U.S. patent application number 15/051076 was filed with the patent office on 2017-01-26 for methods for screening antimicrobial and antiviral compounds and uses thereof.
The applicant listed for this patent is The General Hospital Corporation, Northeastern University. Invention is credited to Frederick M. AUSUBEL, Suresh GOPALAN, Kim LEWIS, Terence I. MOY.
Application Number | 20170020900 15/051076 |
Document ID | / |
Family ID | 39468404 |
Filed Date | 2017-01-26 |
United States Patent
Application |
20170020900 |
Kind Code |
A1 |
AUSUBEL; Frederick M. ; et
al. |
January 26, 2017 |
METHODS FOR SCREENING ANTIMICROBIAL AND ANTIVIRAL COMPOUNDS AND
USES THEREOF
Abstract
The invention features compounds that have antibacterial
activity, their use for the treatment of bacterial infections,
screening methods that use whole animals or plant seedlings to
identify compounds that inhibit a pathogen in the animal or plant,
and screening methods to identify compounds that increase the
lifespan of an organism.
Inventors: |
AUSUBEL; Frederick M.;
(Newton, MA) ; LEWIS; Kim; (Newton, MA) ;
MOY; Terence I.; (Wayland, MA) ; GOPALAN; Suresh;
(Lexington, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The General Hospital Corporation
Northeastern University |
Boston
Boston |
MA
MA |
US
US |
|
|
Family ID: |
39468404 |
Appl. No.: |
15/051076 |
Filed: |
February 23, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12304963 |
Nov 25, 2009 |
9301940 |
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PCT/US07/14127 |
Jun 18, 2007 |
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15051076 |
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60872168 |
Dec 1, 2006 |
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60814465 |
Jun 16, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/427 20130101;
A61K 31/5377 20130101; A61K 31/10 20130101; A61P 31/12 20180101;
A61K 31/655 20130101; A61K 31/426 20130101; A61K 31/496 20130101;
A61K 31/517 20130101; A61K 31/27 20130101; A61P 31/10 20180101;
A61K 31/4184 20130101; A61K 31/44 20130101; A61K 31/221 20130101;
A61K 31/166 20130101; Y02A 50/481 20180101; A61K 31/37 20130101;
A61K 31/198 20130101; C12Q 1/18 20130101; A61K 31/122 20130101;
A61P 31/04 20180101; A61K 31/17 20130101 |
International
Class: |
A61K 31/655 20060101
A61K031/655; A61K 31/198 20060101 A61K031/198; A61K 31/426 20060101
A61K031/426; A61K 31/10 20060101 A61K031/10; A61K 31/17 20060101
A61K031/17; A61K 31/427 20060101 A61K031/427; A61K 31/37 20060101
A61K031/37; A61K 31/4184 20060101 A61K031/4184; A61K 31/122
20060101 A61K031/122; A61K 31/517 20060101 A61K031/517; A61K 31/44
20060101 A61K031/44; A61K 31/496 20060101 A61K031/496; A61K 31/27
20060101 A61K031/27; A61K 31/5377 20060101 A61K031/5377; A61K
31/166 20060101 A61K031/166 |
Goverment Interests
STATEMENT AS TO FEDERALLY FUNDED RESEARCH
[0001] This invention was made in part with Government funding, and
the Government therefore has certain rights in the invention. In
particular, portions of the invention disclosed herein were funded,
in part, by NIH Grant Nos. RO1 AI070863, R21 AI059483, K08
AI63084-01, and RO1 AI072508.
Claims
1-4. (canceled)
5. A pharmaceutical composition comprising a compound of formula
II: ##STR00055## or a salt thereof, and a pharmaceutically
acceptable excipient, wherein each of R.sup.2A, R.sup.2B, R.sup.2C,
R.sup.2D, R.sup.2E, and R.sup.2F is, independently, selected from
H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.2G, OC(O)R.sup.2H, NR.sup.2IR.sup.2J,
NHC(O)R.sup.2K, NHC(S)R.sup.2L, NHC(O)OR.sup.2M, NHC(S)OR.sup.2N,
NHC(O)NHR.sup.2O, NHC(S)NHR.sup.2P, NHC(O)SR.sup.2Q,
NHC(S)SR.sup.2R, NHS(O).sub.2R.sup.2S, C(O)OR.sup.2T, and
C(O)NHR.sup.2U; X.sup.2 is independently selected from OR.sup.2G,
OC(O)R.sup.2H, NR.sup.2IR.sup.2J, NHC(O)R.sup.2K, NHC(S)R.sup.2L,
NHC(O)OR.sup.2M, NHC(S)OR.sup.2N, NHC(O)NHR.sup.2O,
NHC(S)NHR.sup.2P, NHC(O)SR.sup.2Q, NHC(S)SR.sup.2R, and
NHS(O).sub.2R.sup.2S; and each of R.sup.2G, R.sup.2H, R.sup.2I,
R.sup.2J, R.sup.2K, R.sup.2L, R.sup.2M, R.sup.2N, R.sup.2O,
R.sup.2P, R.sup.2Q, R.sup.2R, R.sup.2S, R.sup.2T, and R.sup.2U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl,
C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, and C.sub.1-4
heteroalkyl.
