2, where R can include hydrogen, methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
phosphono [--P(.dbd.O)(OH)(R), where R can include hydrogen,
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
phosphate (--OP(.dbd.O)(OH).sub.2; thiol (--SH); sulfide [--SR,
where R can include methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; disulfide [--SSR, where R can include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers],
sulfinyl [--S(.dbd.O)R, where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; sulfino
(--SO.sub.2H); sulfo (--SO.sub.3H); thiocyanate; isothiocyanate;
carbonothioyl [--C(.dbd.S)R where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
(C.sub.1-4)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such
as ethenyl, propenyl, butenyl, where the double bond can be located
at any position in the alkenyl carbon chain, and including any
alkenyl conformational isomers; where R.sub.23 and R.sub.24 can be
bonded together to form an (C.sub.2-8)alkane ring and/or
(C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring; where R.sub.23 and R.sub.25 can be bonded
together to form an (C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene
ring, such as a five-membered ring or a six-membered ring, where
R.sub.24 and R.sub.26 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene ring, such as a
five-membered ring or a six-membered ring; where R.sub.27 and
R.sub.30 can be bonded together to form an (C.sub.2-8)alkane ring
and/or (C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring; where R.sub.28 and R.sub.29 can be bonded
together to form an (C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene
ring, such as a five-membered ring or a six-membered ring; where
R.sub.27 and R.sub.30 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene ring, such as a
five-membered ring or a six-membered ring; and where R.sub.29 and
R.sub.30 can be bonded together to form an (C.sub.2-8)alkane ring
and/or (C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring.
[0071] The compounds of the invention (e.g., niclosamide compounds;
emricasan compounds; CDK inhibitors; proteasome inhibitors;
compounds in Tables 7-10 and FIGS. 9-22, or a prodrug, metabolite,
or derivative thereof) may be administered to the subject in
combinations of two or more. For example, in some embodiments, the
at least one compound comprises a combination of the niclosamide
compound and the emricasan compound, and the niclosamide compound
and the emricasan compound are administered simultaneously,
together within the same composition or in separate compositions.
In other embodiments, the at least one compound comprises a
combination of the niclosamide compound and the emricasan compound,
and wherein the niclosamide compound and the emricasan compound are
administered consecutively in any order.
[0072] In some embodiments, the at least one compound comprises the
CDK inhibitor, and the CDK inhibitor has a structure shown in FIG.
11 (PHA-690509 or a derivative thereof; Formula 6), FIG. 12
(Alvocidib or a derivative thereof; Formula 7), FIG. 13 (PHA-793887
or a derivative thereof; Formula 8), FIG. 14 (Dinaciclib or a
derivative thereof; Formula 9), FIG. 15 (Seliciclib or a derivative
thereof; Formula 10), or a pharmaceutically acceptable salt of any
of the foregoing.
[0073] In some embodiments, the CDK inhibitor has the structure
shown in FIG. 11 (Formula 6, which encompasses the CDK inhibitor
PHA-690509 and derivatives of PHA-690509):
##STR00006##
or a pharmaceutically acceptable salt thereof, where R.sub.7,
R.sub.8, R.sub.9, R.sub.10 can be independently selected from the
group consisting of H; F; Cl; Br; I; OH; ketone (.dbd.O); ether
[--OR, where R can include methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such
as ethenyl, propenyl, butenyl, where the double bond can be located
at any position in the alkenyl carbon chain, and including any
alkenyl conformational isomers]; acyl halide (--COX); carbonyl
[--COR, where R can include methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such
as ethenyl, propenyl, butenyl, where the double bond can be located
at any position in the alkenyl carbon chain, and including any
alkenyl conformational isomers]; aldehyde (--CHO); carbonate ester
[--OCOOR, where R can include methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such
as ethenyl, propenyl, butenyl, where the double bond can be located
at any position in the alkenyl carbon chain, and including any
alkenyl conformational isomers]; carboxyl (--COOH); amide
[--CONR'R'', where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; amines [--NR'R'', where R' and R'' can be
independently selected from hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; cyanate
(--OCN); isocynate (--NCO); nitrate (--ONO.sub.2); nitrile (--CN);
isonitrile (--NC); nitroso (--NO); oxime (--CH.dbd.NOH); borono
--B(OH).sub.2; boronare [--B(OR')(R''), where R' and R'' can be
independently selected from hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; borinate
[--B(R')(OR''), where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; phosophino [--PR.sub.2, where R can
include hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; phosphono [--P(.dbd.O)(OH)(R), where R can
include hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; phosphate (--OP(.dbd.O)(OH).sub.2; thiol
(--SH); sulfide [--SR, where R can include methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; disulfide
[--SSR, where R can include methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such
as ethenyl, propenyl, butenyl, where the double bond can be located
at any position in the alkenyl carbon chain, and including any
alkenyl conformational isomers], sulfinyl [--S(.dbd.O)R, where R
can include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; sulfino (--SO.sub.2H); sulfo
(--SO.sub.3H); thiocyanate; isothiocyanate; carbonothioyl
[--C(.dbd.S)R where R can include methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
(C.sub.1-4)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; and (C.sub.2-4)alkenyl,
such as ethenyl, propenyl, butenyl, where the double bond can be
located at any position in the alkenyl carbon chain, and including
any alkenyl conformational isomers; and where R.sub.8 and R.sub.9
can be bonded together to form a (C.sub.2-8)lactam ring with or
without a double carbon-carbon bond.
[0074] In some embodiments, the CDK inhibitor has the structure
shown in FIG. 12 (Formula 7, encompassing the CDK inhibitor
Alvocidib and derivatives of Alvocidib):
##STR00007##
or a pharmaceutically acceptable salt thereof, where R.sub.11,
R.sub.12, R.sub.13, R.sub.14 and R.sub.15 can be independently
selected from a group consisting of H; F; Cl; Br; I; OH; ketone
(.dbd.O); ether [--OR, where R can include methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; acyl
halide (--COX); carbonyl [--COR, where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; aldehyde
(--CHO); carbonate ester [--OCOOR, where R can include methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' can be
independently selected from hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; amines
[--NR'R'', where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; cyanate (--OCN); isocynate (--NCO);
nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC); nitroso
(--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; boronare
[--B(OR')(R''), where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; borinate [--B(R')(OR''), where R' and R''
can be independently selected from hydrogen, methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
phosophino [--PR.sub.2, where R can include hydrogen, methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
phosphono [--P(.dbd.O)(OH)(R), where R can include hydrogen,
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
phosphate (--OP(.dbd.O)(OH).sub.2; thiol (--SH); sulfide [--SR,
where R can include methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; disulfide [--SSR, where R can include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers],
sulfinyl [--S(.dbd.O)R, where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; sulfino
(--SO.sub.2H); sulfo (--SO.sub.3H); thiocyanate; isothiocyanate;
carbonothioyl [--C(.dbd.S)R where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
(C.sub.1-4)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such
as ethenyl, propenyl, butenyl, where the double bond can be located
at any position in the alkenyl carbon chain, and including any
alkenyl conformational isomers; where R.sub.11 and R.sub.15 can be
bonded together to form an (C.sub.2-8)alkane ring and/or
(C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring; and where R.sub.13 and R.sub.14 can be bonded
together to form an (C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene
ring, such as a five-membered ring or a six-membered ring.
[0075] In some embodiments, the CDK inhibitor has the structure
shown in FIG. 13 (Formula 8, which encompasses the CDK inhibitor
PHA-793887 and derivatives of PHA-793887:
##STR00008##
or a pharmaceutically acceptable salt thereof, where R.sub.16 and
R.sub.17 can be independently selected from a group consisting of
H; F; Cl; Br; I; OH; ketone (.dbd.O); ether [--OR, where R can
include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; acyl halide (--COX); carbonyl [--COR,
where R can include methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; aldehyde (--CHO); carbonate ester
[--OCOOR, where R can include methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such
as ethenyl, propenyl, butenyl, where the double bond can be located
at any position in the alkenyl carbon chain, and including any
alkenyl conformational isomers]; carboxyl (--COOH); amide
[--CONR'R'', where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; amines [--NR'R'', where R' and R'' can be
independently selected from hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; cyanate
(--OCN); isocynate (--NCO); nitrate (--ONO.sub.2); nitrile (--CN);
isonitrile (--NC); nitroso (--NO); oxime (--CH.dbd.NOH); borono
--B(OH).sub.2; boronare [--B(OR')(R''), where R' and R'' can be
independently selected from hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; borinate
[--B(R')(OR''), where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; phosophino [--PR.sub.2, where R can
include hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; phosphono [--P(.dbd.O)(OH)(R), where R can
include hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; phosphate (--OP(.dbd.O)(OH).sub.2; thiol
(--SH); sulfide [--SR, where R can include methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; disulfide
[--SSR, where R can include methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such
as ethenyl, propenyl, butenyl, where the double bond can be located
at any position in the alkenyl carbon chain, and including any
alkenyl conformational isomers], sulfinyl [--S(.dbd.O)R, where R
can include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; sulfino (--SO.sub.2H); sulfo
(--SO.sub.3H); thiocyanate; isothiocyanate; carbonothioyl
[--C(.dbd.S)R where R can include methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
(C.sub.1-4)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; and (C.sub.2-4)alkenyl,
such as ethenyl, propenyl, butenyl, where the double bond can be
located at any position in the alkenyl carbon chain, and including
any alkenyl conformational isomers.
[0076] In some embodiments, the CDK inhibitor has the structure
shown in FIG. 14 (Formula 9, which encompasses the CDK inhibitor
Dinaciclib and derivatives of Dinaciclib):
##STR00009##
or a pharmaceutically acceptable salt thereof, where R.sub.18 and
R.sub.19 can be selected from the group consisting of H; F; Cl; Br;
I; OH; ketone (.dbd.O); ether [--OR, where R can include methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
acyl halide (--COX); carbonyl [--COR, where R can include methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
aldehyde (--CHO); carbonate ester [--OCOOR, where R can include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' can be
independently selected from hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; amines
[--NR'R'', where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; cyanate (--OCN); isocynate (--NCO);
nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC); nitroso
(--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; boronare
[--B(OR')(R''), where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; borinate [--B(R')(OR''), where R' and R''
can be independently selected from hydrogen, methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
phosophino [--PR.sub.2, where R can include hydrogen, methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
phosphono [--P(.dbd.O)(OH)(R), where R can include hydrogen,
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
phosphate (--OP(.dbd.O)(OH).sub.2; thiol (--SH); sulfide [--SR,
where R can include methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; disulfide [--SSR, where R can include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers],
sulfinyl [--S(.dbd.O)R, where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; sulfino
(--SO.sub.2H); sulfo (--SO.sub.3H); thiocyanate; isothiocyanate;
carbonothioyl [--C(.dbd.S)R where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
(C.sub.1-4)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; and (C.sub.2-4)alkenyl,
such as ethenyl, propenyl, butenyl, where the double bond can be
located at any position in the alkenyl carbon chain, and including
any alkenyl conformational isomers.
[0077] In some embodiments, the CDK inhibitor as the structure
shown in FIG. 15 (Formula 10, which encompasses the CDK inhibitor
Selicicib and derivatives of Selicicib:
##STR00010##
or a pharmaceutically acceptable salt thereof, where R.sub.20,
R.sub.21, R.sub.22 can be selected from the group consisting of H;
F; Cl; Br; I; OH; ketone (.dbd.O); ether [--OR, where R can include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
acyl halide (--COX); carbonyl [--COR, where R can include methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
aldehyde (--CHO); carbonate ester [--OCOOR, where R can include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' can be
independently selected from hydrogen, methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; amines
[--NR'R'', where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; cyanate (--OCN); isocynate (--NCO);
nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC); nitroso
(--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; boronare
[--B(OR')(R''), where R' and R'' can be independently selected from
hydrogen, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl,
isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl,
propenyl, butenyl, where the double bond can be located at any
position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; borinate [--B(R')(OR''), where R' and R''
can be independently selected from hydrogen, methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
phosophino [--PR.sub.2, where R can include hydrogen, methyl,
ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
phosphono [--P(.dbd.O)(OH)(R), where R can include hydrogen,
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers];
phosphate (--OP(.dbd.O)(OH).sub.2; thiol (--SH); sulfide [--SR,
where R can include methyl, ethyl, n-propyl, isopropyl, n-butyl,
sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl, such as
ethenyl, propenyl, butenyl, where the double bond can be located at
any position in the alkenyl carbon chain, and including any alkenyl
conformational isomers]; disulfide [--SSR, where R can include
methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl,
tert-butyl; (C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl,
where the double bond can be located at any position in the alkenyl
carbon chain, and including any alkenyl conformational isomers],
sulfinyl [--S(.dbd.O)R, where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers]; sulfino
(--SO.sub.2H); sulfo (--SO.sub.3H); thiocyanate; isothiocyanate;
carbonothioyl [--C(.dbd.S)R where R can include methyl, ethyl,
n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl;
(C.sub.2-4)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, and including any alkenyl conformational isomers];
(C.sub.1-4)alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; and (C.sub.2-4)alkenyl,
such as ethenyl, propenyl, butenyl, where the double bond can be
located at any position in the alkenyl carbon chain, and including
any alkenyl conformational isomers.
[0078] In some embodiments, the at least one compound comprises the
CDK inhibitor, and the CDK inhibitor has a structure shown in FIG.
16, or a pharmaceutically acceptable salt thereof.
[0079] In some embodiments, the at least one compound comprises a
combination of the CDK inhibitor and the emricasan compound, and
wherein the CDK inhibitor and the emricasan compound are
administered simultaneously, together within the same composition
or in separate compositions. In other embodiments, the at least one
compound comprises a combination of the CDK inhibitor and an
emricasan compound, and wherein the CDK inhibitor and the emricasan
compound are administered consecutively in any order.
[0080] In some embodiments, the at least one compound is selected
from among SN-38, Echinomycin, (S)-(+)-Camptothecin, BI-2536,
10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Dinaciclib, Vincristine, Idarubicin, Thapsigargin, Lexibulin,
Ixazomib, Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine,
RGB-286147, Vinorelbine, Bardoxolone methyl, Cycloheximide,
Actinomycin D, AZD-7762, PF-184, CHIR-124, Cyanein, Triptolide,
KX-01, PF-477736, Epirubicin, Mycophenolate (mycophenolic acid),
Daunorubicin, PIK-75, Vindesine, Torin-2, 7-Hydroxystaurosporine,
CGP-60474, Floxuridine, Go-6976, OSU-03012, a prodrug of any of the
foregoing, a metabolite of any of the foregoing, a derivative of
any of the foregoing, or a pharmaceutically acceptable salt of any
of the foregoing.
[0081] The methods of the invention may be used to treat an
existing Flavivirus infection in a subject, or the methods of the
invention may be used prophylactically to prevent a Flavivirus
infection in a subject. As used herein, in this context, the term
"prevent" or "prevention" is inclusive of delaying the onset of
infection and/or one or more symptoms of infection, and precluding
the occurrence or reoccurrence of infection and/or one or more
symptoms of infection. Thus, in some embodiments, the subject has
the Flavivirus infection at the time the at least one compound is
administered, and the at least one compound is administered as
therapy.
[0082] In some embodiments, the methods further comprise, prior to
administering the at least one compound to the subject, identifying
the subject as having the Flavivirus infection. The identifying
step may comprise assaying a biological sample (e.g., blood,
saliva, or urine) obtained from the subject for the presence of
Flavivirus nucleic acids or Flavivirus proteins (e.g., Zika virus
nucleic acids or Zika virus proteins). In some embodiments,
assaying includes the use of reverse transcriptase-polymerase chain
reaction (RT-PCR), immunological assay (e.g., ELISA), or
Plaque-reduction neutralization testing (PRNT).
[0083] One or more compounds of the invention (also referred to
herein as the active ingredients) are administered by any route
appropriate to the condition to be treated. Suitable routes include
oral, rectal, nasal, topical (including buccal and sublingual),
vaginal and parenteral (including subcutaneous, intramuscular,
intravenous, intradermal, intrathecal and epidural), and the like.
It will be appreciated that the preferred route may vary with for
example the condition of the subject. In some embodiments, at least
one compound of the invention is administered orally, nasally,
rectally, parenterally, subcutaneously, intramuscularly, or
intravascularly (e.g., intravenously).
[0084] In some embodiments, the methods further comprise
administering an additional agent for treating or preventing
Flavivirus infection, or a symptom thereof, in the same formulation
as the at least one compound, or in a separate formulation before,
during, or after administration of the at least one compound. The
additional agent may be one or more compounds of the invention, or
one or more different agents, or both.
[0085] In some embodiments, at least one compound of the invention
is administered to the subject in a composition, wherein the
composition comprises the at least one compound and a
pharmaceutically acceptable buffer, carrier, or diluent.
[0086] Another aspect of the invention concerns a composition
comprising a combination of two or more of the following:
[0087] (a) a niclosamide compound, or
[0088] (b) an emricasan compound, or
[0089] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0090] (d) a proteasome inhibitor, or
[0091] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
[0092] Another aspect of the invention concerns a composition
comprising:
[0093] (a) a niclosamide compound, or
[0094] (b) an emricasan compound, or
[0095] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0096] (d) a proteasome inhibitor, or
[0097] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing; and
[0098] an additional agent effective for the treatment of one or
more symptoms of Flavivirus infection. In some embodiments, the
Flavivirus infection is Zika virus infection.
[0099] Another aspect of the invention concerns a packaged dosage
formulation comprising at least one compound in a pharmaceutically
acceptable dosage in one or more packages, packets, or containers,
wherein the at least one compound comprises:
[0100] (a) a niclosamide compound, or
[0101] (b) an emricasan compound, or
[0102] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0103] (d) a proteasome inhibitor, or
[0104] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
[0105] In some embodiments, the packaged dosage formulation
comprises a combination of two or more of the foregoing compounds
from (a), (b), (c), (d), or (e).
[0106] Another aspect of the invention concerns a kit comprising,
in one or more containers, at least one compound comprising:
[0107] (a) a niclosamide compound, or
[0108] (b) an emricasan compound, or
[0109] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0110] (d) a proteasome inhibitor, or
[0111] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
[0112] In some embodiments, the kit comprises a combination of two
or more of the foregoing compounds from (a), (b), (c), (d), or (e).
In some embodiments, the kit further comprises an additional agent
effective for the treatment or prevention of Flavivirus virus
infection. In some embodiments, the kit further comprises an
additional agent effective for the treatment of one or more
symptoms of Flavivirus infection.
[0113] As with the methods of the invention, in the compositions,
packaged dosage formulations, and kits of the invention, the
niclosamide compound may be: (a) niclosamide, (b) a niclosamide
derivative, (c) a metabolite or prodrug of (a) or (b), or (d) a
pharmaceutically acceptable salt of (a), (b), or (c); the emricasan
compound may comprise: (a) emricasan, (b) an emricasan derivative,
(c) a metabolite or prodrug of (a) or (b), or (d) a
pharmaceutically acceptable salt of (a), (b), or (c); the CDK
inhibitor may comprise, for example: a compound having the
structure shown in FIG. 11 (PHA-690509 or a derivative thereof),
FIG. 12 (Alvocidib or a derivative thereof), FIG. 13 (PHA-793887 or
a derivative thereof), FIG. 14 (Dinaciclib or a derivative
thereof), FIG. 15 (Seliciclib or a derivative thereof), or a
pharmaceutically acceptable salt of any of the foregoing, or a
compound having a structure shown in FIG. 16, or a pharmaceutically
acceptable salt thereof; and a proteasome inhibitor may comprise,
for example, the proteasome inhibitors in FIGS. 19A to 19E-2.