6. A kit comprising: (i) a pharmaceutical composition of claim 5
and (ii) instructions for administering the composition to a
subject for the treatment of a microbial or viral infection.
7. A method of treating a microbial or viral infection in a
subject, said method comprising administering to said subject a
pharmaceutical composition of claim 5 in an amount effective to
treat said infection.
8. The method of claim 7, wherein said infection is a bacterial
infection selected from community-acquired pneumonia, upper and
lower respiratory tract infection, skin and soft tissue infection,
bone and joint infection, hospital-acquired lung infection, acute
bacterial otitis media, bacterial pneumonia, complicated infection,
noncomplicated infection, pyelonephritis, intra-abdominal
infection, deep-seated abcess, bacterial sepsis, central nervous
system infection, bacteremia, wound infection, peritonitis,
meningitis, infections after burn, urogenital tract infection,
gastro-intestinal tract infection, pelvic inflammatory disease,
endocarditis, and intravascular infection.
9-12. (canceled)
13. A pharmaceutical composition comprising a compound of formula
IV: ##STR00056## or a salt thereof, and a pharmaceutically
acceptable excipient, wherein each of R.sup.4A, R.sup.4B, R.sup.4C,
R.sup.4D, R.sup.4E, and R.sup.4F is, independently, selected from
H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.4G, OC(O)R.sup.4H, NR.sup.4IR.sup.4J,
NHC(O)R.sup.4K, NHC(S)R.sup.4L, NHC(O)OR.sup.4M, NHC(S)OR.sup.4N,
NHC(O)NHR.sup.4O, NHC(S)NHR.sup.4P, NHC(O)SR.sup.4O,
NHC(S)SR.sup.4R, NHS(O).sub.2R.sup.4, C(O)OR.sup.4T, and
C(O)NHR.sup.4U; X.sup.4 is --S(O)-- or --S(O).sub.2--; and each of
R.sup.4G, R.sup.4H, R.sup.4I, R.sup.4J, R.sup.4K, R.sup.4L,
R.sup.4M, R.sup.4N, R.sup.4O, R.sup.4P, R.sup.4Q, R.sup.4R,
R.sup.4S, R.sup.4T, and R.sup.4U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl.
14. A kit comprising: (i) a pharmaceutical composition of claim 13
and (ii) instructions for administering the composition to a
subject for the treatment of a microbial or viral infection.
15. A method of treating a microbial or viral infection in a
subject, said method comprising administering to said subject a
pharmaceutical composition of claim 13 in an amount effective to
treat said infection.
16. The method of claim 15, wherein said infection is a bacterial
infection selected from community-acquired pneumonia, upper and
lower respiratory tract infection, skin and soft tissue infection,
bone and joint infection, hospital-acquired lung infection, acute
bacterial otitis media, bacterial pneumonia, complicated infection,
noncomplicated infection, pyelonephritis, intra-abdominal
infection, deep-seated abcess, bacterial sepsis, central nervous
system infection, bacteremia, wound infection, peritonitis,
meningitis, infections after burn, urogenital tract infection,
gastro-intestinal tract infection, pelvic inflammatory disease,
endocarditis, and intravascular infection.
17-20. (canceled)
21. A pharmaceutical composition comprising a compound of formula
VI: ##STR00057## or a salt thereof, and a pharmaceutically
acceptable excipient, wherein each of R.sup.6A, R.sup.6B, R.sup.6C,
R.sup.6D, R.sup.6E, and R.sup.6F is, independently, selected from
H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.6G, OC(O)R.sup.6H, NR.sup.6IR.sup.6J,
NHC(O)R.sup.6K, NHC(S)R.sup.6L, NHC(O)OR.sup.6M, NHC(S)OR.sup.6N,
NHC(O)NHR.sup.6O, NHC(S)NHR.sup.6P, NHC(O)SR.sup.6Q,
NHC(S)SR.sup.6R, NHS(O).sub.2R.sup.6S, C(O)OR.sup.6T, and
C(O)NHR.sup.6U; each of X.sup.6A and X.sup.6B is, independently,
selected from O and S; and each of R.sup.6G, R.sup.6H, R.sup.6I,
R.sup.6J, R.sup.6K, R.sup.6L, R.sup.6M, R.sup.6N, R.sup.6O,
R.sup.6P, R.sup.6Q, R.sup.6R, R.sup.6S, R.sup.6T, and R.sup.6U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl,
C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, and C.sub.1-4
heteroalkyl.
22. A kit comprising: (i) a pharmaceutical composition of claim 21
and (ii) instructions for administering the composition to a
subject for the treatment of a microbial or viral infection.
23. A method of treating a microbial or viral infection in a
subject, said method comprising administering to said subject a
pharmaceutical composition of claim 21 in an amount effective to
treat said infection.
24. The method of claim 23, wherein said infection is a bacterial
infection selected from community-acquired pneumonia, upper and
lower respiratory tract infection, skin and soft tissue infection,
bone and joint infection, hospital-acquired lung infection, acute
bacterial otitis media, bacterial pneumonia, complicated infection,
noncomplicated infection, pyelonephritis, intra-abdominal
infection, deep-seated abcess, bacterial sepsis, central nervous
system infection, bacteremia, wound infection, peritonitis,
meningitis, infections after burn, urogenital tract infection,
gastro-intestinal tract infection, pelvic inflammatory disease,
endocarditis, and intravascular infection.
25-48. (canceled)
49. A pharmaceutical composition comprising a compound of formula
XIII: ##STR00058## or a salt thereof, and a pharmaceutically
acceptable excipient, wherein each of R.sup.13A, R.sup.13B, and
R.sup.13C is, independently, selected from H, halide, nitro,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.13G,
OC(O)R.sup.13H, NR.sup.13I, R.sup.13J, NHC(O)R.sup.13K,
NHC(S)R.sup.13L, NHC(O)OR.sup.13M, NHC(S)OR.sup.13N,
NHC(O)NHR.sup.13O, NHC(S)NHR.sup.13P, NHC(O)SR.sup.13Q,
NHC(S)SR.sup.13R, NHS(O).sub.2R.sup.13S, C(O)OR.sup.13T, and
C(O)NHR.sup.13U; and each of R.sup.13G, R.sup.13H, R.sup.13I,
R.sup.13J, R.sup.13K, R.sup.13L, R.sup.13M, R.sup.13N, R.sup.13O,
R.sup.13P, R.sup.13Q, R.sup.13R, R.sup.13S, R.sup.13T, and
R.sup.13U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl,
and C.sub.1-4 heteroalkyl.
50. A kit comprising: (i) a pharmaceutical composition of claim 49
and (ii) instructions for administering the composition to a
subject for the treatment of a microbial or viral infection.
51. A method of treating a microbial or viral infection in a
subject, said method comprising administering to said subject a
pharmaceutical composition of claim 49 in an amount effective to
treat said infection.
52. The method of claim 51, wherein said infection is a bacterial
infection selected from community-acquired pneumonia, upper and
lower respiratory tract infection, skin and soft tissue infection,
bone and joint infection, hospital-acquired lung infection, acute
bacterial otitis media, bacterial pneumonia, complicated infection,
noncomplicated infection, pyelonephritis, intra-abdominal
infection, deep-seated abcess, bacterial sepsis, central nervous
system infection, bacteremia, wound infection, peritonitis,
meningitis, infections after burn, urogenital tract infection,
gastro-intestinal tract infection, pelvic inflammatory disease,
endocarditis, and intravascular infection.
53-207. (canceled)
Description
BACKGROUND OF THE INVENTION
[0002] This invention relates to the field of antimicrobials and
antivirals for the treatment of infections and compounds that
increase longevity.