[0114] Various techniques may be used to increase bioavailability
of the compounds of the invention (e.g., compounds of in Tables
7-10, and FIGS. 9-22, niclosamide compounds, emricasan compounds,
CDK inhibitors, and protease inhibitors). For example, prodrugs of
compounds can be prepared. Prodrugs employ various physical and
chemical modifications to improve features of the active drug, and
in some embodiments may be viewed as pharmacologically inactive
prodrug functional groups that undergo a chemical transformation or
enzymatic cleavage to liberate the active parent drug and produce
the desired effect in the body. Utilizing a prodrug approach can
yield benefits such as enhanced solubility, improved selective
targeting of drugs to anatomical sites, protection from rapid
metabolism and elimination, reduction toxic effects of an active
drug on other parts of the body, and enhanced patient
compliance.
[0115] Non-limiting examples of techniques useful for enhancing the
bioavailability of BCS Class II drugs include use of co-solvents,
hydrotropy, micronization, change in dielectric constant of
solvent, amorphous forms, chemical modification of the drug, use of
surfactants, inclusion complex, alteration of pH of solvent, use of
hydrates or solvates, use of soluble prodrugs, application of
ultrasonic waves, functional polymer technology, controlled
precipitation technology, evaporative precipitation in aqueous
solution, use of precipitation inhibitors, solvent deposition,
precipitation, selective adsorption on insoluble carriers, size
reduction technologies, lipid based delivery systems, micellar
technologies, porous micro particle technology, solid dispersion
technique, and various types of solid dispersion systems.
[0116] For example, a method for synthesizing a phosphate prodrug
of niclosamide (p-niclosamide) with improved water solubility is
described in Pan J-X et al., "Niclosamide, an antihelminthic agent,
demonstrates antitumor activity by blocking multiple signaling
pathways of cancer stem cells," Chin J Cancer, 2012, 31(4):178-184,
which is incorporated by reference in its entirety (see, for
example, Scheme 1 at page 183). This method can be used to prepare
pro-drugs of niclosamide compounds and other compounds of the
invention, such as compounds in Table 7 and FIG. 16.
[0117] Other methods for enhancement of bioavailability (e.g., by
enhancement of solubility) are described in Reddy M S et al.,
"Solubility enhancement of fenofibrate, a BCS class II drug, by
self emulsifying drug delivery systems," International Research
Journal of Pharmacy, 2011, 2(11): 173-177; Khamkar G S, "Self micro
emulsifying drug delivery system (SMEED) o/w microemulsion for BCS
Class II drugs: an approach to enhance oral bioavailability,"
International Journal of Pharmacy and Pharmaceutical Sciences,
2011, 3(3):1-3; Elgart A et al., "Improved oral bioavailability of
BCS class 2 compounds by self nano-emulsifying drug delivery
systems (SNEDDS): the underlying mechanisms for amiodarone and
talinolol", Pharm Res., 2013 December; 30(12):3029-44; Singh N. et
al. "Techniques for bioavailability enhancement of BCS class II
drugs: a review," International Journal of Pharmaceutical and
Chemical Science, 2013, 2(2):1092-1101; Elkihel L et al.,
"Synthesis and orally macrofilaricidal evaluation of niclosamide
lymphotropic prodrugs," Arzneimittelforschung, 1994, 44(11):
1259-64; and Kansara H. et al., "Techniques used to enhance
bioavailability of BCS class II drugs: a review," Int. J. Drug Dev.
& Res., 2015, 7(1):82-93, which are each incorporated herein by
reference in its entirety.
[0118] International Patent Publication WO 2016/004166 (Wang G et
al.), which is incorporated herein by reference in its entirety,
describes boron-based prodrugs of phenol- or aromatic hydroxyl
group-containing therapeutic molecules, including niclosamide
(Table 1, page 15). The boron-prodrug platform can be utilized to
make prodrugs of compounds disclosed herein.
[0119] International Patent Publication WO 2009/143297 (Naweed
Muhammad et al.), which is incorporated herein by reference in its
entirety, describes carbonate prodrugs which comprise a carbonic
phosphoric anhydride prodrug moiety attached to the hydroxyl or
carboxyl group of a parent drug moiety.
[0120] Chemical reactions, reactants, and reagents that may be
utilized to enhance solubility and make prodrugs of compounds are
described in March's Advanced Organic Chemistry, 7.sup.th edition,
2013, Michael B. Smith, which is incorporated herein by reference
in its entirety.
[0121] Compounds, and compositions comprising them, useful in the
methods of the subject invention can be formulated according to
known methods for preparing pharmaceutically useful compositions.
Formulations are described in detail in a number of sources which
are well known and readily available to those skilled in the art.
For example, Remington's Pharmaceutical Science by E. W. Martin
describes formulations which can be used in connection with the
subject invention. In general, the compositions of the subject
invention will be formulated such that an effective amount of at
least one compound of the invention is combined with a suitable
carrier or diluent in order to facilitate effective administration
of the composition. The compositions used in the present methods
can also be in a variety of forms. These include, for example,
solid, semi-solid, and liquid dosage forms, such as tablets, pills,
powders, liquid solutions or suspension, suppositories, injectable
and infusible solutions, and sprays. The preferred form depends on
the intended mode of administration and therapeutic application.
The compositions also preferably include conventional
pharmaceutically acceptable carriers and diluents which are known
to those skilled in the art. Examples of carriers or diluents for
use with the subject peptides and polynucleotides include, but are
not limited to, water, saline, oils including mineral oil, ethanol,
dimethyl sulfoxide, gelatin, cyclodextrans, magnesium stearate,
dextrose, cellulose, sugars, calcium carbonate, glycerol, alumina,
starch, and equivalent carriers and diluents, or mixtures of any of
these. Formulations of the compounds of the invention can also
comprise suspension agents, protectants, lubricants, buffers,
preservatives, and stabilizers. To provide for the administration
of such dosages for the desired therapeutic treatment,
pharmaceutical compositions of the invention will advantageously
comprise between about 0.1% and 45%, and especially, 1 and 15% by
weight of the total of one or more of the peptide or polynucleotide
based on the weight of the total composition including carrier or
diluent.
[0122] The compounds of the subject invention can also be
administered utilizing liposome technology, slow release capsules,
implantable pumps, and biodegradable containers. These delivery
methods can, advantageously, provide a uniform dosage over an
extended period of time.
[0123] The compounds of the invention can also be modified by the
addition of chemical groups, such as PEG (polyethylene glycol).
PEGylated compounds can exhibit extended half-lives in vivo in
comparison to compounds that are not PEGylated when administered in
vivo. Compounds can also be modified to improve cell membrane
permeability. In one embodiment, cell membrane permeability can be
improved by attaching a lipophilic moiety, such as a steroid, to
the compound. In another embodiment, compounds of the invention can
be coupled to a cell-penetrating peptide (CPP). CPPs are typically
short peptides that are highly cationic and typically include
several arginine and/or lysine amino acids. CPPs can be classified
as hydrophilic, amphiphilic, or periodic sequence.
[0124] The subject invention also concerns a packaged dosage
formulation comprising in one or more containers at least compound,
and/or composition of the subject invention formulated in a
pharmaceutically acceptable dosage. The package can contain
discrete quantities of the dosage formulation, such as tablet,
capsules, lozenge, and powders. The quantity of compound in a
dosage formulation and that can be administered to a patient can
vary from about 1 mg to about 5000 mg, or about 1 mg to about 2000
mg, or more typically about 1 mg to about 500 mg, or about 5 mg to
about 250 mg, or about 10 mg to about 100 mg.
[0125] Optionally, the methods include, prior to administration of
at least one compound of the invention, or re-administration of at
least one compound of the invention, determining whether the
subject has a Flavivirus infection (e.g., a Zika virus infection)
or one or more symptoms consistent with a Flavivirus infection. In
the case of Zika virus, during the first week after onset of
symptoms, viral RNA can often be identified in serum; thus, Zika
virus disease can be diagnosed by performing reverse
transcriptase-polymerase chain reaction (RT-PCR) on serum. Urine
and saliva samples may also be used for detection of Zika virus
(Gourinat A-C et al. (2015) Emerg Infect Dis, vol. 21, no. 1, pp.
84-86; and Musso D et al. (2015) J Clin Virol, vol. 68, pp.
53-55).
[0126] Virus-specific IgM and neutralizing antibodies typically
develop toward the end of the first week of illness; cross-reaction
with related flaviviruses (e.g., dengue and yellow fever viruses)
is common and may be difficult to discern. Plaque-reduction
neutralization testing (PRNT) can be performed to measure
virus-specific neutralizing antibodies and discriminate between
cross-reacting antibodies in primary flavivirus infections.
[0127] In the case of Zika virus, some infected individuals will
not know they have the disease because they will not have symptoms.
The most common symptoms of Zika virus infection are fever,
maculo-papular rash (often spreading from face to body), joint
pain, retro-orbital pain, or conjunctivitis (red eyes). Other
common symptoms include general non-specific such as myalgia,
asthenia, and headache. The incubation period (the time from
exposure to symptoms) for Zika virus disease is not known, but is
likely to be a few days to a week. The illness is usually mild with
symptoms lasting for several days to a week after being bitten by
an infected mosquito. The Zika virus usually remains in the blood
of an infected person for approximately a week but it can be found
longer in some individuals.
[0128] Treatment methods optionally include steps of advising that
the subject get plenty of rest and drink fluids for hydration and
administration of agents that alleviate symptoms of Flavivirus
infection (e.g., Zika virus infection), such as those that reduce
fever and pain (e.g., acetaminophen and/or paracetamol). The
methods may include administration of the fluids to the subject for
hydration.
[0129] The subject may be any age or gender. In some embodiments,
the subject is male. In some embodiments, the subject is female. In
some embodiments, the subject is a post-pubescent female. In some
embodiments, the subject is a post-pubescent, pre-menopausal
female. In some embodiments, the subject is a non-pregnant female.
In some embodiments, the subject is a pregnant female. In some
embodiments, the subject has Guillain-Barre syndrome or another
condition that is associated with ZIKV infection.
[0130] The invention further provides kits, including at least one
compound of the invention (e.g., compound of Tables 7-10, or FIGS.
9-22, or prodrug, or metabolite, or derivative or pharmaceutically
acceptable salt of a compound of Tables 7-10 or FIGS. 9-22; a
niclosamide compound; emricasan compound; CDK inhibitor; proteasome
inhibitor, or a combination of two or more of the foregoing) and
pharmaceutical formulations, packaged into suitable packaging
material, optionally in combination with instructions for using the
kit components, e.g., instructions for performing a method of the
invention. In one embodiment, a kit includes an amount of at least
one compound of the invention, and instructions for administering
at least one compound of the invention to a subject in need of
treatment on a label or packaging insert. In further embodiments, a
kit includes an article of manufacture, for delivering at least one
compound of the invention into a subject locally, regionally or
systemically, for example.
[0131] As used herein, the term "packaging material" refers to a
physical structure housing the components of the kit. The packaging
material can maintain the components in a sterile state, and can be
made of material commonly used for such purposes (e.g., paper,
corrugated fiber, glass, plastic, foil, ampules, etc.). The label
or packaging insert can include appropriate printed and/or digital
instructions, for example, for practicing a method of the
invention, e.g., treating a Flavivirus infection (e.g., Zika virus
infection), an assay for identifying a subject having a Flavivirus
infection (e.g., Zika virus infection), etc. Thus, in additional
embodiments, a kit includes a label or packaging insert including
instructions for practicing a method of the invention in solution,
in vitro, in vivo, or ex vivo.
[0132] Instructions can therefore include instructions for
practicing any of the methods of the invention described herein.
For example, pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration to a subject to treat a Flavivirus infection (e.g.,
Zika virus infection). Instructions may additionally include
indications of a satisfactory clinical endpoint or any adverse
symptoms that may occur, storage information, expiration date, or
any information required by regulatory agencies such as the Food
and Drug Administration or European Medicines Agency for use in a
human subject.
[0133] The instructions may be digital or on "printed matter,"
e.g., on paper or cardboard within the kit, on a label affixed to
the kit or packaging material, or attached to a vial or tube
containing a component of the kit. Instructions may comprise voice
or video tape and additionally be included on a computer readable
medium, such as a disk (diskette or hard disk), optical CD such as
CD- or DVD-ROM/RAM, magnetic tape, electrical storage media such as
RAM and ROM and hybrids of these such as magnetic/optical storage
media.
[0134] Kits can additionally include a buffering agent, a
preservative, or an agent for stabilizing at least one compound of
the invention. The kit can also include components for assaying for
the presence of Zika virus or other Flavivirus, e.g., an antibody
or antibody fragment specific for a Zika virus or other Flavivirus
antigen, one or more primers specific for Zika virus or other
Flavivirus nucleic acids, a control sample or a standard. Each
component of the kit can be enclosed within an individual container
or in a mixture and all of the various containers can be within
single or multiple packages.
[0135] Kits can include packaging material that is
compartmentalized to receive one or more containers such as vials,
tubes, and the like, each of the container(s) including one of the
separate elements to be used in a method described herein.
Packaging materials for use in packaging pharmaceutical products
include, by way of example only U.S. Pat. Nos. 5,323,907, 5,052,558
and 5,033,252. Examples of pharmaceutical packaging materials
include, but are not limited to, blister packs, bottles, tubes,
pumps, bags, vials, light-tight sealed containers, syringes,
bottles, and any packaging material suitable for a selected
formulation and intended mode of administration and treatment.
[0136] A kit may include one or more additional containers, each
with one or more of various materials desirable from a commercial
and user standpoint for use of the compounds for treating or
preventing Zika virus or other Flavivirus infection. Non-limiting
examples of such materials include, but not limited to, buffers,
diluents, carrier, package, container, vial and/or tube labels
listing contents and/or instructions for use, and package inserts
with instructions for use.
[0137] A label can be on or associated with a container containing
a compound of the invention. A label can be on a container when
letters, numbers or other characters forming the label are
attached, molded or etched into the container itself, a label can
be associated with a container when it is present within a
receptacle or carrier that also holds the container, e.g., as a
package insert. A label can be used to indicate that the contents
are to be used for a specific therapeutic application. The label
can also indicate directions for use of the contents, such as in
the methods described herein.
[0138] In some embodiments of the kit, the compound(s) of the
invention can be presented in a pack or dispenser device which can
contain one or more unit dosage forms containing a compound
disclosed herein. The pack can for example contain metal or plastic
foil, such as a blister pack. The pack or dispenser device can be
accompanied by instructions for administration. The pack or
dispenser can also be accompanied with a notice associated with the
container in form prescribed by a governmental agency regulating
the manufacture, use, or sale of pharmaceuticals, which notice is
reflective of approval by the agency of the form of the drug for
human or veterinary administration. Such notice, for example, can
be the labeling approved by the U.S. Food and Drug Administration
for prescription drugs, or the approved product insert.
Compositions containing a compound provided herein formulated in a
compatible pharmaceutical carrier can also be prepared, placed in
an appropriate container, and labeled for treatment of an indicated
condition.
[0139] Another aspect of the invention concerns an in vitro
screening method to identify compounds as potential treatments for
Flavivirus infection (e.g., Zika virus infection), as described
herein. The screening method comprises determining the presence or
absence of caspase activity and determining whether the compounds
reduce virally-induced apoptosis and/or suppress viral
replication.
EXEMPLIFIED EMBODIMENTS
[0140] Examples of claimed embodiments of the invention include,
but are not limited to:
Embodiment 1
[0141] A method for treating or preventing Flavivirus infection in
a human or non-human animal subject, said method comprising
administering an effective amount of at least one compound to a
subject in need thereof, wherein the at least one compound inhibits
Flavirus infection or suppresses Flavivirus-induced caspase-3
activity, and wherein the at least one compound comprises:
[0142] (a) a niclosamide compound, or
[0143] (b) an emricasan compound, or
[0144] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0145] (d) a proteasome inhibitor, or
[0146] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
Embodiment 2
[0147] The method of embodiment 1, wherein the at least one
compound comprises a combination of two or more of the
compounds.
Embodiment 3
[0148] The method of embodiment 1 or 2, wherein the Flavivirus
infection is Zika virus infection.
Embodiment 4
[0149] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises a compound in Table 7, or a prodrug,
metabolite, or derivative thereof, or a pharmaceutically acceptable
salt of any of the foregoing.
Embodiment 5
[0150] The method of any one of embodiments 1 to 4, wherein the at
least one compound comprises the niclosamide compound, and the
niclosamide compound comprises:
[0151] (a) niclosamide,
[0152] (b) a niclosamide derivative,
[0153] (c) a prodrug or metabolite of (a) or (b), or
[0154] (d) a pharmaceutically acceptable salt of (a), (b), or
(c).
Embodiment 6
[0155] The method of embodiment 5, wherein the niclosamide or
niclosamide derivative has the structure of Formula 1:
##STR00011##
or a pharmaceutically acceptable salt thereof, wherein R.sub.1,
R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6 are independently
selected from the group consisting of a H; F; Cl; Br; I; OH; ketone
(.dbd.O); (C.sub.1-6)alkyl; (C.sub.2-6)alkenyl; (C.sub.2-6)alkynyl;
ether [--OR, where R can include (C.sub.1-6)alkyl; aralkyl;
alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl;
cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl;
amino; alkylamino; arylamino; dialkylamino; alkylarylamino;
diarylamino; acylamino; hydroxyl; alkoxy; alkoxyalkyl; aryloxy;
arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy;
benzyloxy; alkaryl; arylalkyl; carbamate; amino; alkylamino;
arylamino; dialkylamino; alkylarylamino; diarylamino; heteroalkyl;
alkyltriphenylphosphonium; heterocyclyl; acyl halide [--COX, where
X is selected from F, Cl, Br, and I]; carbonyl [--COR, where R
selected from (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl]; aldehyde
(--CHO); ester [--OC(.dbd.O)R, --ROC(.dbd.O)R', RC(.dbd.O)OR',
--C(.dbd.O)OR', where R and R' is selected from
(C.sub.1-14)alkyland (C.sub.2-14)alkenyl; carbonate ester [--OCOOR,
where R is selected from (C.sub.1-6)alkyland (C.sub.1-6)alkenyl;
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' is selected
from hydrogen; alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl;
amines [--NR'R'', where R' and R'' is selected from hydrogen;
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl; cyanate (--OCN); isocynate
(--NCO); nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC);
nitroso (--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; borono
and boronate [--B(OR')(R''), where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; borinate [--B(R')(OR''),
where R' and R'' is selected from H; (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; phosophino [--PR.sub.2, where R is selected
from H; (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphate
[--OP(.dbd.O)(OR).sub.2, where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphono
[--RP(.dbd.O)(OH), where R is selected from (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; thiol (--SH); thioalkyl; alkylthio; sulfide
[--SR, where R is selected from (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; disulfide [--SSR, where R is selected from
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl, sulfonamide; sulfinyl
[--S(.dbd.O)R, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; sulfino (--SO.sub.2H); sulfo (--SO.sub.3H);
thiocyanate; isothiocyanate; carbonothioyl [--C(.dbd.S)R where R is
selected from (C.sub.1-6)alkyland (C.sub.2-6)alkenyl; wherein
R.sub.2 and R.sub.4 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-s)alkene ring; wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.4 and R.sub.6 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; and wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring.