[0003] The growing problem of antibiotic resistant bacteria (see
Chambers H. F., Emerg. Infect. Dis. 7:178-182 (2001); Hecht D. W.,
Clin. Infect. Dis. 39:92-97 (2004); Jacobs M. R., Am. J. Med. 117
Suppl. 3A:3S-15S (2004); Molbak K., Clin. Infect. Dis. 41:1613-1620
(2005); Shah et al., Res. Microbiol. 155:409-421 (2004);
Wisplinghoff et al., Clin. Infect. Dis. 39:309-317 (2004); and
Zinner S. H., Expert Rev. Anti. Infect. Ther. 3:907-913 (2005)) and
the imminent threat of biowarfare agents (see Lane et al., Nat.
Med. 7:1271-1273 (2001)) points to a need for new anti-infective
therapies. However, the rate of new antimicrobial and antiviral
discovery is unlikely to meet the expected need for the foreseeable
future (see Boggs et al., Clin. Microbiol. Infect. 10 Suppl.
4:32-36 (2004); Bush K., Clin. Microbiol. Infect. 10 Suppl. 4:10-17
(2004); Dougherty et al., Curr. Pharm. Des. 8:1119-1135 (2002);
Schmid M. B., Nat. Rev. Microbiol. 2:739-746 (2004); Silver L. L.,
IDrugs 8:651-655 (2005); and Walsh C., Nat. Rev. Microbiol. 1:65-70
(2003)). Specific problems include the over-mining of cultivable
microorganisms (see Osburne et al., ASM News 66:411-417 (2000)), a
high background of toxic compounds or compounds with poor
pharmacokinetic properties in (2004); and Lipinski et al., Nature
432:855-861 (2004)), and the inability of most synthetic leads to
penetrate across the multi-drug resistance (MDR) barrier of
Gram-negative bacteria (see Li et al., Drugs 64:159-204 (2004)).
The increased use of in vitro assays for small-molecule discovery
that bear little resemblance to the biological systems in which the
drugs need to function may also be responsible for the decline in
the rate of drug discovery (see Lipinski et al., Nature 432:855-861
(2004); Horrobin D. F., Nat. Rev. Drug Discov. 2:151-154 (2003);
Williams M., Curr. Opin. Investig. Drugs 5:29-33 (2004)). Efforts
to identify new antifungal compounds have been hindered by the fact
that most compounds that have antifungal activity in vitro are also
toxic to mammalian cells.
[0004] Plant pathogenic diseases are also of concern because they
cause damage to plants and plant products. Phytopathogens produce
disease in plants by any number of methods including: (1) consuming
host cell nutrients; (2) killing or disrupting host cell metabolism
through toxins, enzymes, or growth-regulators; (3) affecting
photosynthesis by inducing chlorosis (e.g., by degrading
chloroplasts); and (4) blocking conductive tissues and interfering
with normal physiological processes.
[0005] Crop plants, ornamentals, trees, and shrubs are especially
vulnerable to diseases caused by bacteria, fungi, and viruses.
Phytopathogenic bacteria, for example, cause the development of
many disease symptoms including leaf spots and blights, soft-rots,
wilts, overgrowths, scabs, and cankers. Bacterial diseases occur
most commonly on vegetables (and some ornamentals) that have fleshy
storage tissues, such as potatoes, carrots, onions, iris, or
hyacinth. They may also occur in plants bearing fleshy fruit (such
as cucumber, squash, eggplant, or tomato), as well as in leafy
plants (such as cabbage, celery, lettuce, or spinach). Plant
bacterial diseases occur throughout the world and cause serious
damage to crops in the field, in transit, and in storage.
[0006] A facile bioassay compatible with high throughput screening
technologies that simultaneously evaluated libraries of chemical
compounds for antimicrobial and antiviral activity and host
toxicity in the context of a live-animal, or plant seedling,
infection model could solve some of the main obstacles in current
antimicrobial discovery. There is a need in the art for the
development of new antimicrobial and antiviral compounds.
SUMMARY OF THE INVENTION
[0007] We have discovered compounds that have antibacterial
activity. These compounds can be useful for the treatment of
bacterial infection in animals and plants. In addition, we
discovered a screening method that uses whole animals or plant
seedlings to identify compounds that inhibit a pathogen in the
animal or plant seedling. The screening method can also be used to
identify compounds that increase longevity of an organism.
[0008] The invention features a pharmaceutical composition
including a compound of formula (I), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00001##
In formula (I) each of R.sup.1A, R.sup.1B, R.sup.1C, R.sup.1D,
R.sup.1E, and R.sup.1F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.1G, OC(O)R.sup.1H, NR.sup.1IR.sup.1J, NHC(O)R.sup.1K,
NHC(S)R.sup.1L, NHC(O)OR.sup.1M, NHC(S)OR.sup.1N, NHC(O)NHR.sup.1O,
NHC(S)NHR.sup.1P, NHC(O)SR.sup.1Q, NHC(S)SR.sup.1R,
NHS(O).sub.2R.sup.1S, C(O)OR.sup.1T, and C(O)NHR.sup.1U; and each
of R.sup.1G, R.sup.1H, R.sup.1I, R.sup.1J, R.sup.1K, R.sup.1L,
R.sup.1M, R.sup.1N, R.sup.1O, R.sup.1P, R.sup.1Q, R.sup.1R,
R.sup.1S, R.sup.1T, and R.sup.1U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.1A and R.sup.1D are H; each of R.sup.1B,
R.sup.1C, R.sup.1E, and R.sup.1F is, independently, selected from
H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.1G, OC(O)R.sup.1H, NR.sup.1IR.sup.1J,
NHC(O)R.sup.1K, NHC(S)R.sup.1L, NHC(O)OR.sup.1M, NHC(S)OR.sup.1N,
NHC(O)NHR.sup.1O, NHC(S)NHR.sup.1P, NHC(O)SR.sup.1Q,
NHC(S)SR.sup.1R, NHS(O).sub.2R.sup.1S, C(O)OR.sup.1T, and
C(O)NHR.sup.1U; and each of R.sup.1G, R.sup.1H, R.sup.1I, R.sup.1J,
R.sup.1K, R.sup.1L, R.sup.1M, R.sup.1N, R.sup.1O, R.sup.1P,
R.sup.1Q, R.sup.1R, R.sup.1S, R.sup.1T, and R.sup.1U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and C.sub.1-4 heteroalkyl.
[0009] The invention further features a pharmaceutical composition
including a compound of formula (II), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00002##
In formula (II) each of R.sup.2A, R.sup.2B, R.sup.2C, R.sup.2D,
R.sup.2E, and R.sup.2F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.2G, OC(O)R.sup.2H, NR.sup.2IR.sup.2J, NHC(O)R.sup.2K,
NHC(S)R.sup.2L, NHC(O)OR.sup.2M, NHC(S)OR.sup.2N, NHC(O)NHR.sup.2O,
NHC(S)NHR.sup.2P, NHC(O)SR.sup.2Q, NHC(S)SR.sup.2R,
NHS(O).sub.2R.sup.2S, C(O)OR.sup.2T, and C(O)NHR.sup.2U; X.sup.2 is
independently selected from OR.sup.2G, OC(O)R.sup.2H,
NR.sup.2IR.sup.2J, NHC(O)R.sup.2K, NHC(S)R.sup.2L, NHC(O)OR.sup.2M,
NHC(S)OR.sup.2N, NHC(O)NHR.sup.2O, NHC(S)NHR.sup.2P,
NHC(O)SR.sup.2Q, NHC(S)SR.sup.2R, and NHS(O).sub.2R.sup.2S; and
each of R.sup.2G, R.sup.2H, R.sup.2I, R.sup.2J, R.sup.2K, R.sup.2L,
R.sup.2M, R.sup.2N, R.sup.2O, R.sup.2P, R.sup.2Q, R.sup.2R,
R.sup.2S, R.sup.2T, and R.sup.2U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.2A and R.sup.2D are H; each of R.sup.2B,
R.sup.2C, R.sup.2E, and R.sup.2F is, independently, selected from
H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.2G, OC(O)R.sup.2H, NR.sup.2IR.sup.2J,
NHC(O)R.sup.2K, NHC(S)R.sup.2L, NHC(O)OR.sup.2M, NHC(S)OR.sup.2N,
NHC(O)NHR.sup.2O, NHC(S)NHR.sup.2P, NHC(O)SR.sup.2Q,
NHC(S)SR.sup.2R, NHS(O).sub.2R.sup.2S, C(O)OR.sup.2T, and
C(O)NHR.sup.2U; X.sup.2 is independently selected from OR.sup.2G,
OC(O)R.sup.2H, NR.sup.2IR.sup.2, NHC(O)R.sup.2K, NHC(S)R.sup.2L,
NHC(O)OR.sup.2M, NHC(S)OR.sup.2N, NHC(O)NHR.sup.2O,
NHC(S)NHR.sup.2P, NHC(O)SR.sup.2Q, NHC(S)SR.sup.2R, and
NHS(O).sub.2R.sup.2S; and each of R.sup.2G, R.sup.2H, R.sup.2I,
R.sup.2J, R.sup.2K, R.sup.2L, R.sup.2M, R.sup.2N, R.sup.2O,
R.sup.2P, R.sup.2Q, R.sup.2R, R.sup.2S, R.sup.2T, and R.sup.2U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, and C.sub.1-4
heteroalkyl.