Embodiment 7
[0156] The method of embodiment 5, wherein the niclosamide or
niclosamide derivative has the structure of Formula 2:
##STR00012##
or a pharmaceutically acceptable salt thereof, where R.sub.7,
R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12 are independently
selected from the group consisting of a H; F; Cl; Br; I; OH; ketone
(.dbd.O); (C.sub.1-6)alkyl; (C.sub.2-6)alkenyl; (C.sub.2-6)alkynyl;
ether [--OR, where R can include (C.sub.1-6)alkyl; aralkyl;
alkaryl; halogenated alkyl; heteroalkyl; aryl; heterocyclyl;
cycloalkyl; cycloalkenyl; cycloalkynyl; hydroxyalkyl; aminoalkyl;
amino; alkylamino; arylamino; dialkylamino; alkylarylamino;
diarylamino; acylamino; hydroxyl; alkoxy; alkoxyalkyl; aryloxy;
arylalkoxy; acyloxy; nitro; carbamoyl; trifluoromethyl; phenoxy;
benzyloxy; alkaryl; arylalkyl; carbamate; amino; alkylamino;
arylamino; dialkylamino; alkylarylamino; diarylamino; heteroalkyl;
alkyltriphenylphosphonium; heterocyclyl; acyl halide [--COX, where
X is selected from F, Cl, Br, and I]; carbonyl [--COR, where R
selected from (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl]; aldehyde
(--CHO); ester [--OC(.dbd.O)R, --ROC(.dbd.O)R', RC(.dbd.O)OR',
--C(.dbd.O)OR', where R and R' is selected from
(C.sub.1-14)alkyland (C.sub.2-14)alkenyl; carbonate ester [--OCOOR,
where R is selected from (C.sub.1-6)alkyland (C.sub.1-6)alkenyl;
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' is selected
from hydrogen; alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl;
amines [--NR'R'', where R' and R'' is selected from hydrogen;
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl; cyanate (--OCN); isocynate
(--NCO); nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC);
nitroso (--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; borono
and boronate [--B(OR')(R''), where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; borinate [--B(R')(OR''),
where R' and R'' is selected from H; (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; phosophino [--PR.sub.2, where R is selected
from H; (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphate
[--OP(.dbd.O)(OR).sub.2, where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphono
[--RP(.dbd.O)(OH), where R is selected from (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; thiol (--SH); thioalkyl; alkylthio; sulfide
[--SR, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; disulfide [--SSR, where R is selected from
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl, sulfonamide; sulfinyl
[--S(.dbd.O)R, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; sulfino (--SO.sub.2H); sulfo (--SO.sub.3H);
thiocyanate; isothiocyanate; carbonothioyl [--C(.dbd.S)R where R is
selected from (C.sub.1-6)alkyland (C.sub.2-6)alkenyl; wherein
R.sub.7 and R.sub.10 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; and wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.8 and R.sub.10 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.10 and R.sub.11 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring; and wherein
R.sub.4 and R.sub.5 can be bonded together to form an
(C.sub.1-8)alkane ring and/or (C.sub.2-8)alkene ring.
Embodiment 8
[0157] The method of embodiment 5, wherein the niclosamide or
niclosamide derivative has the structure of Formula 3:
##STR00013##
or a pharmaceutically acceptable salt thereof, where R.sub.13 can
include (C.sub.1-14)alkyl, such as methyl, ethyl, n-propyl,
isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl; and
(C.sub.2-14)alkenyl, such as ethenyl, propenyl, butenyl, where the
double bond can be located at any position in the alkenyl carbon
chain, including any alkenyl conformational isomers.
Embodiment 9
[0158] The method of embodiment 5, wherein the niclosamide or
niclosamide derivative has the structure of Formula 4:
##STR00014##
or a pharmaceutically acceptable salt thereof.
Embodiment 10
[0159] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises an emricasan compound, and wherein the
emricasan compound comprises:
[0160] (a) emricasan,
[0161] (b) an emricasan derivative,
[0162] (c) a prodrug or metabolite of (a) or (b), or
[0163] (d) a pharmaceutically acceptable salt of (a), (b), or
(c).
Embodiment 11
[0164] The method of embodiment 10, wherein the emricasan or
emricasan derivative has the structure of Formula 5:
##STR00015##
or a pharmaceutically acceptable salt thereof, where R.sub.23,
R.sub.24, R.sub.25, R.sub.26, R.sub.27, R.sub.28, R.sub.29,
R.sub.30, R.sub.31, R.sub.32, R.sub.33, and R.sub.34 are
independently selected from the group consisting of a H; F; Cl; Br;
I; OH; ketone (.dbd.O); (C.sub.1-6)alkyl; (C.sub.2-6)alkenyl;
(C.sub.2-6)alkynyl; ether [--OR, where R can include
(C.sub.1-6)alkyl; aralkyl; alkaryl; halogenated alkyl; heteroalkyl;
aryl; heterocyclyl; cycloalkyl; cycloalkenyl; cycloalkynyl;
hydroxyalkyl; aminoalkyl; amino; alkylamino; arylamino;
dialkylamino; alkylarylamino; diarylamino; acylamino; hydroxyl;
alkoxy; alkoxyalkyl; aryloxy; arylalkoxy; acyloxy; nitro;
carbamoyl; trifluoromethyl; phenoxy; benzyloxy; alkaryl; arylalkyl;
carbamate; amino; alkylamino; arylamino; dialkylamino;
alkylarylamino; diarylamino; heteroalkyl;
alkyltriphenylphosphonium; heterocyclyl; acyl halide [--COX, where
X is selected from F, Cl, Br, and I]; carbonyl [--COR, where R
selected from (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl]; aldehyde
(--CHO); ester [--OC(.dbd.O)R, --ROC(.dbd.O)R', RC(.dbd.O)OR',
--C(.dbd.O)OR', where R and R' is selected from
(C.sub.1-14)alkyland (C.sub.2-14)alkenyl; carbonate ester [--OCOOR,
where R is selected from (C.sub.1-6)alkyland (C.sub.1-6)alkenyl;
carboxyl (--COOH); amide [--CONR'R'', where R' and R'' is selected
from hydrogen; alkyl, such as methyl, ethyl, n-propyl, isopropyl,
n-butyl, sec-butyl, isobutyl, tert-butyl; (C.sub.2-4)alkenyl;
amines [--NR'R'', where R' and R'' is selected from hydrogen;
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl; cyanate (--OCN); isocynate
(--NCO); nitrate (--ONO.sub.2); nitrile (--CN); isonitrile (--NC);
nitroso (--NO); oxime (--CH.dbd.NOH); borono --B(OH).sub.2; borono
and boronate [--B(OR')(R''), where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; borinate [--B(R')(OR''),
where R' and R'' is selected from H; (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; phosophino [--PR.sub.2, where R is selected
from H; (C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphate
[--OP(.dbd.O)(OR).sub.2, where R is selected from H;
(C.sub.1-6)alkyl; and (C.sub.2-6)alkenyl; phosphono
[--RP(.dbd.O)(OH), where R is selected from (C.sub.1-6)alkyl and
(C.sub.2-6)alkenyl; thiol (--SH); thioalkyl; alkylthio; sulfide
[--SR, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; disulfide [--SSR, where R is selected from
(C.sub.1-6)alkyland (C.sub.2-6)alkenyl, sulfonamide; sulfinyl
[--S(.dbd.O)R, where R is selected from (C.sub.1-6)alkyland
(C.sub.2-6)alkenyl; sulfino (--SO.sub.2H); sulfo (--SO.sub.3H);
thiocyanate; isothiocyanate; carbonothioyl [--C(.dbd.S)R where R is
selected from (C.sub.1-6)alkyland (C.sub.2-6)alkenyl; wherein
R.sub.23 and R.sub.24 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.23 and R.sub.25 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene ring; where
R.sub.24 and R.sub.26 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene ring; wherein
R.sub.27 and R.sub.30 can be bonded together to form an
(C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene; wherein R.sub.28
and R.sub.29 can be bonded together to form an (C.sub.2-8)alkane
ring and/or (C.sub.2-8)alkene ring; and wherein R.sub.27 and
R.sub.30 can be bonded together to form an (C.sub.2-8)alkane ring
and/or (C.sub.2-8)alkene ring, such as a five-membered ring or a
six-membered ring; and wherein R.sub.29 and R.sub.30 can be bonded
together to form an (C.sub.2-8)alkane ring and/or (C.sub.2-8)alkene
ring.
Embodiment 12
[0165] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises a combination of the niclosamide
compound and the emricasan compound, and wherein the niclosamide
compound and the emricasan compound are administered
simultaneously, together within the same composition or in separate
compositions.
Embodiment 13
[0166] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises a combination of the niclosamide
compound and the emricasan compound, and wherein the niclosamide
compound and the emricasan compound are administered consecutively
in any order.
Embodiment 14
[0167] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises the CDK inhibitor, and the CDK
inhibitor has a structure shown in FIG. 11 (PHA-690509 or a
derivative thereof), FIG. 12 (Alvocidib or a derivative thereof),
FIG. 13 (PHA-793887 or a derivative thereof), FIG. 14 (Dinaciclib
or a derivative thereof), FIG. 15 (Seliciclib or a derivative
thereof), or a pharmaceutically acceptable salt of any of the
foregoing.
Embodiment 15
[0168] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises the CDK inhibitor, and the CDK
inhibitor has a structure shown in FIG. 16, or a pharmaceutically
acceptable salt thereof.
Embodiment 16
[0169] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises a CDK inhibitor, and the CDK inhibitor
is selected from among Alvocidib, Kenpaullone, Olomoucine,
Purvalanol A, Purvalanol B, Seliciclib, NU-6027, Indirubin,
Flavopiridol, AT7519, PD-0332991, SNS-032, PHA-793887, PHA-690509,
RGB-286147, BS-194, BS-181, AZD-5438, R-547, Dinaciclib, Milciclib,
BMS-265246, 7-Hydroxystaurosporine, CGP-60474, CDK9 inhibitor,
NU-6102, Fascaplysin, Cdk4/6 Inhibitor IV, or a prodrug,
metabolite, derivative, or pharmaceutically acceptable salt of any
of the foregoing.
Embodiment 17
[0170] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises a combination of the CDK inhibitor and
the emricasan compound, and wherein the CDK inhibitor and the
emricasan compound are administered simultaneously, together within
the same composition or in separate compositions.
Embodiment 18
[0171] The method of any one of embodiments 1 to 3, wherein the at
least one compound comprises a combination of the CDK inhibitor and
an emricasan compound, and wherein the CDK inhibitor and the
emricasan compound are administered consecutively in any order.
Embodiment 19
[0172] The method of any one of embodiments 1 to 18, wherein the
subject has the Flavivirus infection at the time of said
administering, and the at least one compound is administered as
therapy.
Embodiment 20
[0173] The method of 19, further comprising, prior to said
administering, identifying the subject as having the Flavivirus
infection.
Embodiment 21
[0174] The method of embodiment 20, wherein said identifying
comprises assaying a biological sample (e.g., blood, saliva, or
urine) obtained from the subject for the presence of Flavivirus
nucleic acids or Flavivirus proteins.
Embodiment 22
[0175] The method of embodiment 21, wherein said assaying comprises
use of reverse transcriptase-polymerase chain reaction (RT-PCR),
immunological assay (e.g., ELISA), or Plaque-reduction
neutralization testing (PRNT).
Embodiment 23
[0176] The method of any one of embodiments 1 to 18, wherein the
subject does not have the Flavivirus infection at the time of said
administering, and the at least one compound is administered as
prophylaxis.
Embodiment 24
[0177] The method of any one of embodiments 1 to 23, wherein the at
least one compound is administered orally, nasally, rectally,
parenterally, subcutaneously, intramuscularly, or intravascularly
(e.g., intravenously).
Embodiment 25
[0178] The method of any one of embodiments 1 to 24, further
comprising administering an additional agent for treating or
preventing Flavivirus infection, or a symptom thereof, in the same
formulation as at least one compound, or in a separate formulation
before, during, or after administration of the at least one
compound.
Embodiment 26
[0179] The method of any one of embodiments 1 to 25, wherein said
administering comprises administering a composition to the subject,
wherein the composition comprises the at least one compound and a
pharmaceutically acceptable buffer, carrier, or diluent.
Embodiment 27
[0180] A method for inhibiting Flavivirus infection in human or
non-human animal cells in vitro or in vivo, said method comprising
contacting an effective amount of at least one compound to a human
or non-human animal cell in vitro or in vivo before or after
exposure of the cell to Flavivirus, wherein the at least one
compound inhibits Flavirus infection or suppresses
Flavivirus-induced caspase-3 activity, and wherein the at least one
compound comprises:
[0181] (a) a niclosamide compound, or
[0182] (b) an emricasan compound, or
[0183] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0184] (d) a proteasome inhibitor, or
[0185] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
Embodiment 28
[0186] The method of embodiment 27, wherein the at least one
compound comprises a combination of two or more of the
compounds.
Embodiment 29
[0187] The method of embodiment 27 or 28, wherein the Flavivirus is
Zika virus.
Embodiment 30
[0188] A composition comprising a combination of two or more of the
following:
[0189] (a) a niclosamide compound, or
[0190] (b) an emricasan compound, or
[0191] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0192] (d) a proteasome inhibitor, or
[0193] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
Embodiment 31
[0194] A composition comprising:
[0195] (a) a niclosamide compound, or
[0196] (b) an emricasan compound, or
[0197] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0198] (d) a proteasome inhibitor, or
[0199] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing, and an additional agent
effective for the treatment of one or more symptoms of Flavivirus
infection.
Embodiment 32
[0200] The composition of embodiment 31, wherein the Flavivirus
infection is Zika virus infection.
Embodiment 33
[0201] A packaged dosage formulation comprising at least one
compound in a pharmaceutically acceptable dosage in one or more
packages, packets, or containers, wherein the at least one compound
comprises: [0202] (a) a niclosamide compound, or [0203] (b) an
emricasan compound, or [0204] (c) a cyclin-dependent kinase (CDK)
inhibitor, or [0205] (d) a proteasome inhibitor, or [0206] (e) a
compound selected from among Teriflunomide, Hydroxocobalamin,
Ensulizole, Tenonitrozole, Isoliquiritigenin, Nitazoxanide,
Febuxostat, Leflunomide, Vidofludimus, SB-366791, Emodin, Diphenyl
isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
Embodiment 34
[0207] The packaged dose formulation of embodiment 33, wherein the
packaged dose formulation comprises a combination of two or more of
the compounds.
Embodiment 35
[0208] A kit comprising, in one or more containers, at least one
compound comprising:
[0209] (a) a niclosamide compound, or
[0210] (b) an emricasan compound, or
[0211] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0212] (d) a proteasome inhibitor, or
[0213] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
Embodiment 36
[0214] The kit of embodiment 35, wherein the packaged dose
formulation comprises a combination of two or more of the
compounds.
Embodiment 37
[0215] The kit of embodiment 35 or 36, further comprising an
additional agent effective for the treatment or prevention of
Flavivirus virus infection.
Embodiment 38
[0216] The kit of embodiment 35 or 36, further comprising an
additional agent effective for the treatment of one or more
symptoms of Flavivirus infection.
Definitions
[0217] The terms "compounds of the invention" or "compounds of the
present invention" (unless specifically identified otherwise), and
grammatical variations thereof, refer to the compounds and classes
of compounds disclosed herein, such as the compounds in Tables
7-10, and FIGS. 9-22, or a prodrug, metabolite, or derivative
thereof; niclosamide compounds; emricasan compounds;
cyclin-dependent kinase (CDK) inhibitors, individually or
combinations of two or more, including pharmaceutically acceptable
salts of any of the foregoing, as well as all stereoisomers
(including diastereoisomers and enantiomers), rotamers, tautomers
and isotopically labeled compounds (including deuterium
substitutions), as well as inherently formed moieties (e.g.,
polymorphs, solvates and/or hydrates). For purposes of this
invention, solvates and hydrates are generally considered
compositions.
[0218] For example, in some embodiments, a compound of the
invention is:
[0219] (a) a niclosamide compound, or
[0220] (b) an emricasan compound, or
[0221] (c) a cyclin-dependent kinase (CDK) inhibitor, or
[0222] (d) a proteasome inhibitor, or
[0223] (e) a compound selected from among Teriflunomide,
Hydroxocobalamin, Ensulizole, Tenonitrozole, Isoliquiritigenin,
Nitazoxanide, Febuxostat, Leflunomide, Vidofludimus, SB-366791,
Emodin, Diphenyl isophthalate, Benzoylpas, Fenobam, Indobufen,
2-(2H-Benzotriazol-2-yl)-4-methylphenol, Tiaprofenic acid,
Flufenamic acid, Vitamin B12, Cinanserin,
5-Nitro-2-(3-phenylpropylamino)benzoic acid, Veliflapon,
Thiabendazole, SIB 1893, Anethole trithione, Naringenin,
Phenazopyridine, Fanetizole, Terazosin, Diacerein, CAY10505,
Hesperetin, Suprofen, Ketorolac tromethamine, Piperine,
Pirarubicin, Piraxostat, Albendazole oxide, Tyrphostin AG 494,
Genistin, Fenbufen, Apatinib, RITA, BF-170 hydrochloride, OSI-930,
Tribromsalan, Pifexole, Formononetin, Ebselen, Tranilast,
Benzylparaben, 2-Ethoxylethyl-p-methoxycinnamate, Baicalein,
Nemorubicin, Rutaecarpine, 2-Methyl-6-(phenylethynyl)pyridine
(MPEP), 5,7-Dihydroxyflavone, Vitamin B12, Pipofezine, Flurbiprofen
axetil, 2-Amino-6-nitrobenzothiazole, Malachite green oxalate,
Enfenamic acid, Fenaminosulf, AS-252424, Phenserine, Epalrestat,
Alizarin, Dalcetrapib, SN-38, Echinomycin, (S)-(+)-Camptothecin,
BI-2536, 10-hydroxycamptothecin, Topotecan, Delanzomib, Volasertib,
Ispinesib, Paclitaxel, FK-506, Emetine, AVN-944, Digoxin,
Vincristine, Idarubicin, Thapsigargin, Lexibulin, Ixazomib,
Cephalomannine, Mitoxantrone, MLN-2238, Demecolcine, Vinorelbine,
Bardoxolone methyl, Cycloheximide, Actinomycin D, AZD-7762, PF-184,
CHIR-124, Cyanein, Triptolide, KX-01, PF-477736, Epirubicin,
Mycophenolate (mycophenolic acid), Daunorubicin, PIK-75, Vindesine,
Torin-2, Floxuridine, Go-6976, OSU-03012, or a prodrug, metabolite,
or derivative of any of the foregoing, or a pharmaceutically
acceptable salt of any of the foregoing.
[0224] In some embodiments, the compound of the invention is any
compound in Tables 7-10, or FIGS. 9-22, or a prodrug, metabolite,
or derivative thereof. In some embodiments, the compound of the
invention is a pharmaceutically acceptable salt of a compound of
Tables 7-10, or FIGS. 9-22, or prodrug, or metabolite, or
derivative of a compound of Tables 7-10 or FIGS. 9-22.
[0225] In some embodiments, a compound of the invention is a
niclosamide compound. In some embodiments, the niclosamide compound
is niclosamide, or a pharmaceutically acceptable salt thereof. In
some embodiments, the niclosamide compound is a prodrug,
metabolite, or derivative of niclosamide. In some embodiments, the
niclosamide compound is a compound having a chemical structure of
any of FIGS. 9A-D, or a pharmaceutically acceptable salt thereof.
In some embodiments, the niclosamide compound is a prodrug of
niclosamide or a prodrug of a compound having a chemical structure
of any of FIGS. 9A-D. In some embodiments, the compound of the
invention is a derivative of niclosamide, such as p-niclosamide or
an acyl derivative of niclosamide such as DK-520.
[0226] In some embodiments, a compound of the invention is an
emricasan compound. In some embodiments, the emricisan compound is
emricasan, or a pharmaceutically acceptable salt thereof. In some
embodiments, the emricasan compound is a prodrug, metabolite, or
derivative of emricasan. In some embodiments, the emricasan
compound is a compound having the chemical structure of FIG. 10, or
a pharmaceutically acceptable salt thereof. In some embodiments,
the emricasan compound is a prodrug of emricasan or a prodrug of a
compound having the chemical structure of FIG. 10.
[0227] In some embodiments, a compound of the invention is a CDK
inhibitor. A CDK inhibitor is an agent, such as a peptide or
chemical compound, that inhibits the function of one or more
cyclin-dependent kinases. The CDK inhibitor may be a broad CDK
inhibitor, targeting multiple types of CDKs or may be a specific
CDK inhibitor, targeting a specific type of CDK (see, for example,
[50-52], which are incorporated herein by reference in their
entirety). In some embodiments, the CDK inhibitor is PHA-690509,
Alvocidib, PHA-793887, Dinaciclib, Seliciclib, a compound of FIG.
16, or a pharmaceutically acceptable salt of any of the foregoing.