[0010] The invention also features a pharmaceutical composition
including a compound of formula (III), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00003##
In formula (III) each of R.sup.3A, R.sup.3B, and R.sup.3C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.3G, OC(O)R.sup.3H,
NR.sup.3IR.sup.3J, NHC(O)R.sup.3K, NHC(S)R.sup.3L, NHC(O)OR.sup.3M,
NHC(S)OR.sup.3N, NHC(O)NHR.sup.3O, NHC(S)NHR.sup.3P,
NHC(O)SR.sup.3Q, NHC(S)SR.sup.3R, NHS(O).sub.2R.sup.3S,
C(O)OR.sup.3T, and C(O)NHR.sup.3U; X.sup.3 is independently
selected from OR.sup.3G, OC(O)R.sup.3H, NR.sup.3IR.sup.3J,
NHC(O)R.sup.3K, NHC(S)R.sup.3L, NHC(O)OR.sup.3M, NHC(S)OR.sup.3N,
NHC(O)NHR.sup.3O, NHC(S)NHR.sup.3P, NHC(O)SR.sup.3Q,
NHC(S)SR.sup.3R, and NHS(O).sub.2R.sup.3S; and each of R.sup.3G,
R.sup.3H, R.sup.3I, R.sup.3J, R.sup.3K, R.sup.3L, R.sup.3M,
R.sup.3N, R.sup.3O, R.sup.3P, R.sup.3Q, R.sup.3R, R.sup.3S,
R.sup.3T, and R.sup.3U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.3A is H; each of R.sup.3B and R.sup.3C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.3G, OC(O)R.sup.3H,
NR.sup.3IR.sup.3J, NHC(O)R.sup.3K, NHC(S)R.sup.3L, NHC(O)OR.sup.3M,
NHC(S)OR.sup.3N, NHC(O)NHR.sup.3O, NHC(S)NHR.sup.3P,
NHC(O)SR.sup.3Q, NHC(S)SR.sup.3R, NHS(O).sub.2R.sup.3S,
C(O)OR.sup.3T, and C(O)NHR.sup.3U; X.sup.3 is independently
selected from OR.sup.3G, OC(O)R.sup.3H, NR.sup.3IR.sup.3J,
NHC(O)R.sup.3K, NHC(S)R.sup.3L, NHC(O)OR.sup.3M, NHC(S)OR.sup.3N,
NHC(O)NHR.sup.3O, NHC(S)NHR.sup.3P, NHC(O)SR.sup.3Q,
NHC(S)SR.sup.3R, and NHS(O).sub.2R.sup.3S; and each of R.sup.3G,
R.sup.3H, R.sup.3I, R.sup.3J, R.sup.3K, R.sup.3L, R.sup.3M,
R.sup.3N, R.sup.3O, R.sup.3P, R.sup.3Q, R.sup.3R, R.sup.3S,
R.sup.3T, and R.sup.3U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.1-4 heteroalkyl.