In some embodiments, the CDK inhibitor is a prodrug, metabolite, or
derivative of PHA-690509, Alvocidib, PHA-793887, Dinaciclib,
Seliciclib, or of a compound of FIG. 16. In some embodiments, the
CDK inhibitor is a compound having the chemical structure of FIG.
11, FIG. 12, FIG. 13, FIG. 14, FIG. 15, or a pharmaceutically
acceptable salt of any of the foregoing. In some embodiments, the
CDK inhibitor is a prodrug of PHA-690509, Alvocidib, PHA-793887,
Dinaciclib, Seliciclib, or a prodrug of a compound of FIG. 16. In
some embodiments, the CDK inhibitor is a prodrug of a compound
having the chemical structure of FIG. 11, FIG. 12, FIG. 13, FIG.
14, or FIG. 15.
[0228] In some embodiments, a compound of the invention is a
proteasome inhibitor. A proteasome inhibitor is an agent, such as a
peptide or chemical compound, that inhibites the action of one or
more types of proteasome. (see, for example, [53, 54], which are
incorporated herein by reference in their entirety). In some
embodiments, the proteasome inhibitor Epirubicin, Ixazoomib,
Doxorubicin, Carflizomib, Bortezomib, Delanzomib, Oprozomib,
NPI-206, MG-115, Niclosamide, or pharmaceutically acceptable salt
of any of the foregoing.
[0229] The term "a," "an," "the" and similar terms used in the
context of the present invention (especially in the context of the
claims) are to be construed to cover both the singular and plural
unless otherwise indicated herein or clearly contradicted by the
context. Thus, for example, reference "a cell" or "a compound"
should be construed to cover both a singular cell or singular
compound and a plurality of cells and a plurality of compounds
unless indicated otherwise or clearly contradicted by the
context.
[0230] The term "isolated," when used as a modifier of a
composition, means that the compositions are made by the hand of
man or are separated from their naturally occurring in vivo
environment. Generally, compositions so separated are substantially
free of one or more materials with which they normally associate
with in nature, for example, one or more protein, nucleic acid,
lipid, carbohydrate, cell membrane. A "substantially pure" molecule
can be combined with one or more other molecules. Thus, the term
"substantially pure" does not exclude combinations of compositions.
Substantial purity can be at least about 60% or more of the
molecule by mass. Purity can also be about 70% or 80% or more, and
can be greater, for example, 90% or more. Purity can be determined
by any appropriate method, including, for example, UV spectroscopy,
chromatography (e.g., HPLC, gas phase), gel electrophoresis (e.g.,
silver or coomassie staining) and sequence analysis (for nucleic
acid and peptide).
[0231] The present invention includes derivatives of identified
compounds, also referred to herein as pharmaceutically active
derivatives. "Pharmaceutically active derivative" refers to any
compound that upon administration to the subject or cell, is
capable of providing directly or indirectly, the activity disclosed
herein. The term "indirectly" also encompasses prodrugs which may
be converted to the active form of the drug via endogenous enzymes
or metabolism. The prodrug is a derivative of the compounds
according to the invention and presenting Flavivirus (e.g., Zika
virus) inhibitory activity and/or protective activity against
effects of Flavivirus (e.g., Zika virus) that has a chemically or
metabolically decomposable group, and a compound that may be
converted into a pharmaceutically active compound according to the
invention in vivo by solvolysis under physiological conditions. The
prodrug is converted into a compound according to the present
invention by a reaction with an enzyme, gastric acid or the like
under a physiological condition in the living body, e.g., by
oxidation, reduction, hydrolysis or the like, each of which is
carried out enzymatically. These compounds can be produced from
compounds of the invention according to well-known methods. The
term "indirectly" also encompasses metabolites of compounds
according to the invention. Chemical reactions, reactants, and
reagents useful for making derivatives can be found, for example,
in March's Advanced Organic Chemistry, 7.sup.th edition, 2013,
Michael B. Smith, which is incorporated herein by reference in its
entirety.
[0232] The term "metabolite" refers to all molecules derived from
any of the compounds according to the invention in a cell or
organism, preferably mammal. Pharmaceutically active metabolites of
the compounds of the invention may be administered to a subject or
contacted with a cell in vitro or in vivo.
[0233] The term "prodrug" refers to a chemical compound that can be
converted by the body (i.e., biotransformed) to another chemical
compound that has pharmacological activity. The prodrug may itself
have pharmacological activity before conversion, or be inactive
before conversion and activated upon conversion. Active prodrugs or
inactive prodrugs of compounds of the invention may be administered
to a subject or contacted with a cell in vitro or in vivo. Instead
of administering a drug directly, a prodrug may be used instead to
improve how a drug is absorbed, distributed, metabolized, and
excreted (ADME). For example, a prodrug may be used to improve
bioavailability when a drug itself is poorly absorbed from the
gastrointestinal tract, or to improve how selectively the drug
interacts with cells or processes that are not its intended target,
which can reduce adverse or unintended effects of a drug. Major
types of prodrugs include, but are not limited to, type I prodrugs,
which are biotransformed inside cells (intracellularly), and type
II prodrugs, which are biotransformed outside cells
(extracellularly), such as in digestive fluids or in the body's
circulatory system. These types can be further categorized into
subtypes based on factors such as whether the intracellular
bioactivation location is also a site of therapeutic action, or
whether or not bioactivation occurs in the gastrointestinal fluids
or in the circulation system (Wu, Kuei-Meng, "A New Classification
of Prodrugs: Regulatory Perspectives, Pharmaceuticals, 2009,
2(3):77-81, which is incorporated by reference herein in its
entirety).
[0234] Pharmaceutical formulations include "pharmaceutically
acceptable" and "physiologically acceptable" carriers, diluents or
excipients. As used herein the terms "pharmaceutically acceptable"
and "physiologically acceptable" include solvents (aqueous or
non-aqueous), solutions, emulsions, dispersion media, coatings,
isotonic and absorption promoting or delaying agents, compatible
with pharmaceutical administration. Such formulations can be
contained in a liquid; emulsion, suspension, syrup or elixir, or
solid form; tablet (coated or uncoated), capsule (hard or soft),
powder, granule, crystal, or microbead. Supplementary compounds
(e.g., preservatives, antibacterial, antiviral and antifungal
agents) can also be incorporated into the compositions. The phrase
"pharmaceutically acceptable" indicates that the substance or
composition must be compatible chemically and/or toxicologically,
with the other ingredients comprising a formulation, and/or the
mammal being treated therewith.
[0235] The phrase "effective amount", in the context of a subject,
means an amount of at least one compound of the invention that (i)
treats or prevents the particular disease, condition, or disorder
(e.g., Zika or other Flavivirus infection) in a subject, (ii)
attenuates, ameliorates, or eliminates one or more symptoms of the
particular disease, condition, or disorder (e.g., Zika or other
Flavivirus infection) in a subject, or (iii) prevents or delays the
onset of one or more symptoms of the particular disease, condition,
or disorder described herein (e.g., Zika or other Flavivirus
infection) in a subject.
[0236] The phrase "effective amount", in the context of a cell in
vitro or in vivo, means an amount of at least one compound of the
invention that (i) treats or prevents the particular disease,
condition, or disorder (e.g., Zika or other Flavivirus infection)
in a cell, (ii) attenuates, ameliorates, or eliminates one or more
effects of the particular disease, condition, or disorder (e.g.,
Zika or other Flavivirus infection) in a cell, or (iii) prevents or
delays the onset of one or more effects of the particular disease,
condition, or disorder described herein (e.g., Zika or other
Flavivirus infection) in a subject.
[0237] Niclosamide compounds, proteasome inhibitors, and CDK
inhibitors of the invention can inhibit or reduce Zika virus or
other Flavivirus replication (e.g., Zika virus infection), and thus
represent one class of compounds of the invention. Emricasan
compounds of the invention are pan-caspase inhibitors, which
inhibit Flavivirus-induced increase in caspase-3 activity, and
reduce cell death, and represent another class of compounds of the
invention. A combination of one class of compounds (e.g.,
Niclosamide compound, and/or CDK inhibitor, and/or proteasome
inhibitor) with the other class of compounds (e.g., Emricasan
compound) can exhibit a synergistic effect in protecting neural
cells from Flavivirus-induced (e.g., Zika virus-induced) cell
death.
[0238] As used herein, a subject is "in need of" a treatment if
such human or non-human animal subject would benefit biologically,
medically or in quality of life from such treatment (preferably, a
human). In some embodiments, the subject has a Flavivirus infection
and is in need of therapy. In other embodiments, the subject does
not have a Flavivirus infection and is in need of prophylaxis. In
some embodiments, the subject in need of prophylaxis is at risk of
becoming infected with the Flavivirus. In some embodiments, the
subject is at increased risk of becoming infected with the
Flavivirus relative to others in the population.
[0239] As used herein, the term "inhibit", "inhibition" or
"inhibiting" refers to the reduction or suppression of a given
condition, symptom, or disorder, or disease (e.g., Zika virus or
other Flavivirus infection, or Zika viral or other Flavivirus load
or titer), or a significant decrease in the baseline activity of a
biological activity or process (inhibits or suppresses Zika or
other Flavivirus infection, or inhibits or suppresses Zika or other
Flavivirus replication, or inhibits or suppresses Zika-induced or
other Flavivirus-induced neural cell death, or inhibits or
suppresses infection-induced caspase-3 activity in neural cell
types).
[0240] As used herein, the terms "subject", "patient", and
"individual" refer to a human or non-human animal. Typically, the
animal is a mammal. A subject also refers to for example, primates
(e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits,
rats, mice, fish, birds and the like. In certain embodiments, the
subject is a primate. In yet other embodiments, the subject is a
human. The subject may be any age or gender. For example, in some
embodiments, the subject is a female. In some embodiments, the
subject is a post-pubescent female or a post-pubescent female. In
some embodiments, the subject is a pregnant female.
[0241] As used herein, the term "treat", "treating" or "treatment"
of any disease or disorder refers in one embodiment, to
ameliorating the disease or disorder (i.e., slowing or arresting or
reducing the development of the disease or at least one of the
clinical symptoms thereof). In another embodiment "treat",
"treating" or "treatment" refers to alleviating or ameliorating at
least one physical parameter including those which may not be
discernible by the subject. In yet another embodiment, "treat",
"treating" or "treatment" refers to modulating the disease or
disorder, either physically, (e.g., stabilization of a discernible
symptom), physiologically, (e.g., stabilization of a physical
parameter), or both. In yet another embodiment, "treat", "treating"
or "treatment" refers to prophylaxis (preventing or delaying the
onset or development or progression of the disease or
disorder).
[0242] As used herein, the term "administration" is intended to
include, but is not limited to, the following delivery methods:
topical, oral, parenteral, subcutaneous, transdermal, transbuccal,
intravascular (e.g., intravenous or intra-arterial), intramuscular,
subcutaneous, intranasal, and intra-ocular administration.
Administration can be local at a particular anatomical site, such
as a site of infection, or systemic.
[0243] As used herein, the term "contacting" in the context of
contacting a cell with at least one compound of the invention in
vitro or in vivo means bringing at least one compound into contact
with the cell, or vice-versa, or any other manner of causing the
compound and the cell to come into contact. In those embodiments of
the method for inhibiting Flavivirus infection in human or
non-human animal cells in vitro or in vivo, when a cell is
contacted with a compound in vivo, the compound is administered to
a subject, and the administration may occur by any route (e.g.,
topical, oral, parenteral, subcutaneous, transdermal, transbuccal,
intravascular (e.g., intravenous or intra-arterial), intramuscular,
subcutaneous, intranasal, and intra-ocular administration).
[0244] The compounds of the present invention can be formulated
into pharmaceutically-acceptable salt forms.
Pharmaceutically-acceptable salts of the compounds of the invention
can be prepared using conventional techniques. "Pharmaceutically
acceptable salt" includes both acid and base addition salts. A
pharmaceutically acceptable salt of any one of the compounds
described herein is intended to encompass any and all
pharmaceutically suitable salt forms. Preferred pharmaceutically
acceptable salts described herein are pharmaceutically acceptable
acid addition salts and pharmaceutically acceptable base addition
salts.
[0245] "Pharmaceutically acceptable acid addition salt" refers to
those salts which retain the biological effectiveness and
properties of the free bases, which are not biologically or
otherwise undesirable, and which are formed with inorganic acids
such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric
acid, phosphoric acid, hydroiodic acid, hydrofluoric acid,
phosphorous acid, and the like. Also included are salts that are
formed with organic acids such as aliphatic mono- and dicarboxylic
acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,
alkanedioic acids, aromatic acids, aliphatic and, aromatic sulfonic
acids, etc. and include, for example, acetic acid, trifluoroacetic
acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid,
maleic acid, malonic acid, succinic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,
salicylic acid, and the like. Exemplary salts thus include
sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates,
phosphates, monohydrogenphosphates, dihydrogenphosphates,
metaphosphates, pyrophosphates, chlorides, bromides, iodides,
acetates, trifluoroacetates, propionates, caprylates, isobutyrates,
oxalates, malonates, succinate suberates, sebacates, fumarates,
maleates, mandelates, benzoates, chlorobenzoates, methylbenzoates,
dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates,
phenylacetates, citrates, lactates, malates, tartrates,
methanesulfonates, and the like. Also contemplated are salts of
amino acids, such as arginates, gluconates, and galacturonates
(see, for example, Berge S. M. et al., "Pharmaceutical Salts,"
Journal of Pharmaceutical Science, 66:1-19 (1997), which is hereby
incorporated by reference in its entirety). Acid addition salts of
basic compounds may be prepared by contacting the free base forms
with a sufficient amount of the desired acid to produce the salt
according to methods and techniques with which a skilled artisan is
familiar.
[0246] "Pharmaceutically acceptable base addition salt" refers to
those salts that retain the biological effectiveness and properties
of the free acids, which are not biologically or otherwise
undesirable. These salts are prepared from addition of an inorganic
base or an organic base to the free acid. Pharmaceutically
acceptable base addition salts may be formed with metals or amines,
such as alkali and alkaline earth metals or organic amines. Salts
derived from inorganic bases include, but are not limited to,
sodium, potassium, lithium, ammonium, calcium, magnesium, iron,
zinc, copper, manganese, aluminum salts and the like. Salts derived
from organic bases include, but are not limited to, salts of
primary, secondary, and tertiary amines, substituted amines
including naturally occurring substituted amines, cyclic amines and
basic ion exchange resins, for example, isopropylamine,
trimethylamine, diethylamine, triethylamine, tripropylamine,
ethanolamine, diethanolamine, 2-dimethylaminoethanol,
2-diethylaminoethanol, dicyclohexylamine, lysine, arginine,
histidine, caffeine, procaine, N,N-dibenzylethylenediamine,
chloroprocaine, hydrabamine, choline, betaine, ethylenediamine,
ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine,
theobromine, purines, piperazine, piperidine, N-ethylpiperidine,
polyamine resins and the like. See Berge et al., supra.
[0247] All patents, patent applications, provisional applications,
and publications referred to or cited herein are incorporated by
reference in their entirety, including all figures and tables, to
the extent they are not inconsistent with the explicit teachings of
this specification.
[0248] Following are examples that illustrate procedures for
practicing the invention. These examples should not be construed as
limiting. All percentages are by weight and all solvent mixture
proportions are by volume unless otherwise noted.
Materials and Methods
[0249] Human Forebrain-Specific NPCs, Astrocytes, Organoids and
Cell Line Cultures.
[0250] The human iPSC line (C1-2 line) was previously generated
from a skin biopsy sample of a healthy male newborn and has been
fully characterized and passaged on MEF feeder layers [41]. The BJ
line were derived from healthy control fibroblasts obtained from
the American Type Culture Collection (CRL-2522, male, neonatal).
All studies followed institutional IRB protocols approved by Johns
Hopkins University School of Medicine and Florida State University.
Human iPSCs were differentiated into forebrain-specific hNPCs
following the previously established protocol [41]. Briefly, human
iPSC colonies were detached from the feeder layer with 1 mg/ml
collagenase treatment for 1 h and suspended in embryonic body (EB)
medium, consisting of FGF-2-free iPSC medium supplemented with 2
.mu.M Dorsomorphin and 2 .mu.M A-83 in non-treated polystyrene
plates for 4 days with a daily medium change. After 4 days, EB
medium was replaced by neural induction medium (NPC medium)
consisting of DMEM/F12, N2 supplement, NEAA, 2 .mu.g/ml heparin,
and 2 .mu.M cyclopamine. Floating EBs were then transferred to
Matrigel-coated 6-well plates at day 7 to form neural tube-like
rosettes. Attached rosettes were kept for 15 d with NPC medium
change every other day. On day 22, rosettes were picked
mechanically and transferred to low attachment plates (Corning) to
form neurospheres in NPC medium containing B27. Neurospheres were
then dissociated with Accutase at 37.degree. C. for 10 min and
placed onto Matrigel-coated 6-well plates at day 24 to form
monolayer hNPCs in NPC medium containing B27. These hNPCs expressed
forebrain-specific progenitor markers, including NESTIN, PAX6,
EMX-1, FOXG1, and OTX2 [41].
[0251] Human BJ iPSC line and forebrain NPCs were derived as
described previously [42,43]. hNPCs were differentiated to
astrocytes and cultured on Matrigel-coated plates in astrocyte
medium (ScienCell). Human iPSC-derived forebrain NPCs were
maintained at high density, grown on Matrigel (BD bioscience) in
NPC media (DMEM/F12, 1.times.N2, 1.times.B27-RA (Invitrogen), 1
.mu.g/ml laminin (Invitrogen) and 20 ng/ml FGF2 (Invitrogen)) and
split approximately 1:3-1:4 every week with Accutase (Millipore).
hNPCs were differentiated to astrocytes and cultured on
Matrigel-coated plates in astrocyte medium (Lonza or ScienCell).
Astrocytes were split 1:3 every week with Accutase and cultured up
to 120 days.
[0252] Forebrain-specific organoids were generated from the C2-1
human iPSC line as previously described [44]. Briefly, human iPSC
colonies were detached 7 days after passage with Collagenase Type
IV, washed with fresh stem cell medium and cultured in a 15 ml
conical tube. On day 1, detached and washed iPSC colonies were
transferred to an ultra-low attachment 6-well plate (Corning
Costar), containing 3 ml of stem cell medium (without FGF-2), plus
2 .mu.M Dorsomorphine (Sigma) and 2 .mu.M A83-01 (Tocris). On days
5-6, half of the medium was replaced with induction medium
consisting of DMEM:F12, 1.times. N2 Supplement (Invitrogen), 10
.mu.g/ml Heparin (Sigma), 1.times. Penicillin/Streptomycin,
1.times. Non-essential Amino Acids, 1.times. Glutamax, 4 ng/ml
WNT-3A (R&D Systems), 1 .mu.M CHIR99021 (Cellagentech), and 1
.mu.M SB-431542 (Cellagentech). On day 7, organoids were embedded
in Matrigel (BD Biosciences) and continued to grow in induction
medium for 6 more days. On day 14, embedded organoids were
mechanically dissociated from Matrigel by pipetting up and down
onto the plate with a 5 ml pipette tip. Typically, 10-20 organoids
were transferred to each well of a 12-well spinning bioreactor
(Spin.OMEGA.) containing differentiation medium, consisting of
DMEM:F12, 1.times.N2 and B27 Supplements (Invitrogen), 1.times.
Penicillin/Streptomycin, 1.times.2-Mercaptoenthanol, 1.times.
Non-essential Amino Acids, 2.5 .mu.g/ml Insulin (Sigma).
[0253] The glioblastoma SNB-19 cell line (part of the National
Cancer Institute 60 human tumor cell line) was a gift from Dr.
David Meckes (Florida State University, Tallahassee, Fla.). SNB-19
cells were maintained at 37.degree. C. in 5% CO.sub.2 in RPMI-60,
1.times. penicillin/streptomycin, and 10% fetal bovine serum
(Invitrogen). The Aedes albopictus C6/36 cell line (ATCC) was
maintained at 28.degree. C. in 5% CO.sub.2.
[0254] Preparation of ZIKV and Cell Infection.
[0255] The MR766-ZIKV stock with the titer of 1.times.10.sup.5
Tissue Culture Infective Dose (TCID)/ml in the form of culture
fluid from an infected rhesus Macaca cell line LLC-MK2, was
originally obtained from ZeptoMetrix (Buffalo, N.Y.). The
FSS13025-ZIKV strain was obtained from Drs. Robert Tesh and
Pei-Yong Shi (University of Texas Medical Branch, Galveston, Tex.).
The PRVABC59 strain was obtained from ATCC (Manassas, Va.).
Original viral stocks were then amplified in Aedes albopictus clone
C6/36 cells. Briefly, C6/36 cells were inoculated with viral
inoculum for one hour at 28.degree. C. in low volume of media (3 ml
per T-75 flask), with rocking every 15 minutes, before addition of
17 ml media. Viral inoculated cells were incubated at 28.degree. C.
for 6-7 days before harvesting of supernatant. C6/36-amplified ZIKV
titer was determined by infecting Vero cells for 48 hours with a
methyl-cellulose overlay and analyzed for focus-forming units per
mL (FFU/ml). In mock infections, an equal volume of spent
uninfected C6/36 culture medium was used. For infections, cells
were seeded into 12-well plates with/without coverslips one day
prior to virus addition. For all cell types, compound was added 1
hour prior to viral addition unless otherwise specified. Cells were
harvested at 24-72 hours post-infection.
[0256] Immunocytochemistry.
[0257] Cells were fixed with 4% paraformaldehyde (Sigma) for 15 min
at room temperature. Samples were permeabilized and blocked with
0.25% Triton X-100 (Sigma) and 10% donkey or goat serum in PBS for
20 min as previously described (Chiang et al., 2011; [41]; Yoon et
al., 2014). Samples were then incubated with primary antibodies at
4.degree. C. overnight, followed by multiple PBST washes and
incubation with secondary antibodies for 1 h at room temperature.
Slides were mounted using VECTASHIELD with DAPI (Vectorlabs,
Burlingame, Calif.). The following primary antibodies were used:
anti-flavivirus group antigen (clone D1-4G2-4-15; mouse; 1:500;
Millipore), and anti-cleaved caspase-3 (Asp15; Rabbit; 1:500; Cell
Signaling Technology). Antibodies were prepared in PBS containing
0.25% Triton X-100 and 10% donkey serum. Images were taken by Zeiss
LSM 700 and 880 confocal microscopes, Olympus BX61, or Zeiss
Axiovert 200M microscope.
[0258] Western Blot.
[0259] Cells were harvested by trypsinization, pelleting, and
subsequent lysis in 1.times. Laemlli buffer and boiled, or directly
lysed in 1.times. Laemlli buffer and boiled. Antibodies used were
anti-ZIKV NS1 (1:2000; BF-1225-36, BioFront Technologies,
Tallahassee, Fla.) or GAPDH (Santa Cruz Biotechnology, Texas).
[0260] NS1 ELISA.
[0261] The anti-ZIKV NS1 ELISA (ZKV-NS1-EK) was obtained from
BioFront Technologies (Tallahassee, Fla.) and used according to the
manufacturer's protocol.
[0262] Compound Libraries.
[0263] The Library of Pharmacologically Active Compounds (LOPAC),
consisting of 1280 compounds, was purchased from Sigma-Aldrich. The
NCATS pharmaceutical collection, a collection consisting of 2816
clinically approved and investigational drugs, was internally
established in 2011 [45]. All compounds were dissolved in DMSO as
10 mM stock solutions, then diluted in DMSO at a 1:3 ratio in
384-well plates, followed by reformatting into 1536-well compound
plates use in high-throughput screening (HTS).
[0264] Caspase 3/7 Assay.
[0265] Caspase-Glo 3/7 assay kit (catalog number G8092; Promega,
Madison, Wis.) was used to detect caspase 3/7 activity. Reagents
were reconstituted as described in the protocol from the
manufacturer. Polystyrene plates (384-well and 1536-well; regular
tissue culture treated and PDL coated) were purchased from Greiner
Bio-One (Monroe, N.C.). Cells were seeded in 384- and 1536-well
assay plates and cultured at 37.degree. C. with 5% CO.sub.2 for 16
hours. ZIKV solution was added to cells, followed by incubation at
37.degree. C. with 5% CO.sub.2 for 6 hours. Caspase-Glo 3/7 was
added to each well, unless otherwise specified, and incubated at
room temperature for 30 minutes. The luminescence intensity of the
assay plates was measured using a ViewLux plate reader
(PerkinElmer). Data were normalized by using the cell-containing
wells without ZIKV as a negative control (0% induction of caspase
3/7 activity) and wells containing ZIKV infected cells that induced
caspase 3/7 activity were used as a positive control (100%
induction of caspase 3/7 activity).
[0266] ATP Content Assay for Cell Viability and Compound
Cytotoxicity.
[0267] The ATPlite luminescence assay system assay kit (catalog
number 6016731; PerkinElmer) was used to determine cell viability.
The reagent was reconstituted and prepared as described by the
manufacturer. In order to measure the cell death caused by ZIKV
infection, cells were cultured for 16 hours at 37.degree. C. with
5% CO.sub.2 in assay plates, followed by the addition of ZIKV
solution and incubation at 37.degree. C. with 5% CO.sub.2 for 72
hours. ATPlite, the ATP monitoring reagent, was then added to the
assay plates and they were incubated for 15 minutes. The resulting
luminescence was measured using the ViewLux plate reader (Perkin
Elmer). Data were normalized using the wells without cells as a
control for 100% cell killing, and cell-containing wells without
ZIKV infection were used as full cell viability (0% cell
killing).
[0268] Large-Scale Compound Screening.
[0269] Human NPCs were seeded onto PDL coated 1536-well assay
plates at 250 cells per 3 .mu.l/well and incubated at 37.degree. C.
in 5% CO.sub.2 for 16 hours. Test compounds dissolved in DMSO were
transferred to assay plates at a volume of 23 nl/well by an
automated pintool workstation (Wako Automation, San Diego, Calif.).
Compounds were incubated with cells for 30 minutes at 37.degree. C.
in 5% CO.sub.2, immediately followed by the addition of 2
.mu.l/well of ZIKV. Incubation time of compound-treated cells with
ZIKV varied based on assay format. Experiments measuring
virus-induced caspase 3/7 activity required a 6 hour incubation of
ZIKV in the presence compounds at 37.degree. C. in 5% CO.sub.2.
Following this incubation, 3.5 .mu.l/well of caspase 3/7 reagent
mixture was added to assay plates. The plates were incubated for 30
minutes at room temperature, and the resultant luminescence signal
was measured using a ViewLux plate reader (Perkin Elmer).
Experiments measuring virus-induced cell death required a 72-hour
incubation of ZIKV in the presence of compounds at 37.degree. C. in
5% CO.sub.2. Following this incubation, 3.5 .mu.l/well of ATP
content detection reagent was added to assay plates, incubated for
30 minutes at the room temperature, and the resultant luminescence
signal was measured in a ViewLux plate reader. Step-by-step assay
protocols are listed in Tables 4 and 5.
[0270] Because compound cytotoxicity could nonspecifically reduce
the caspase activity induced by ZIKV, the inventors also used the
ATP content assay to measure compound cytotoxicity in the absence
of ZIKV infection. The cells were seeded in the same way as
described above in 1536-well assay plates. After a 6-hour
incubation with compounds in the absence of ZIKV, 3.5 .mu.l/well of
ATP content reagent mixture was added to the assay plates and
incubated for 30 minute at room temperature. The luminescence
signal in the assay plates was measured using a ViewLux plate
reader (Table 6). Any compounds that exhibited cytotoxicity were
eliminated as false positive compounds.
[0271] Antiviral Compound Analysis.
[0272] SNB-19 cells, BJ astrocytes, or hNPCs were seeded in 12-well
plates at approximately 3e5 cells/well. The next day, cells were
treated with compound at 1-10.times.IC.sub.50 or indicated
concentration for 1 hour prior to inoculation with ZIKV at
MOI=0.5-1. Cells and supernatant were harvested 24-48 hours post
infection and analyzed by western blot or IFA. Western blot bands
were quantified and IFA images counted using ImageJ (NIH, Bethesda,
Md.).
[0273] Data Analysis and Statistics.
[0274] The primary screen data and concentration response curves
were analyzed using software developed internally [46]. The
concentration-response curves and IC50 values of compound
confirmation data were calculated using Prism software (GraphPad
Software, Inc. San Diego, Calif.). All values are expressed as the
mean.+-.s.d. unless specified otherwise. Western blots and IFA
images were quantified using ImageJ (NIH, Bethesda, Md.).
TABLE-US-00001 TABLE 1 Protocol of caspase 3/7 activity assay.
value Step parameter 384-well 1536-well Description 1 Cell plating
20 .mu.l/well 3 .mu.l/well PDL-coated plates used for
NPCs/astrocytes 2 Incubation overnight at 37.degree. C. with 5%
CO.sub.2 3 Compound addition 10 .mu.l/well 23 .mu.l/well in DMSO
solution 4 Incubation 30 minutes at 37.degree. C. with 5% CO.sub.2
5 Zika virus addition 2 .mu.l/well 6 Incubation 6 hours at
37.degree. C. with 5% CO.sub.2 7 Reagent addition 3 .mu.l/well
Caspase 3/7 assay mixture (Promega) 8 Incubation 30 minutes at room
temperature 9 Plate reading luminescence mode ViewLux plate reader
(PerkinElmer)
TABLE-US-00002 TABLE 2 Protocol of ATP cell viability assay for
Zika virus induced cell death. value Step parameter 384-well
1536-well Description 1 Cell plating 20 .mu.l/well 3 .mu.l/well
PDL-coated plates used for NPCs/astrocytes 2 Incubation overnight
at 37.degree. C. with 5% CO.sub.2 3 Compound addition 10 .mu.l/well
23 .mu.l/well in DMSO solution 4 Incubation 30 minutes at
37.degree. C. with 5% CO.sub.2 5 Zika virus addition 2 .mu.l/well 6
Incubation 3 days at 37.degree. C. with 5% CO.sub.2 7 Reagent
addition 3 .mu.l/well ATP content assay mixture (PerkinElmer) 8
Incubation 30 minutes at room temperature 9 Plate reading
luminescence mode ViewLux plate reader (PerkinElmer)
TABLE-US-00003 TABLE 3 Protocol of ATP content cell viability assay
for compound cytotoxicity. value Step parameter 384-well 1536-well
Description 1 Cell plating 20 .mu.l/well 3 .mu.l/well PDL-coated
plates used for NPCs/astrocytes 2 Incubation Overnight at
37.degree. C. with 5% CO.sub.2 3 Compound addition 10 .mu.l/well 23
.mu.l/well in DMSO solution 4 Incubation 30 minutes at 37.degree.
C. with 5% CO.sub.2 5 Incubation 6 hours at 37.degree. C. with 5%
CO.sub.2 6 Reagent addition 3 .mu.l/well ATP content assay mixture
7 Incubation 30 minutes at room temperature 8 Plate reading
luminescence mode ViewLux plate reader (PerkinElmer)
Note: no Zika virus was added to assay plate in this experiment
that was differed from that described in Table 1 and 2.
Example 1-Anti-Zika Virus Screen and Anti-Zika Compounds
[0275] Although ZIKV only recently became a global health concern,
rapid progress has already been made in understanding its
pathogenesis and developing animal models [10-17]. Following
clinical observations that ZIKV could be found in fetus brains of
infected pregnant women [18,19], the inventors reported that ZIKV
can efficiently infect forebrain-specific human neural progenitor
cells (hNPCs) and attenuate their growth [10]. These results
provide a potential mechanism for ZIKV-induced microcephaly as
hNPCs drive the development of human cortex, which is
underdeveloped in microcephalic fetuses. Furthermore, the inventors
and others have shown that ZIKV infection of brain organoids, 3D
cellular models of human brain development, leads to reduced
ventricular zone thickness, enlarged lumen, and an overall
reduction in organoid size [11,12,15,20], again consistent with
features of microcephaly. These results have also been
recapitulated in mouse models [15,16]. Despite these advancements
in understanding how ZIKV causes developmental abnormalities, there
is currently no drug or vaccine available to treat or prevent ZIKV
infection.
[0276] The inventors performed a drug repurposing screen to
identify compounds that either inhibit ZIKV infection or suppress
infection-induced caspase-3 activity in neural cell types. A
screening of 6,000 compounds that included approved drugs, clinical
trial drug candidates and pharmacologically active compounds,
identified compounds in both categories. Among these, Emricasan, a
pan-caspase inhibitor, potently inhibits ZIKV-induced increase in
caspase-3 activity and reduces cell death. The inventors confirmed
its efficacy to protect human cortical neural progenitors in both
monolayer and organoid cultures. PHA-690509, a cyclin-dependent
kinase (CDK) inhibitor, and four structurally unrelated CDK
inhibitors, inhibit ZIKV replication in human cells in a
dose-dependent manner. In addition, Niclosamide, an FDA approved
category B anthelmintic drug, inhibits ZIKV replication at
sub-micromolar concentrations. Both CDK inhibitors and Niclosamide
are effective in reducing ZIKV replication when added either before
or after ZIKV exposure. Finally, a combination of two classes of
compounds showed a synergistic effect in protecting human neural
cells from ZIKV-induced cell death. The results demonstrate the
effectiveness of this compound screening strategy and provide
compounds for anti-Zika drug development.
[0277] Drug repurposing screens have recently emerged as an
alternative approach to accelerate drug development [21,22].
Following a repurposing phenotypic screen, new indications for
existing drugs may be rapidly identified and clinical trials can be
carried out quickly, which is of critical importance for rapidly
spreading infectious diseases [21,22]. For example, recent drug
repurposing screens have led to discoveries of potential new
candidate therapies for Ebola virus disease [23,24], Giardiasis
[25], Entamoeba histolytica infection [26], malaria gametocytes
[27], Exserohilum rostratum infection [28], and hepatitis C virus
infection [29]. Based on the previous finding that ZIKV infection
of hNPCs results in an increase of caspase-3 activation, followed
by cell death [10], the inventors designed a compound screening
approach using caspase-3 activity as the primary screen assay and a
cell viability assay for confirmation (FIG. 5A). The inventors
identified two classes of effective compounds, one being antiviral
and another capable of protecting neural cells from ZIKV-induced
cell death. Because ZIKV infects neural cells and causes their
death, neuroprotective compounds may provide additional benefits
when combined with drugs that inhibit ZIKV replication.
[0278] The inventors first quantified caspase-3 activity and cell
viability of hNPCs and astrocytes derived from human induced
pluripotent stem cells (iPSCs), as well as glioblastoma SNB-19
cells, after ZIKV infection in a 1536-well plate format (Table 4).
The prototypic ZIKV strain, MR766, was used in the primary screen
because it produced the strongest cell death signal in cell culture
experiments. The signal-to-basal (S/B) ratio and coefficient of
variation (CV) obtained in the caspase-3 activity assay after
6-hour ZIKV exposure were 2.1-fold and 7.0% for hNPCs, 7.0-fold and
5.9% for SNB-19 cells, and 11.0-fold and 9.1% for astrocytes (FIG.
5B). The Z' factors for hNPCs, SNB-19, and human astrocytes were
0.20, 0.68, and 0.72, respectively. Since a Z' factor over 0.5
indicates a robust screening assay [30], the caspase assay using
SNB-19 cells or astrocytes is suitable for high-throughput
screening of compound collections.
[0279] To measure cell viability following ZIKV infection for 3
days, the inventors applied an ATP content assay in a 1536-well
plate format (FIG. 5A and Table 5). Cell viability was reduced by
39%, 82%, and 69% in hNPCs, SNB-19 cells, and human astrocytes,
respectively (FIG. 5C). The Z' factors in these three cell types
were -0.06, 0.37 and 0.32, respectively. Together, the results
indicated the caspase-3 activity assay is a better option for
high-throughput compound screening than the cell viability
assay.
[0280] The inventors carried out a screening campaign using the
caspase-3 activity assay and SNB-19 cells with the Library of
Pharmacologically Active Compounds (LOPAC, 1280 compounds), the
NCATS Pharmaceutical Collection of approved drugs (2816 compounds),
and a collection of clinical candidate compounds (2000 compounds).
Primary hits included a total of 116 compounds that suppressed
ZIKV-induced caspase 3 activity in SNB-19 cells. The inventors also
carried out an independent primary screen using hNPCs with the same
libraries. This second screen resulted in 173 primary hits that
included all 116 compounds from the first caspase-3 screen in
SNB-19 cells. All results of the primary screen of the approved
drug collection and hit confirmation were deposited into the
open-access PubChem database (pubchem.ncbi.nlm.nih.gov).
[0281] Next, the activity of these primary hits in the caspase-3
activity assay was evaluated in ZIKV-infected SNB-19 cells, hNPCs,
and astrocytes in parallel with the compound cytotoxicity assay
(FIGS. 5C-5D and Table 6). Cytotoxic compounds were then eliminated
from the confirmed compound list. Consistent with the screening
design, the inventors identified compounds that reduced
virally-induced caspase activation and apoptosis by either directly
preventing ZIKV-induced cell death or suppressing ZIKV replication
(Table 7).
[0282] Emricasan, a pan-caspase inhibitor, was identified as the
most potent anti-death compound with IC.sub.50 values of 0.13-0.9
.mu.M in both the caspase activity assay and the cell viability
assay in SNB-19 cells against three ZIKV strains: MR766 (1947
Uganda strain), FSS13025 (2010 Cambodian strain), and PRVABC59
(2015 Puerto Rican strain) (FIG. 1A). It was also active in all
three cell types tested (FIG. 5E and Table 8). Finally, Emricasan
reduced the number of active (cleaved) caspase-3-expressing
forebrain-specific hNPCs infected by FSS13025 in both monolayer and
3D organoid cultures (FIGS. 1B-1C). ZIKV antigen persisted in both
2D and 3D cultures after Emricasan treatment (FIGS. 1B-1C).
Therefore, Emricasan displays protective activity for hNPCs, but
does not suppress ZIKV replication.
[0283] Using ZIKV protein NS1 expression as a read-out for testing
anti-ZIKV activity, the inventors identified two compounds that
significantly inhibited ZIKV infection in SNB-19 cells (FIG. 2A).
The first was Niclosamide, an FDA-approved drug for treating worm
infections in both humans and domestic livestock; the other was
PHA-690509, an investigational compound and an inhibitor of
cyclin-dependent kinases (CDKs). Both compounds inhibited all three
strains of ZIKV in a dose-dependent manner, with IC.sub.50 values
of 12 to 36 .mu.M for PHA-690509 and over ten-fold lower at 0.5-2
.mu.M for Niclosamide (FIGS. 2B-2E and FIGS. 6A-6B). To explore the
mechanism of action for these compounds, the inventors performed
time-of-addition experiments (FIG. 3A). Both compounds effectively
inhibited ZIKV infection when added either 1 hour before or 4 hours
after virus inoculation (FIG. 3B). In contrast, a monoclonal
antibody against AXL, a putative ZIKV entry factor [31,32], was
only effective when added prior to inoculation (FIG. 3B). Together,
these results indicate that Niclosamide and PHA-690509 inhibit ZIKV
infection at a post-entry stag.
[0284] Pharmacological CDK inhibitors have been shown to inhibit
replication of diverse viruses in culture, including herpes viruses
and HIV [33], but identification of a CDK inhibitor is particularly
intriguing given the recent observation that ZIKV infection and
cell cycle regulation are closely connected [10]. The inventors
therefore tested four additional structurally distinct CDK
inhibitors for ZIKV inhibition (FIG. 3C). All four CDK inhibitors
effectively inhibited ZIKV (FIG. 3D), whereas four other non-CDK
kinase inhibitors exhibited minimal anti-ZIKV activity in SNB-19
cells (FIGS. 7A-7B). The inventors also examined whether these
compounds could inhibit ZIKV infection in hNPCs and astrocytes
using the clinical isolate from the 2015 Puerto Rico Zika outbreak,
PRVABC59. Niclosamide and PHA-690509 inhibited ZIKV.sup.PR
infection in these central nervous system cells (FIGS. 4A-4B), both
of which are target cells for ZIKV infection in the fetal brain
[34].