[0011] The invention features a pharmaceutical composition
including a compound of formula (IV), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00004##
In formula (IV) each of R.sup.4A, R.sup.4B, R.sup.4C, R.sup.4D,
R.sup.4E, and R.sup.4F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.4G, OC(O)R.sup.4H, NR.sup.4I, R.sup.4J, NHC(O)R.sup.4K,
NHC(S)R.sup.4L, NHC(O)OR.sup.4M, NHC(S)OR.sup.4N, NHC(O)NHR.sup.4O,
NHC(S)NHR.sup.4P, NHC(O)SR.sup.4Q, NHC(S)SR.sup.4R,
NHS(O).sub.2R.sup.4S, C(O)OR.sup.4T, and C(O)NHR.sup.4U; X.sup.4 is
--S(O)-- or --S(O).sub.2--; and each of R.sup.4G, R.sup.4H,
R.sup.4I, R.sup.4J, R.sup.4K, R.sup.4L, R.sup.4M, R.sup.4N,
R.sup.4O, R.sup.4P, R.sup.4Q, R.sup.4R, R.sup.4S, R.sup.4T, and
R.sup.4U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl,
and C.sub.1-4 heteroalkyl. In one particular embodiment, R.sup.4A
and R.sup.4D are H; each of R.sup.4B, R.sup.4C, R.sup.4E, and
R.sup.4F is, independently, selected from H, halide, nitro,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.4G,
OC(O)R.sup.4H, NR.sup.4IR.sup.4J, NHC(O)R.sup.4K, NHC(S)R.sup.4L,
NHC(O)OR.sup.4M, NHC(S)OR.sup.4N, NHC(O)NHR.sup.4O,
NHC(S)NHR.sup.4P, NHC(O)SR.sup.4Q, NHC(S)SR.sup.4R,
NHS(O).sub.2R.sup.4S, C(O)OR.sup.4T, and C(O)NHR.sup.4U; X.sup.4 is
--S(O)-- or --S(O).sub.2--; and each of R.sup.4G, R.sup.4H,
R.sup.4I, R.sup.4J, R.sup.4K, R.sup.4L, R.sup.4M, R.sup.4N,
R.sup.4O, R.sup.4P, R.sup.4Q, R.sup.4R, R.sup.4S, R.sup.4T, and
R.sup.4U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and C.sub.1-4
heteroalkyl.
[0012] The invention further features a pharmaceutical composition
including a compound of formula (V), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00005##
In formula (V) Ar is
##STR00006##
each of R.sup.5A, R.sup.5B, R.sup.5C, R.sup.5D, R.sup.5E, and
R.sup.5F is, independently, selected from H, halide, nitro,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.5G,
OC(O)R.sup.5H, NR.sup.5IR.sup.5J, NHC(O)R.sup.5K, NHC(S)R.sup.5L,
NHC(O)OR.sup.5M, NHC(S)OR.sup.5N, NHC(O)NHR.sup.5O,
NHC(S)NHR.sup.5P, NHC(O)SR.sup.5Q, NHC(S)SR.sup.5R,
NHS(O).sub.2R.sup.5S, C(O)OR.sup.5T, and C(O)NHR.sup.5U; each of
X.sup.5A and X.sup.5B is, independently, selected from O and S;
X.sup.5C is selected from C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl,
C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, and C.sub.1-4
heteroalkyl; and each of R.sup.5G, R.sup.5H, R.sup.5I, R.sup.5J,
R.sup.5K, R.sup.5L, R.sup.5M, R.sup.5N, R.sup.5O, R.sup.5P,
R.sup.5Q, R.sup.5R, R.sup.5S, R.sup.5T, and R.sup.5U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl,
C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, and C.sub.1-4
heteroalkyl. In one particular embodiment, R.sup.5A and R.sup.5D
are H; each of R.sup.5B, R.sup.5C, R.sup.5E, and R.sup.5F is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.5G, OC(O)R.sup.5H,
NR.sup.5IR.sup.5J, NHC(O)R.sup.5K, NHC(S)R.sup.5L, NHC(O)OR.sup.5M,
NHC(S)OR.sup.5N, NHC(O)NHR.sup.5O, NHC(S)NHR.sup.5P,
NHC(O)SR.sup.5Q, NHC(S)SR.sup.5R, NHS(O).sub.2R.sup.5S,
C(O)OR.sup.5T, and C(O)NHR.sup.5U; each of X.sup.5A and X.sup.5B
is, independently, selected from O and S; X.sup.5C is selected from
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.1-4 heteroalkyl; and each of R.sup.5G, R.sup.5H, R.sup.5I,
R.sup.5J, R.sup.5K, R.sup.5L, R.sup.5M, R.sup.5N, R.sup.5O,
R.sup.5P, R.sup.5Q, R.sup.5R, R.sup.5S, R.sup.5T, and R.sup.5U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and C.sub.1-4 heteroalkyl.