[0285] Finally, the inventors tested a combination of these two
classes (anti-death and antiviral) of compounds for synergistic
effects in protecting neural cells from ZIKV-induced death. The
inventors found that in the two-drug combination of Emricasan and
PHA-690509, much lower concentrations of each drug were needed for
inhibiting caspase-3 activity (FIG. 4C). A similar synergistic
effect to preserve neural cell viability after ZIKV infection was
also observed (FIG. 4D). Importantly, Emricasan did not interfere
with PHA's ability to inhibit ZIKV infection in the combination
(FIGS. 8A-8C).
[0286] Emricasan, also named IDN-6556 or PF-03491390, is a
pan-caspase inhibitor that reduces liver injury and fibrosis in a
murine model of non-alcoholic steatohepatitis [35], where
hepatocyte apoptosis is a hallmark of the disease [35]. When
evaluated in clinical trials for chronic Hepatitis C patients,
Emricasan reduced liver injury and was tolerated over 12 weeks
[36,37]. It was reported that the overall (AUC) and maximum (Cmax)
concentration of Emricasan in human blood (oral, BID, .times.4
days) were 1.90 .mu.g/ml (3.35 .mu.M) and 2.36 .mu.g/ml (4.15
.mu.M), respectively. Therefore, Emricasan's plasma concentration
in humans could be suitable for protecting cells from ZIKV
infection-induced death.
[0287] PHA-690509 and the other four CDK inhibitors are all in
clinical trials for other indications. The inventors show that
these additional CDK inhibitors with unrelated structures also
inhibit ZIKV replication, supporting a role for CDKs in the life
cycle of ZIKV in human cells. As flaviviruses are not known to
encode any CDKs, these results suggest that one or more of cellular
CDKs in the host may be important for ZIKV replication. Further
studies on target identification for PHA-690509, including targeted
kinome siRNA or CRISPR screens, may reveal additional insights into
the mechanism of these inhibitors.
[0288] Niclosamide is an FDA-approved drug that is currently
prescribed to treat worm infections, but it has been reported to
inhibit viruses in culture systems [38-40]. Its broad antiviral
activity has been attributed to its ability to neutralize
endo-lysosomal pH and interfere with pH-dependent membrane fusion
[39], which is an essential step in the common virus entry pathway.
The data indicate that Niclosamide suppresses ZIKV infection when
added 4 hours after virus inoculation, suggesting significant
inhibition at a post-entry step, such as replication. Regardless of
the mechanism of action, Niclosamide is a category B drug, for
which no risk to fetus has been found in animal studies; it
therefore represents a promising candidate for a drug to be
repurposed for reducing ZIKV viral load in pregnant women.
[0289] Given the significant time needed to develop and test
effective vaccines, countermeasures against ZIKV, including small
molecule therapeutics, are urgently needed. The inventors have
developed two ultra-high throughput assays that measure
ZIKV-induced caspase 3 activity and reduced cell viability and
screened approximately 6,000 approved drugs and drug candidate
compounds. The inventors' efforts so far have led to the
identification of small molecules that either protect against cell
death or suppress ZIKV replication. The two groups of antiviral
compounds, Niclosamide and CDK inhibitors, likely act via distinct
mechanisms to inhibit ZIKV, and thus present an opportunity for a
combination treatment regimen. The structural diversity of
effective CDK inhibitors provides multiple chemical scaffolds to
which medicinal chemistry could be applied. The findings and tools
provided here will significantly advance the current ZIKV research
and have an immediate impact on the development of anti-ZIKV
therapeutics.
[0290] Further details concerning the screen and identified
compounds can be found in [47], which is incorporated herein by
reference in its entirety.
Supplemental Information--Expanded Materials and Methods
[0291] Cell Lines.
[0292] Human iPSCs were cultured and differentiated into cortical
neural progenitor cells as described previously [41]. The human
iPSC line was previously generated from a skin biopsy sample of a
male newborn (C1-2 line), and has been fully characterized and
passaged on MEF feeder layers [41]. All studies followed
institutional IRB protocols approved by Johns Hopkins University
School of Medicine and Florida State University. Human iPSCs were
differentiated into forebrain-specific hNPCs and immature neurons
following the previously established protocol [41]. Briefly, hiPSC
colonies were detached from the feeder layer with 1 mg/ml
collagenase treatment for 1 h and suspended in embryonic body (EB)
medium, consisting of FGF-2-free iPSC medium supplemented with 2
.mu.M Dorsomorphin and 2 .mu.M A-83 in non-treated polystyrene
plates for 4 days with a daily medium change. After 4 days, EB
medium was replaced by neural induction medium (NPC medium)
consisting of DMEM/F12, N2 supplement, NEAA, 2 .mu.g/ml heparin,
and 2 .mu.M cyclopamine. Floating EBs were then transferred to
Matrigel-coated 6-well plates at day 7 to form neural tube-like
rosettes. Attached rosettes were kept for 15 d with NPC medium
change every other day. On day 22, rosettes were picked
mechanically and transferred to low attachment plates (Corning) to
form neurospheres in NPC medium containing B27. Neurospheres were
then dissociated with Accutase at 37.degree. C. for 10 min and
placed onto Matrigel-coated 6-well plates at day 24 to form
monolayer hNPCs in NPC medium containing B27. These hNPCs expressed
forebrain-specific progenitor markers, including NESTIN, PAX6,
EMX-1, FOXG1, and OTX2 [41].
[0293] The human astrocytes (BJ line) were derived from healthy
control fibroblasts which were obtained from the American Type
Culture Collection (CRL-2522, male, neonatal). hiPSC and forebrain
NPCs were derived as described previously [42,43] hNPCs were
differentiated to astrocytes and cultured on Matrigel-coated plates
in astrocyte medium (ScienCell). Astrocytes were split 1:3 every
week with Accutase and could be expanded up to 120 days in
astrocyte medium. Human astrocytes and neural progenitor cells were
differentiated from human induced pluripotent stem cells as
described previously.
[0294] Human cerebral organoids were culture as described
previously [44]. To generate forebrain-specific organoids, human
iPSC colonies were detached 7 days after passage with Collagenase
Type IV, washed with fresh stem cell medium and cultured in a 15 ml
conical tube. On day 1, detached and washed iPSC colonies were
transferred to an Ultra-Low attachment 6-well plate (Corning
Costar), containing 3 ml of stem cell medium (without FGF-2), plus
2 .mu.M Dorsomorphine (Sigma) and 2 .mu.M A83-01 (Tocris). On days
5-6, half of the medium was replaced with induction medium
consisting of DMEM:F12, 1.times.N2 Supplement (Invitrogen), 10
.mu.g/ml Heparin (Sigma), 1.times. Penicillin/Streptomycin,
1.times. Non-essential Amino Acids, 1.times. Glutamax, 4 ng/ml
WNT-3A (R&D Systems), 1 .mu.M CHIR99021 (Cellagentech), and 1
.mu.M SB-431542 (Cellagentech). On day 7, organoids were embedded
in Matrigel (BD Biosciences) and continued to grow in induction
medium for 6 more days. On day 14, embedded organoids were
mechanically dissociated from Matrigel by pipetting up and down
onto the plate with a 5 ml pipette tip. Typically, 10-20 organoids
were transferred to each well of a 12-well spinning bioreactor
(Spin.OMEGA.) containing differentiation medium, consisting of
DMEM:F12, 1.times.N2 and B27 Supplements (Invitrogen), 1.times.
Penicillin/Streptomycin, 1.times.2-Mercaptoenthanol, 1.times.
Non-essential Amino Acids, 2.5 .mu.g/ml Insulin (Sigma).
[0295] The glioblastoma SNB-19 cell line (part of the National
Cancer Institute 60 human tumor cell line) was a gift from Dr.
David Meckes (FSU, Tallahassee, Fla.). SNB-19 cells were maintained
at 37.degree. C. in 5% CO.sub.2 in RPMI-60, 1.times.
penicillin/streptomycin, and 10% fetal bovine serum (Invitrogen).
The Aedes albopictus C6/36 cell line (ATCC) was maintained at
28.degree. C. in 5% CO.sub.2.
[0296] Human astrocytes and neural progenitor cells were
differentiated from human induced pluripotent stem cells as
described previously [41]. Luminescence caspase 3/7 activity assay
kits (catalog number G8092) were obtained from Promega (Madison,
Wis.). ATPlite 1 step Luminescence Assay System kits (catalog
number 6016731) for the ATP content cell viability assay were
purchased from PerkinElmer. Polystyrene plates (384-well and
1536-well; regular tissue culture treated and PDL coated) were
purchased from Greiner Bio-One (Monroe, N.C.).
[0297] Preparation of ZIKV and Cell Infection.
[0298] The MR766-ZIKV stock with the titer of 1.times.10.sup.5
Tissue Culture Infective Dose (TCID)/ml in the form of culture
fluid from an infected rhesus Macaca cell line LLC-MK2, was
originally obtained from ZeptoMetrix (Buffalo, N.Y.). The
FSS13025-ZIKV strain was shared by Drs. Robert Tesh and Pei-Yong
Shi (University of Texas Medical Branch, Galveston, Tex.). The
PRVABC59 strain was obtained from ATCC (Manassas, Va.). Original
viral stocks were then amplified in Aedes albopictus clone C6/36
cells. Briefly, C6/36 cells were inoculated with viral inoculum for
one hour at 28.degree. C. in low volume of media (3 mL per T-75
flask), with rocking every 15 minutes, before addition of 17 mL
media. Viral inoculated cells were incubated at 28.degree. C. for
6-7 days before harvesting of supernatant. C6/36-amplified ZIKV
titer was determined by infecting vero cells for 48 hours with a
methyl-cellulose overlay and analyzed for focus-forming units per
mL (FFU/mL). In mock infections, an equal volume of spent
uninfected C6/36 culture medium was used. For infections, cells
were seeded into 12-well plates with/without coverslips one day
prior to virus addition. For all cell types, compound was added 1
hr prior to viral addition unless otherwise specified. For neural
progenitor cell infections, viral inoculum was added for 2 hours
before being removed and replaced with fresh media. Cells were
harvested at 24-72 hours post-infection.
[0299] Immunocytochemistry.
[0300] Cells were fixed with 4% paraformaldehyde (Sigma) for 15 min
at room temperature. Samples were permeabilized and blocked with
0.25% Triton X-100 (Sigma) and 10% donkey or goat serum in PBS for
20 min as previously described (Chiang et al., 2011; [41]; Yoon et
al., 2014). Samples were then incubated with primary antibodies at
4.degree. C. overnight, followed by multiple PBST washes and
incubation with secondary antibodies for 1 h at room temperature.
Slides were mounted using VECTASHIELD with DAPI (Vectorlabs,
Burlingame, Calif.). The following primary antibodies were used:
anti-Flavivirus Group Antigen (clone D1-4G2-4-15; mouse; 1:500;
Millipore), and anti-Cleaved caspase-3 (Asp15; Rabbit; 1:500; Cell
Signaling Technology). Antibodies were prepared in PBS containing
0.25% Triton X-100 and 10% donkey serum. Images were taken by Zeiss
LSM 700 and 880 confocal microscopes, Olympus BX61, or Zeiss
Axiovert 200M microscope.
[0301] Western Blot.
[0302] Cells were harvested by trypsinization, pelleting, and
subsequent lysis in 1.times. Laemlli buffer and boiled, or directly
lysed in 1.times. Laemlli buffer and boiled. Antibodies used were
anti-ZIKV NS1 (1:2000; BF-1225-36, BioFront Technologies,
Tallahassee, Fla.) or GAPDH (Santa Cruz Biotechnology, Texas).
[0303] NS1 ELISA.
[0304] The anti-ZIKV NS1 ELISA (ZKV-NS1-EK) was obtained from
BioFront Technologies (Tallahassee, Fla.) and used according to the
manufacturer's protocol.
[0305] Compound Libraries.
[0306] The Library of Pharmacologically Active Compounds (LOPAC),
consisting of 1280 compounds, was purchased from Sigma-Aldrich. The
NCGC pharmaceutical collection, a collection consisting of 2816
clinically approved drugs, was internally established in 2011 [16].
All compounds were dissolved in DMSO as 10 mM stock solutions, then
diluted in DMSO at a 1:3 ratio in 384-well plates, followed by
reformatting into 1536-well compound plates use in high-throughput
screening (HTS).
[0307] Caspase 3/7 Assay.
[0308] Caspase-Glo 3/7 assay kit (Promega) was used to detect
caspase 3/7 activity. Reagents were reconstituted as described in
the protocol from the manufacturer. Cells were seeded in 384- and
1536-well assay plates and cultured at 37.degree. C. with 5%
CO.sub.2 for 16 hours. ZIKV solution was added to cells, followed
by incubation at 37.degree. C. with 5% CO.sub.2 for 6 hours.
Caspase-Glo 3/7 was added to each well at a 1:1 ratio, unless
otherwise specified, and incubated at room temperature for 30
minutes. The luminescence intensity of the assay plates was
measured using a ViewLux plate reader (PerkinElmer). Data was
normalized by using the cell-containing wells without ZIKV as a
negative (0% induction of caspase 3/7 activity) control and wells
containing ZIKV infected cells that induced caspase 3/7 activity
were used as a positive control (100% induction of caspase 3/7
activity).
[0309] ATP Content Assay for Cell Viability and Compound
Cytotoxicity.
[0310] The ATPlite luminescence assay system assay kit
(PerkinElmer) was used to determine cell viability. The reagent was
reconstituted and prepared as described by the manufacture. In
order to measure the cell death caused by ZIKV infection, cells
were cultured for 16 hours at 37.degree. C. with 5% CO.sub.2 in
assay plates, followed by addition of ZIKV solution and incubation
at 37.degree. C. with 5% CO.sub.2 for 72 hours. ATPlite, the ATP
monitoring reagent, was then added to the assay plates and
incubated for 15 minutes. The resulting luminescence was measured
using the ViewLux plate reader (Perkin Elmer). Data was normalized
using the wells without cells as a control for 100% cell killing,
and cell-containing wells without ZIKV infection were used as full
cell viability (0% cell killing).
[0311] Large-Scale Compound Screening.
[0312] Human NPCs were seeded onto PDL coated 1536-well assay
plates at 250 cells per 3 .mu.l/well and incubated at 37.degree. C.
in 5% CO.sub.2 for 16 hours. Test compounds dissolved in DMSO were
transferred to assay plates at a volume of 23 nl/well by an
automated pintool workstation (Wako Automation, San Diego, Calif.).
Compounds were incubated with cells for 30 minutes at 37.degree. C.
in 5% CO.sub.2, immediately followed by the addition of 2
.mu.l/well of ZIKV. Incubation time of compound-treated cells with
ZIKV varied based on assay format. Experiments measuring virus
induced caspase 3/7 activity required a 6 hour incubation of ZIKV
in the presence compounds at 37.degree. C. in 5% CO.sub.2.
Following this incubation, 3 .mu.l/well of caspase 3/7 reagent
mixture was added to assay plates, incubated for 30 minute at room
temperature, and the resultant luminescence signal was measured
using a ViewLux plate reader (Perkin Elmer). Experiments measuring
the virus induced cell death required a 72 hour incubation of ZIKV
in the presence of compounds at 37.degree. C. in 5% CO.sub.2.
Following this incubation, 3 .mu.l/well of ATP content detection
reagent was added to assay plates, incubated for 30 minute at room
temperature, and the resultant luminescence signal was measured in
a ViewLux plate reader. Step-by-step assay protocols are listed in
Table 1 and Table 2.
[0313] Because compound cytotoxicity could nonspecifically reduce
the caspase activity induced by ZIKV, the inventors also used the
ATP content assay to measure compound cytotoxicity in the absence
of ZIKV infection. The cells were seeded in the same way as
described above in 1536-well assay plates. After a 6 hour
incubation with compounds in the absence of ZIKV, 3 .mu.l/well of
ATP content reagent mixture was added to the assay plates and
incubated for 30 minute at room temperature. The luminescence
signal in the assay plates was measured using a ViewLux plate
reader (Table 3). Any compounds that exhibited cytotoxicity were
eliminated as false positive compounds.
[0314] Antiviral Compound Analysis.
[0315] SNB-19, BJ astrocyte, or NPCs were seeded in 12-well plates
at approximately 3e5 cells/well. The next day, cells were treated
with compound at 1-10.times.IC50 or indicated concentration for 1
hour prior to inoculation with virus at MOI=0.5-1. Cells and
supernatant were harvested 24-48 hours post infection and analyzed
by western blot or IFA. Western blot bands were quantified and IFA
images counted using ImageJ (NIH, Bethesda, Md.).
[0316] Data Analysis and Statistics.
[0317] The primary screen data and curve fitting were analyzed
using software developed internally [17]. The
concentration-response curves and IC50 values of compound
confirmation data were calculated using Prism software (GraphPad
Software, Inc. San Diego, Calif.). All values are expressed as the
mean+/-SD (n.gtoreq.3) unless it was specified. Western blots and
IFA images were quantified using ImageJ (NIH, Bethesda, Md.).
TABLE-US-00004 TABLE 4 Protocol of caspase 3/7 activity assay.
Value Step Parameter 384-well 1536-well Description 1 Cell plating
20 .mu.l/well 3 .mu.l/well PDL-coated plates used for
hNPCs/astrocytes 2 Incubation overnight at 37.degree. C. with 5%
CO.sub.2 3 Compound addition 6 .mu.l/well 0.023 .mu.l/well in DMSO
solution 4 Incubation 30 minutes at 37.degree. C. with 5% CO.sub.2
5 Zika virus addition 4 .mu.l/well 2 .mu.l/well 6 Incubation 6
hours at 37.degree. C. with 5% CO.sub.2 7 Reagent addition 30
.mu.l/well 3.5 .mu.l/well Caspase 3/7 assay mixture 8 Incubation 30
minutes at room temperature 9 Plate reading luminescence mode
ViewLux plate reader
TABLE-US-00005 TABLE 5 Protocol of ATP cell viability assay for
ZIKV-induced cell death. Value Step parameter 384-well 1536-well
Description 1 Cell plating 20 .mu.l/well 3 .mu.l/well PDL-coated
plates used for hNPCs/astrocytes 2 Incubation overnight at
37.degree. C. with 5% CO.sub.2 3 Compound addition 6 .mu.l/well
0.023 .mu.l/well in DMSO solution 4 Incubation 30 minutes at
37.degree. C. with 5% CO.sub.2 5 Zika virus addition 4 .mu.l/well 2
.mu.l/well 6 Incubation 3 days at 37.degree. C. with 5% CO.sub.2 7
Reagent addition 30 .mu.l/well 3.5 .mu.l/well ATP content assay
mixture 8 Incubation 15 minutes at room temperature 9 Plate reading
luminescence mode ViewLux plate reader
TABLE-US-00006 TABLE 6 Protocol of ATP content cell viability assay
for compound cytotoxicity. Value Step parameter 384-well 1536-well
Description 1 Cell plating 24 .mu.l/well 5 .mu.l/well PDL-coated
plates used for NPCs/astrocytes 2 Incubation Overnight at
37.degree. C. with 5% CO.sub.2 3 Compound addition 6 .mu.l/well
0.023 .mu.l/well in DMSO solution 4 Incubation 30 minutes at
37.degree. C. with 5% CO.sub.2 5 Incubation 6 hours at 37.degree.
C. with 5% CO.sub.2 6 Reagent addition 30 .mu.l/well 3.5 .mu.l/well
ATP content assay mixture 7 Incubation 15 minutes at room
temperature 8 Plate reading luminescence mode ViewLux plate
reader
[0318] Note: no Zika virus was added to assay plate in this
experiment.
TABLE-US-00007 TABLE 7 List of compounds including 35 confirmed
compounds determined in the caspase 3/7 assay with hNPCs, SNB-19
cells, and human astrocytes. Caspase 3 Cell viability Caspase 3
Cell viability (IC50, uM) (IC50, uM) (efficacy %) (efficacy, %)
Astro- SNB- Astro- SNB- Astro- SNB- Astro- SNB- SampleID Name
Library cyte 19 NPCs cyte 19 NPCs cyte 19 NPCs cyte 19 NPCs
NCGC00346477-01 Emricasan C 0.46 0.62 0.37 4.11 0.87 3.88 77.1 97.6
85.3 98.7 194 54.4 NCGC00263218-02 Teriflunomide C 1.30 1.03 0.92
-- -- -- 86.9 93.1 94.5 0.0 0.0 -86.9 NCGC00346732-02
Hydroxocobalamin C 0.97 1.46 0.87 -- -- -- 83.4 85.7 67.4 0.0 0.0
0.0 NCGC00166262-03 Ensulizole B 1.95 1.84 1.38 -- -- -- 82.7 73.3
80.6 0.0 0.0 0.0 NCGC00160655-03 Tenonitrozole B 2.06 1.95 1.30 --
-- -- 88.8 81.7 81.5 0.0 0.0 -47.2 NCGC00090504-08
Isoliquiritigenin A 3.08 2.18 1.84 -- 3.08 -- 95.1 96.9 89.0 0.0
36.3 -46.1 NCGC00090774-03 Nitazoxanide B 2.00 2.24 1.26 31.6 23.7
-- 90.3 86.4 82.3 53.6 62.1 -68.9 NCGC00182059-02 Febuxostat B 2.31
2.91 3.88 -- 3.66 -- 84.8 104 83.2 0.0 68.4 -43.8 NCGC00015610-12
Leflunomide B 4.35 3.27 1.38 -- -- -- 98.1 106 97.8 0.0 0.0 -55.6
NCGC00345056-01 Vidofludimus C 2.91 3.66 2.45 -- -- -- 101 101 87.8
0.0 0.0 0.0 NCGC00094344-03 SB-366791 A 4.11 3.66 2.91 -- -- --
93.9 93.3 89.9 0.0 0.0 -42.0 NCGC00015420-08 Emodin A 3.27 4.35
2.59 -- -- -- 86.4 83.3 82.1 0.0 0.0 0.0 NCGC00164168-02 Diphenyl B
6.68 4.73 4.47 -- -- -- 104 92.8 89.4 0.0 0.0 0.0 isophthalate
NCGC00164513-02 Benzoylpas B 5.81 6.90 4.89 -- -- -- 94.9 92.8 85.4
0.0 0.0 0.0 NCGC00092384-03 Fenobam B 9.20 7.31 5.48 -- 1.73 --
97.2 110 108 0.0 60.5 0.0 NCGC00253758-01 Indobufen B 6.52 7.74
6.15 -- -- -- 86.3 85.8 81.3 0.0 0.0 0.0 NCGC00166243-03 2-(2H- B
9.20 8.69 4.89 -- -- -- 68.5 80.1 77.6 0.0 0.0 0.0 Benzotriazol-
2-yl)-4- methylphenol NCGC00263191-01 PHA-690509 C 13.0 9.20 19.5
7.74 10.9 -- 71.6 74.6 73.0 81.9 165 -60.6 NCGC00179573-03
Tiaprofenic acid B 6.15 10.3 8.69 -- -- -- 88.2 96.2 86.7 0.0 0.0
0.0 NCGC00016490-12 Flufenamic acid B 8.69 10.9 17.3 -- -- -- 71.9
61.5 68.1 0.0 0.0 0.0 NCGC00249888-01 Vitamin B12 B 8.69 11.6 6.90
-- -- -- 77.1 101 94.7 0.0 0.0 0.0 NCGC00024599-04 Cinanserin B
15.5 12.3 17.3 -- 19.5 -- 80.2 92.0 80.4 0.0 56.9 0.0
NCGC00015740-10 5-Nitro-2- A 9.75 13.0 8.69 -- -- -- 82.1 82.9 59.8
0.0 0.0 0.0 (3-phenyl- propylamino) benzoic acid NCGC00250411-01
Veliflapon C 9.20 13.8 9.75 -- -- -- 65.9 70.0 74.3 0.0 0.0 -36.0
NCGC00016410-13 Thiabendazole B 10.3 14.6 9.75 -- -- -- 81.0 89.4
83.4 0.0 0.0 0.0 NCGC00025046-05 SIB 1893 A 12.3 15.5 8.69 -- -- --
96.2 105 95.2 0.0 0.0 0.0 NCGC00167471-02 Anethole trithione B 13.8
15.5 13.0 -- -- -- 62.7 85.5 70.3 0.0 0.0 0.0 NCGC00016457-03
Naringenin C 17.3 15.5 15.5 -- -- -- 65.2 66.3 63.4 0.0 0.0 -32.5
NCGC00018130-09 Phenazopyridine B 15.5 16.4 11.6 -- 27.5 -- 69.9
73.7 72.5 0.0 35.3 -38.1 hydrochloride NCGC00160438-02 Fanetizole B
10.9 17.3 14.6 -- -- -- 72.9 78.1 81.8 0.0 0.0 0.0 NCGC00016026-08
Terazosin B 12.3 17.3 10.9 -- -- -- 88.9 77.3 84.4 0.0 0.0 0.0
hydrochloride NCGC00018274-05 Diacerein B 15.5 17.3 15.5 30.8 -- --
72.9 63.2 56.5 35.2 0.0 0.0 NCGC00346625-01 CAY10505 C 16.4 17.3
10.9 -- -- -- 80.1 91.2 83.5 0.0 0.0 0.0 NCGC00163540-03 Hesperetin
C 16.4 17.3 10.9 -- -- -- 72.7 86.0 79.6 0.0 0.0 -52.8
NCGC00094916-06 Suprofen B 19.5 17.3 15.5 -- -- -- 64.5 65.6 59.1
0.0 0.0 0.0 NCGC00185990-01 Ketorolac B 21.8 17.3 10.3 -- -- --
61.7 84.5 75.0 0.0 0.0 0.0 tromethamine NCGC00094872-08 Piperine C
20.6 19.5 13.8 -- -- -- 73.9 75.5 66.0 0.0 0.0 -44.4
NCGC00344543-01 Pirarubicin B 13.8 20.6 -- -- -- -- 59.8 63.9 51.5
0.0 0.0 -87.7 NCGC00263135-01 Piraxostat C 8.91 21.1 15.8 63.1 5.01
-- 70.5 63.5 67.1 34.7 60.3 -39.7 NCGC00253760-01 Albendazole oxide
B 15.5 21.8 12.3 -- -- -- 78.8 86.0 88.1 0.0 0.0 -62.1
NCGC00016019-06 Tyrphostin AG 494 A 18.4 21.8 17.3 -- 30.8 -- 72.5
55.6 72.8 0.0 45.6 -62.9 NCGC00346737-01 Genistin C 25.9 23.1 17.3
-- -- -- 60.2 58.1 58.6 0.0 0.0 -80.6 NCGC00016834-07 Fenbufen B
21.8 24.5 19.5 -- -- -- 81.5 76.3 72.5 0.0 0.0 0.0 NCGC00249393-01
Apatinib C 19.5 24.5 -- -- -- -- 57.9 61.2 0.0 0.0 0.0 0.0
NCGC00159570-01 RITA C 27.5 0.22 14.6 5.81 -- -- 41.2 101 50.9 81.7
0.0 -91.4 NCGC00015735-08 Niclosamide B 1.84 2.06 1.84 1.73 1.95 --
96.8 93.9 87.5 51.2 97.3 -77.4 NCGC00162305-03 BF-170 A 4.61 6.15
4.11 6.15 -- -- 118 104 92.7 65.8 0.0 -61.1 hydrochloride
NCGC00263159-01 OSI-930 C 8.20 6.90 6.90 -- 3.08 -- 91.5 114 96.9
0.0 71.2 -56.2 NCGC00159476-09 Tribromsalan B 6.90 7.74 13.8 -- --
-- 102 107 108 0.0 0.0 -35.7 NCGC00014684-04 Pifexole B 6.90 8.20
4.61 -- -- -- 86.3 83.6 92.2 0.0 0.0 -35.3 NCGC00017269-06
Formononetin C 14.6 12.3 10.9 -- -- -- 94.7 104 88.3 0.0 0.0 -56.0
NCGC00015412-13 Ebselen A 8.20 17.3 8.20 -- -- -- 93.8 117 109 0.0
0.0 -82.0 NCGC00018185-07 Tranilast C 17.3 17.3 19.5 -- -- -- 71.6
70.3 107 0.0 0.0 -54.5 NCGC00164155-02 Benzylparaben B 17.3 19.5
15.5 -- -- -- 62.8 91.8 56.0 0.0 0.0 0.0 NCGC00249899-01
2-Ethoxylethyl-p- B -- 27.5 18.4 -- -- -- 0.0 53.0 56.0 0.0 0.0 0.0
methoxycinnamate NCGC00017236-08 Baicalein C -- 29.1 18.4 -- -- --
0.0 50.6 57.5 0.0 0.0 -69.3 NCGC00319019-01 Nemorubicin B 15.5 30.8
-- 0.87 0.41 -- 42.6 53.1 82.9 146 117 -57.2 NCGC00015892-05
Rutaecarpine A 4.89 5.18 4.61 -- -- -- 89.5 86.4 87.5 0.0 0.0 -44.7
NCGC00015682-02 MPEP A 5.81 5.18 4.11 -- -- -- 118 98.6 96.1 0.0
0.0 0.0 NCGC00016456-12 5,7- C 15.5 8.69 5.48 -- -- -- 55.1 42.7
51.0 0.0 0.0 -69.3 Dihydroxyflavone NCGC00249888-04 Vitamin B12 B
6.73 13.4 11.3 -- 0.12 -- 62.7 43.0 59.1 0.0 67.3 0.0
NCGC00160646-02 Pipofezine B 11.3 15.1 12.0 -- -- -- 56.3 31.5 47.7
0.0 0.0 0.0 NCGC00182710-02 Flurbiprofen axetil B 21.8 19.5 -- --
-- -- 65.5 45.7 0.0 0.0 0.0 0.0 NCGC00091632-02 2-Amino-6- B 39.8
53.1 56.2 -- -- -- 54.1 46.1 57.1 0.0 0.0 0.0 nitrobenzothiazole
NCGC00013058-02 Malachite B 17.3 -- 21.8 -- -- -- 54.0 0.0 62.7 0.0
0.0 -89.7 green oxalate NCGC00262948-01 Enfenamic acid B 25.9 --
21.8 -- -- 7.74 58.0 49.5 47.1 0.0 0.0 58.5 NCGC00091080-03
Fenaminosulf B 35.5 -- -- -- 15.8 -- 58.5 0.0 0.0 0.0 36.7 -53.0
NCGC00185997-02 AS-252424 A 1.73 2.18 1.64 -- -- -- 37.5 38.0 52.3
0.0 0.0 0.0 NCGC00163250-02 Phenserine A 25.9 19.5 25.9 -- -- --
47.4 38.4 51.5 0.0 0.0 -105 NCGC00164613-03 Epalrestat C 19.5 20.6
15.5 -- 5.48 -- 93.3 79.2 72.1 0.0 82.1 -47.9 NCGC00095227-06
Alizarin B 30.8 21.8 21.8 -- -- -- 47.9 40.9 58.5 0.0 0.0 0.0
NCGC00263083-01 Dalcetrapib C -- -- 35.5 47.3 35.5 -- 0.0 50.2 50.2
45.7 110 -43.6 Note: the full name of compound can be found in the
Pubchem website (pubchem.ncbi.nlm.nih.gov) using the Sample ID
number in the first column. A: LOPAC library (Sigma-Aldrich). B:
Approved drug library. C: Clinical drug candidate library.
TABLE-US-00008 TABLE 8 IC.sub.50 values of selected compounds for
improving cell viability in hNPCs, astrocytes and SNB-19 cells
following ZIKV infection. IC.sub.50 (.mu.M) Compound ID Compound
name Collection* hNPCs Astrocytes SNB-19 NCGC00346477 Emricasan C
3.88 4.11 0.87 NCGC00263191 PHA-690509 C -- 7.47 10.1 NCGC00015735
Niclosamide B -- 1.73 1.95 Note: *compound is from "A" - LOPAC
library, "B" - approved drug library, and "C" - clinical drug
candidate library. "--": n.a.
[0319] FIGS. 17A-C show toxicity analysis of CDKis on human
astrocyte viability and hNPC proliferation. FIGS. 18A to 18G-2 show
the antiviral activity of emetine against ZIKV infection. FIGS. 19A
to 19E-2 show the antiviral activity of proteasome inhibitors
against ZIKV and dengue virus (DENV) infection
Example 2--Further Anti-Zika Screen and Anti-Zika Virus
Compounds
[0320] The inventors identified the compounds of Table 9 from a
library of compounds used in a large scale compound screen
measuring cell-death inhibition in ZIKV infected cells. Each
compound having both a NS1 IC50 value below 10.sup.6 M (1 uM) and a
ratio of Toxicity IC.sub.50 (cell-killing activity) to NS1
IC.sub.50 (virus-inhibiting activity) of 4 or greater was selected
as an effective compound.
[0321] A one-step NS1 TR-FRET (Time-resolved fluorescence resonance
energy transfer) assay for a screen of anti-ZIKV compounds was
carried out. Human embryonic kidney cell line HEK293 cells were
seeded into 384-well plates and infected with ZIKV MR766 at a MOI
of 0.5 for 2 days. The FRET assay was performed with two clones of
anti-NS1 antibody (ab) differentially labelled with donor and
acceptor fluorophores. Different combinations of antibody dilutions
were tested to achieve high and producible signal to noise
ratios.
[0322] Because this assay is measuring NS1, which a virally-encoded
protein, this screen is focused on compounds that have antiviral
activities as opposed to compounds that can block ZIKV-induced cell
death. The advantage of this FRET assay over the NS1 ELISA assay
[47] is it has been adapted to the 384-well format and does not
require a washing step. These are advantages as a screening
assay.
[0323] Each compound having both a NS1 IC.sub.50 value below
10.sup.-6 M (1 uM) and a ratio of Toxicity IC.sub.50 (cell-killing
activity) to NS1 IC.sub.50 (virus-inhibiting activity) of 4 or
greater was selected as an effective compound. The compounds
identified as effective from the screen are listed in Table 9 and
their chemical structures shown in Table 10.
TABLE-US-00009 TABLE 9 Tox IC50/NS1 Name NS1-IC50 Tox IC50 IC50
ratio 1. SN-38 1.13E-08 No 2. Echinomycin 1.74E-08 No 3.
(S)-(+)-Camptothecin 2.64E-08 No 4. BI-2536 4.14E-08 No 5.
10-hydroxycamptothecin 4.66E-08 No 6. Topotecan hydrochloride
5.14E-08 No 7. Delanzomib 5.85E-08 3.00E-07 5.1 8. Volasertib
6.48E-08 No 9. Ispinesib 6.74E-08 3.75E-05 555.9 10. PACLITAXEL
8.94E-08 No 11. FK-506 1.04E-07 No 12. Emetine 1.14E-07 No 13.
AVN-944 1.36E-07 2.00E-05 147.0 14. Digoxin 1.52E-07 6.00E-05 394.2
15. Dinaciclib 1.54E-07 No 16. Vincristine sulfate 1.56E-07 No 17.
Idarubicin hydrochloride 1.68E-07 No 18. Thapsigargin 1.71E-07
4.00E-05 234.5 19. Lexibulin hydrochloride 1.71E-07 No 20. Ixazomib
citrate 1.96E-07 1.58E-06 8.1 21. Cephalomannine 2.21E-07 No 22.
Mitoxantrone 2.32E-07 5.00E-05 215.4 23. MLN-2238 2.34E-07 2.13E-06
9.1 24. Demecolcine 2.38E-07 No 25. RGB-286147 2.68E-07 No 26.
Vinorelbine tartrate 2.70E-07 No 27. Bardoxolone methyl 2.82E-07
2.68E-06 9.5 28. Cycloheximide 2.87E-07 No 29. Actinomycin D
3.52E-07 No 30. AZD-7762 3.66E-07 No 31. PF-184 3.88E-07 No 32.
CHIR-124 4.05E-07 3.25E-06 8.0 33. Cyanein 4.32E-07 No 34.
Triptolide 4.65E-07 No 35. KX-01 4.81E-07 1.93E-05 40.2 36.
PF-477736 4.89E-07 No 37. Epirubicin hydrochloride 4.92E-07 No 38.
Mycophenolate mofetil 5.29E-07 No 39. Daunorubicin 5.36E-07 No 40.
Mycophenolic acid 5.39E-07 No 41. PIK-75 6.16E-07 No 42. Vindesine
sulfate salt 6.64E-07 No 43. Torin-2 7.24E-07 No 44.
7-Hydroxystaurosporine 7.60E-07 1.02E-04 134.0 45. CGP-60474
7.94E-07 6.00E-05 75.6 46. Floxuridine 9.18E-07 6.00E-05 65.4 47.
Go-6976 9.44E-07 2.02E-05 21.4 48. OSU-03012 9.92E-07 1.00E-05
10.1
TABLE-US-00010 TABLE 10 Structures Name Structure 1. SN-38
##STR00016## 2. Echinomycin ##STR00017## 3. (S)-(+)-Camptothecin
##STR00018## 4. BI-2536 ##STR00019## 5. 10-hydroxycamptothecin
##STR00020## 6. Topotecan hydrochloride ##STR00021## 7. Delanzomib
##STR00022## 8. Volasertib ##STR00023## 9. Ispinesib ##STR00024##
10. PACLITAXEL ##STR00025## 11. FK-506 ##STR00026## 12. Emetine
##STR00027## 13. AVN-944 ##STR00028## 14. Digoxin ##STR00029## 15.
Dinaciclib ##STR00030## 16. Vincristine sulfate ##STR00031## 17.
Idarubicin hydrochloride ##STR00032## 18. Thapsigargin ##STR00033##
19. Lexibulin hydrochloride ##STR00034## 20. Ixazomib citrate
##STR00035## 21. Cephalomannine ##STR00036## 22. Mitoxantrone
##STR00037## 23. MLN-2238 ##STR00038## 24. Demecolcine ##STR00039##
25. RGB-286147 ##STR00040## 26. Vinorelbine tartrate ##STR00041##
27. Bardoxolone methyl ##STR00042## 28. Cycloheximide ##STR00043##
29. Actinomycin D ##STR00044## 30. AZD-7762 ##STR00045## 31. PF-184
##STR00046## 32. CHIR-124 ##STR00047## 33. Cyanein ##STR00048## 34.
Triptolide ##STR00049## 35. KX-01 ##STR00050## 36. PF-477736
##STR00051## 37. Epirubicin hydrochloride ##STR00052## 38.
Mycophenolate mofetil ##STR00053## 39. Daunorubicin ##STR00054##
40. Mycophenolic acid ##STR00055## 41. PIK-75 ##STR00056## 42.
Vindesine sulfate salt ##STR00057## 43. Torin-2 ##STR00058## 44.
7-Hydroxystaurosporine ##STR00059## 45. CGP-60474 ##STR00060## 46.
Floxuridine ##STR00061## 47. Go-6976 ##STR00062## 48. OSU-03012
##STR00063##
Example 3--Antiviral Activity of Niclosamide Derivatives
[0324] A scheme for the synthesis of the Niclosamide derivative
5-Chloro-N-(2-chloro-4-nitrophenyl)-2-(phosphonooxy)-benzamide
(also referred to as p-niclosamide) is shown in FIG. 20.
[0325] Niclosamide monophosphate 4 can be easily made from
commercially available niclosamide 1 in three steps:
phosphorylation of 1 followed by TMSBr mediated deprotection of
phosphate esters 2 gave trimethylsilyl phosphate 3, subsequent
treatment with methanol or water provided desired compound 4.
[0326] Step a: Niclosamide 1 (2 mmol) was mixed with 10 mL of
anhydrous CH.sub.3CN. Then at - 10 OC was added CCl.sub.4 (10
mmol), DIPEA (4 mmol) and DMAP (0.5 mmol). The reaction mixture was
then stirred at this temperature for 10 minutes. Then diethyl
phosphate (3 mmol) was added dropwise and the resulting mixture was
stirred at -10.degree. C. until reaction was complete. At 0.degree.
C., the reaction was quenched with 50 mL of 0.5 mol/L
KH.sub.2PO.sub.4. The resulting mixture was warmed to room
temperature. The mixture was extracted with three 50 mL portions of
ethyl acetate. The combined organic layer was then washed with 50
mL of water and brine. The organic extract was dried over magnesium
sulphate and concentrated, providing 2 as a pale yellow solid,
which was directly used in the next step.
[0327] Step b: Under inert atmosphere, to a stirred solution of 2
(2 mmol) in anhydrous CHCl.sub.3 at room temperature was added
(CH.sub.3).sub.3SiBr (10 mmol). The reaction mixture was
concentrated under vacuum after overnight stirring at room
temperature. The resulting 3 was directly used in the next
step.
[0328] Step c: At 0.degree. C., 3 was mixed with 6 mL dry MeOH. The
solution was then warm to rt and stirred at rt. After 30 minutes,
LC-MS found no starting material left. The mixture was
concentrated, the resulting white solid was treated twice with 10
mL distilled H.sub.2O, followed by 5 mL dry MeOH. The resulting
white precipitate was then dried under high vacuum overnight,
providing 255 mg 4 as a white solid (31%, over three steps).
.sup.1H NMR (400 MHz, DMSO-d.sub.6) .delta. 10.50 (s, 1H), 8.48 (d,
J=9.1 Hz, 1H), 8.41 (d, J=2.7 Hz, 1H), 8.29 (dd, J=2.6, 9.1 Hz,
1H), 7.83 (dd, J=0.9, 2.8 Hz, 1H), 7.68 (dd, J=2.8, 8.9 Hz, 1H),
7.55 (dd, J=1.1, 8.9 Hz, 1H), 3.17 (s, 2H). LC/MS (Long Gradient 4%
to 100% Acetonitrile (0.05% TFA) over 7 minutes, Agilent Eclipse
XDB-C18 3 micron 3.times.75 mm): (electrospray+ve), m/z 407.0
(MH).sup.+, t.sub.R=3.971, UV.sub.254=100%.
[0329] FIGS. 21 and 22 show the antiviral activity of niclosamide
derivatives n-Niclosamide and DK-520, respectively, against ZIKV
infection in human glioblastoma SNB-19 cells.
Example 4--Evaluation of Compounds in Mice In Vivo
[0330] Animal models useful for Zika virus have been reported in
the literature (see, for example, Morrison T E et al., J. Virol.,
2017, "Animal Models of Zika Virus Infection, Pathogenesis, and
Immunity," 91(8):1-15; Dowall S D et al., PLOS Neglected Tropical
Diseases, 2016, "A Susceptible Mouse Model for Zika Virus
Infection," 10(5):e0004658; Koid F et al., Front Microbiol., 2016,
"Development of a Zika Virus Infection Model in Cynomolgus
Macaques," 7: 2028; Dudley D M et al., Nat Commun., 2016, "A rhesus
macaque model of Asian-lineage Zika virus infection," 28; 7:12204,
which are incorporated herein by reference in their entirety.
[0331] To further assess the activity of compounds of the invention
against ZIKV infection, compounds will be tested in mice. NOD SCID
mice have a spontaneous homozygous scid mutation (Prkdc.sup.scid).
They are characterized by an absence of functional T cells and B
cells, thus having severe combined immune deficiency (Bosma M J et
al., Annu Rev Immunol, 1991, "The SCID mouse mutant: definition,
characterization, and potential uses", 9: 323-350). Ifngr KO
(AG129) mice have an IFN-alpha/beta receptor (Ifngr) deficiency
(homozygous Ifngr1.sup.tm1Agt). These mice have normal T cell
responses but are defective in natural resistance, evidenced by an
increased susceptibility to viral infection (Huang, S W et al.,
Science, 1993, "Immune response in mice that lack the
interferon-gamma receptor," 259(5102):1742-1745). SJL is an inbred
strain that contains a naturally-occurring dysferlin mutation and
was originally developed from three different sources of Swiss
Webster mice. CD-1 is an outbred mouse line most commonly used in
toxicology and carcinogenicity bioassays.
[0332] In Vivo Infection:
[0333] Pregnant mice of SCID, AG129, SJL or CD-1 strain, will be
infected through footpad injection with ZIKV-containing saline on
day 6-14 of gestation. Footpad injection is a combination of
intradermal and subcutaneous injections used primarily in models of
immunization and to administer certain types of pathogens. This
route of injection has been successfully utilized to infect certain
strains of immune deficient mice (Lazear H M J et al., Cell Host
Microbe, 2016, "A Mouse Model of Zika Virus Pathogenesis"
19(5):720-30).
[0334] For safe and effective footpad injection, a published
protocol that uses a hands-free method to deliver infectious
material will be followed (Long K M et al., Methods Mol Biol, 2013,
"Safe and effective mouse footpad inoculation" 1031: 97-100).
Briefly, after inserting the animal into a commercial mouse
restrainer, the foot to be injected (hind foot) will be pulled to
the outside of the restrainer with the ventral side of the foot
facing upwards. Straight, atraumatic forceps will be used to grasp
the foot just above the toes. A needle (25G or smaller) will be
laid on top of the foot with bevel side up and inserted just under
the skin between the walking pads and the heel. A volume of 10-50
.mu.l (volume dependent on titer of solution; a dose of
10.sup.3-10.sup.5 infectious particles per animal) of ZIKV or
saline will be injected in a slow motion into each animal. 3-5
seconds after the delivery, the needle will be withdrawn slowly
from the injection site. Firm pressure will be maintained on the
injection site for a few seconds once the needle is removed, so
that no injected material should be leaking from the injection
site. After removing needle, the foot with the forceps will be
released and the mouse returned to its cage.
[0335] A potential alternative to footpad injection is injecting
into the hock rather than the foot; however, previous studies have
used the footpad route (for replication of technique). Also, no
adjuvant or irritating chemical is being used, only 1 foot will be
injected using a non-adjuvant diluent and no repeat injections will
occur.
[0336] Niclosamide and Niclosamide Derivatives
[0337] Niclosamide is an FDA-approved drug for use in humans to
treat worm infections and is well tolerated. It has low toxicity in
mammals with an oral LD50 of 45 mg/g in rats. The potency of
Niclosamide on inhibition of ZIKV replication is in the
submicromolar range, whereas clinically it can be reached at
micromolar levels.
Prospective Treatment Protocol:
[0338] Vehicle: saline
[0339] Route of Administration: IP injection; food feed or oral
garvage.
[0340] Proposed dosage: 20-100 mg/Kg
[0341] Number of doses: up to 7
[0342] Interval between doses: 1 day
[0343] Pre-treatment of animals: not necessary
Emricasan and Emricasan Derivatives
[0344] Emricasan, also named IDN-6556 or PF-03491390, is an
inhibitor of activated caspases with sub- to nano-molar activity in
vitro. Emricasan is currently being evaluated in Phase II clinical
trials to reduce hepatic injury and liver fibrosis caused by
chronic HCV infection, and has been well tolerated in human trials
without significant adverse events. It was reported that overall
and maximum concentrations of Emricasan (oral delivery, BID,
.times.4 days) in human blood were 1.90 .mu.g/ml (3.35 .mu.M) and
2.36 .mu.g/ml (4.15 .mu.M), respectively. Therefore, the reported
human plasma concentration of Emricasan is about 10-fold higher
than the IC50 for inhibition of increased caspase-3 activity and
cell death caused by ZIKV infection in vitro.
Prospective Treatment Protocol:
[0345] Vehicle: saline
[0346] Route of Administration: IP injection, food feed or oral
garvage.
[0347] Proposed dosage: 5-50 mg/Kg
[0348] Number of doses: up to 7
[0349] Interval between doses: 1 day
[0350] Pre-treatment of animals: not necessary
CDK Inhibitors and Derivatives
[0351] Many CDKis are being evaluated in clinical trials for
various cancers, Cytic Fibrosis, and Cushings Disease, but some of
these compounds may not be suitable for pregnant women because of
potentially hazardous effects on the fetus. In the in vitro
analyses herein, treatment with PHA-690509 or Seleciclib partially
rescued ZIKV-induced reduction of hNPC proliferation and the
treatment itself exhibited a minimal effect on hNPC proliferation
in brain organoids, which model early human brain development in
vitro. These CDKis and their derivatives may be useful for treating
non-pregnant humans who face an increased risk of Guillain-Barre
syndrome and other conditions following ZIKV infection. For
example, viral RNA and infectious virus have been detected in the
semen of men weeks after acute symptoms have resolved. A reduction
in viral load in men can potentially cut down transmission
risk.
Prospective Treatment Protocol:
[0352] Vehicle: saline
[0353] Route of Administration: IP injection; food feed or oral
garvage.
[0354] Proposed dosage: 20-100 mg/Kg
[0355] Number of doses: up to 7
[0356] Interval between doses: 1 day
[0357] Pre-treatment of animals: not necessary
[0358] It should be understood that the examples and embodiments
described herein are for illustrative purposes only and that
various modifications or changes in light thereof will be suggested
to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the
appended claims. In addition, any elements or limitations of any
invention or embodiment thereof disclosed herein can be combined
with any and/or all other elements or limitations (individually or
in any combination) or any other invention or embodiment thereof
disclosed herein, and all such combinations are contemplated with
the scope of the invention without limitation thereto.
REFERENCES
[0359] 1. Dick, G. W., Kitchen, S. F. & Haddow, A. J. Zika
virus. I. Isolations and serological specificity. Trans R Soc Trop
Med Hyg 46, 509-520 (1952). [0360] 2. Duffy, M. R., et al. Zika
virus outbreak on Yap Island, Federated States of Micronesia. N
Engl J Med 360, 2536-2543 (2009). [0361] 3. Cao-Lormeau, V. M., et
al. Zika virus, French polynesia, South pacific, 2013. Emerg Infect
Dis 20, 1085-1086 (2014). [0362] 4. Musso, D. Zika Virus
Transmission from French Polynesia to Brazil. Emerg Infect Dis 21,
1887 (2015). [0363] 5. Heymann, D. L., et al. Zika virus and
microcephaly: why is this situation a PHEIC? Lancet 387, 719-721
(2016). [0364] 6. Mlakar, J., et al. Zika Virus Associated with
Microcephaly. N Engl J Med 374, 951-958 (2016). [0365] 7.
Rasmussen, S. A., Jamieson, D. J., Honein, M. A. & Petersen, L.
R. Zika Virus and Birth Defects--Reviewing the Evidence for
Causality. N Engl J Med 374, 1981-1987 (2016). [0366] 8.
Cao-Lormeau, V. M., et al. Guillain-Barre Syndrome outbreak
associated with Zika virus infection in French Polynesia: a
case-control study. Lancet 387, 1531-1539 (2016). [0367] 9. Araujo,
L. M., Ferreira, M. L. & Nascimento, O. J. Guillain-Barre
syndrome associated with the Zika virus outbreak in Brazil. Arq
Neuropsiquiatr 74, 253-255 (2016). [0368] 10. Tang, H., et al. Zika
Virus Infects Human Cortical Neural Progenitors and Attenuates
Their Growth. Cell Stem Cell 18, 587-590 (2016). [0369] 11. Garcez,
P. P., et al. Zika virus impairs growth in human neurospheres and
brain organoids. Science 352, 816-818 (2016). [0370] 12. Qian, X.,
et al. Brain-Region-Specific Organoids Using Mini-bioreactors for
Modeling ZIKV Exposure. Cell 165, 1238-1254 (2016). [0371] 13.
Rossi, S. L., et al. Characterization of a Novel Murine Model to
Study Zika Virus. Am J Trop Med Hyg (2016). [0372] 14. Lazear, H.
M., et al. A Mouse Model of Zika Virus Pathogenesis. Cell Host
Microbe (2016). [0373] 15. Cugola, F. R., et al. The Brazilian Zika
virus strain causes birth defects in experimental models. Nature
534, 267-271 (2016). [0374] 16. Li, C., et al. Zika Virus Disrupts
Neural Progenitor Development and Leads to Microcephaly in Mice.
Cell Stem Cell (2016). [0375] 17. Miner, J. J., et al. Zika Virus
Infection during Pregnancy in Mice Causes Placental Damage and
Fetal Demise. Cell 165, 1081-1091 (2016). [0376] 18. Mlakar, J., et
al. Zika Virus Associated with Microcephaly. N Engl J Med (2016).
[0377] 19. Driggers, R. W., et al. Zika Virus Infection with
Prolonged Maternal Viremia and Fetal Brain Abnormalities. N Engl J
Med (2016). [0378] 20. Dang, J., et al. Zika Virus Depletes Neural
Progenitors in Human Cerebral Organoids through Activation of the
Innate Immune Receptor TLR3. Cell Stem Cell (2016). [0379] 21.
Zheng, W., Thorne, N. & McKew, J. C. Phenotypic screens as a
renewed approach for drug discovery. Drug Discov Today 18,
1067-1073 (2013). [0380] 22. Sun, W., Sanderson, P. E. & Zheng,
W. Drug combination therapy increases successful drug
repositioning. Drug Discov Today (2016). [0381] 23. Kouznetsova,
J., et al. Identification of 53 compounds that block Ebola
virus-like particle entry via a repurposing screen of approved
drugs. Emerg Microbes Infect 3, e84 (2014). [0382] 24. Johansen, L.
M., et al. A screen of approved drugs and molecular probes
identifies therapeutics with anti-Ebola virus activity. Sci Transl
Med 7, 290ra289 (2015). [0383] 25. Chen, C. Z., et al.
High-throughput Giardia lamblia viability assay using
bioluminescent ATP content measurements. Antimicrob Agents
Chemother 55, 667-675 (2011). [0384] 26. Debnath, A., et al. A
high-throughput drug screen for Entamoeba histolytica identifies a
new lead and target. Nat Med 18, 956-960 (2012). [0385] 27. Sun,
W., et al. Chemical signatures and new drug targets for
gametocytocidal drug development. Sci Rep 4, 3743 (2014). [0386]
28. Sun, W., et al. Rapid identification of antifungal compounds
against Exserohilum rostratum using high throughput drug
repurposing screens. PLoS One 8, e70506 (2013). [0387] 29. He, S.,
et al. Repurposing of the antihistamine chlorcyclizine and related
compounds for treatment of hepatitis C virus infection. Sci Transl
Med 7, 282ra249 (2015). [0388] 30. Zhang, J. H., Chung, T. D. &
Oldenburg, K. R. A Simple Statistical Parameter for Use in
Evaluation and Validation of High Throughput Screening Assays. J
Biomol Screen 4, 67-73 (1999). [0389] 31. Hamel, R., et al. Biology
of Zika Virus Infection in Human Skin Cells. J Virol 89, 8880-8896
(2015). [0390] 32. Nowakowski, T. J., et al. Expression Analysis
Highlights AXL as a Candidate Zika Virus Entry Receptor in Neural
Stem Cells. Cell Stem Cell 18, 591-596 (2016). [0391] 33. Schang,
L. M., St Vincent, M. R. & Lacasse, J. J. Five years of
progress on cyclin-dependent kinases and other cellular proteins as
potential targets for antiviral drugs. Antivir Chem Chemother 17,
293-320 (2006). [0392] 34. Driggers, R. W., et al. Zika Virus
Infection with Prolonged Maternal Viremia and Fetal Brain
Abnormalities. N Engl J Med 374, 2142-2151 (2016). [0393] 35.
Barreyro, F. J., et al. The pan-caspase inhibitor Emricasan
(IDN-6556) decreases liver injury and fibrosis in a murine model of
non-alcoholic steatohepatitis. Liver Int 35, 953-966 (2015). [0394]
36. Pockros, P. J., et al. Oral IDN-6556, an antiapoptotic caspase
inhibitor, may lower aminotransferase activity in patients with
chronic hepatitis C. Hepatology 46, 324-329 (2007). [0395] 37.
Shiffman, M. L., et al. Clinical trial: the efficacy and safety of
oral PF-03491390, a pancaspase inhibitor--a randomized
placebo-controlled study in patients with chronic hepatitis C.
Aliment Pharmacol Ther 31, 969-978 (2010). [0396] 38. Wu, C. J., et
al. Inhibition of severe acute respiratory syndrome coronavirus
replication by niclosamide. Antimicrob Agents Chemother 48,
2693-2696 (2004). [0397] 39. Jurgeit, A., et al. Niclosamide is a
proton carrier and targets acidic endosomes with broad antiviral
effects. PLoS Pathog 8, e1002976 (2012). [0398] 40. Fang, J., et
al. Identification of three antiviral inhibitors against Japanese
encephalitis virus from library of pharmacologically active
compounds 1280. PLoS One 8, e78425 (2013). [0399] 41. Wen, Z., et
al. Synaptic dysregulation in a human iPS cell model of mental
disorders. Nature 515, 414-418 (2014). [0400] 42. Brennand, K. J.,
et al. Modelling schizophrenia using human induced pluripotent stem
cells. Nature 473, 221-225 (2011). [0401] 43. Topol, A., et al.
Increased abundance of translation machinery in stem cell-derived
neural progenitor cells from four schizophrenia patients. Transl
Psychiatry 5, e662 (2015). [0402] 44. Qian, X., et al.
Brain-region-specific oragnoids using min-bioreactors for modeling
ZIKV exposure. Cell (2016). [0403] 45. Huang, R., et al. The NCGC
pharmaceutical collection: a comprehensive resource of clinically
approved drugs enabling repurposing and chemical genomics. Sci
Transl Med 3, 80ps16 (2011). [0404] 46. Wang, Y., Jadhav, A.,
Southal, N., Huang, R. & Nguyen, D. T. A grid algorithm for
high throughput fitting of dose-response curve data. Curr Chem
Genomics 4, 57-66 (2010). [0405] 47. Xu et al. Identification of
small-molecule inhibitors of Zika virus infection and induced
neural cell death via a drug repurposing screen. Nature Medicine,
22(10):1101-1110 (2016). [0406] 48. Pan J-X et al., "Niclosamide,
an antihelminthic agent, demonstrates antitumor activity by
blocking multiple signaling pathways of cancer stem cells," Chin J
Cancer, 2012, 31(4):178-184. [0407] 49. Mook R A et al.,
Structure-activity studies of Wnt/.mu.-catenin inhibition in the
Niclosamide chemotype: Identification of derivatives with improved
drug exposure. Bioorg Med Chem.; 23(17):5829-38 (2015). [0408] 50.
Malumbres, M et al. CDK inhibitors in cancer therapy: What is
next?. Trends in Pharmacological Sciences. 29 (1): 16-21 (2008).
[0409] 51. Asghar U et al., The history and future of targeting
cyclin-dependent kinases in cancer therapy. Nat Rev Drug Discov;
14(2): 130-146 (2015). [0410] 52. Sanchez-Martinez C et al., Cyclin
dependent kinase (CDK) inhibitors as anticancer drugs. Bioorganic
& Medicinal Chemistry Letters, Volume 25, Issue 17, Pages
3420-3435 (2015). [0411] 53. Manasanch E E et al., Proteasome
inhibitors in cancer therapy. Nature Reviews Clinical Oncology. 14,
417-433 (2017). [0412] 54. Dou Q P et al., Overview of Proteasome
Inhibitor-Based Anti-cancer Therapies: Perspective on Bortezomib
and Second Generation Proteasome Inhibitors versus Future
Generation Inhibitors of Ubiquitin-Proteasome System. Curr Cancer
Drug Targets. 14(6): 517-536 (2014).
* * * * *