[0013] The invention also features a pharmaceutical composition
including a compound of formula (VI), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00007##
In formula (VI) each of R.sup.6A, R.sup.6B, R.sup.6C, R.sup.6D,
R.sup.6E, and R.sup.6F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.6G, OC(O)R.sup.6H, NR.sup.6IR.sup.6J, NHC(O)R.sup.6K,
NHC(S)R.sup.6L, NHC(O)OR.sup.6M, NHC(S)OR.sup.6N, NHC(O)NHR.sup.6O,
NHC(S)NHR.sup.6P, NHC(O)SR.sup.6Q, NHC(S)SR.sup.6R,
NHS(O).sub.2R.sup.6S, C(O)OR.sup.6T, and C(O)NHR.sup.6U; each of
X.sup.6A and X.sup.6B is, independently, selected from O and S; and
each of R.sup.6G, R.sup.6H, R.sup.6I, R.sup.6J, R.sup.6K, R.sup.6L,
R.sup.6M, R.sup.6N, R.sup.6O, R.sup.6P, R.sup.6Q, R.sup.6R,
R.sup.6S, R.sup.6T, and R.sup.6U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.6A and R.sup.6D are H; each of R.sup.6B,
R.sup.6C, R.sup.6E, and R.sup.6F is, independently, selected from
H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.6G, OC(O)R.sup.6H, NR.sup.6IR.sup.6J,
NHC(O)R.sup.6K, NHC(S)R.sup.6L, NHC(O)OR.sup.6M, NHC(S)OR.sup.6N,
NHC(O)NHR.sup.6O, NHC(S)NHR.sup.6P, NHC(O)SR.sup.6Q,
NHC(S)SR.sup.6R, NHS(O).sub.2R.sup.6S, C(O)OR.sup.6T, and
C(O)NHR.sup.6U; each of X.sup.6A and X.sup.6B is O; and each of
R.sup.6G, R.sup.6H, R.sup.6I, R.sup.6J, R.sup.6K, R.sup.6L,
R.sup.6M, R.sup.6N, R.sup.6O, R.sup.6P, R.sup.6Q, R.sup.6R,
R.sup.6S, R.sup.6T, and R.sup.6U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.1-4 heteroalkyl.
[0014] The invention features a pharmaceutical composition
including a compound of formula (VII), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00008##
In formula (VII) each of R.sup.7A, R.sup.7B, R.sup.7C, R.sup.7D,
R.sup.7E, and R.sup.7F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.7G, OC(O)R.sup.7H, NR.sup.7IR.sup.7J, NHC(O)R.sup.7K,
NHC(S)R.sup.7L, NHC(O)OR.sup.7M, NHC(S)OR.sup.7N, NHC(O)NHR.sup.7O,
NHC(S)NHR.sup.7P, NHC(O)SR.sup.7Q, NHC(S)SR.sup.7R,
NHS(O).sub.2R.sup.7S, C(O)OR.sup.7T, and C(O)NHR.sup.7U; X.sup.7 is
independently selected from OR.sup.7G, OC(O)R.sup.7H,
NR.sup.7IR.sup.7J, NHC(O)R.sup.7K, NHC(S)R.sup.7L, NHC(O)OR.sup.7M,
NHC(S)OR.sup.7N, NHC(O)NHR.sup.7O, NHC(S)NHR.sup.7P,
NHC(O)SR.sup.7Q, NHC(S)SR.sup.7R, and NHS(O).sub.2R.sup.7S; and
each of R.sup.7G, R.sup.7H, R.sup.7I, R.sup.7J, R.sup.7K, R.sup.7L,
R.sup.7M, R.sup.7N, R.sup.7O, R.sup.7P, R.sup.7Q, R.sup.7R,
R.sup.7S, R.sup.7T, and R.sup.7U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.7A is H; each of R.sup.7B, R.sup.7C, R.sup.7D,
R.sup.7E, and R.sup.7F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.7G, OC(O)R.sup.7H, NR.sup.7IR.sup.7J, NHC(O)R.sup.7K,
NHC(S)R.sup.7L, NHC(O)OR.sup.7M, NHC(S)OR.sup.7N, NHC(O)NHR.sup.7O,
NHC(S)NHR.sup.7P, NHC(O)SR.sup.7Q, NHC(S)SR.sup.7R,
NHS(O).sub.2R.sup.7S, C(O)OR.sup.7T, and C(O)NHR.sup.7U; X.sup.7 is
independently OR.sup.7G; and each of R.sup.7G, R.sup.7H, R.sup.7I,
R.sup.7J, R.sup.7K, R.sup.7L, R.sup.7M, R.sup.7N, R.sup.7O,
R.sup.7P, R.sup.7Q, R.sup.7R, R.sup.7S, R.sup.7T, and R.sup.7U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and C.sub.1-4 heteroalkyl.
[0015] The invention further features a pharmaceutical composition
including a compound of formula (VIII), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00009##
In formula (VIII) each of R.sup.8A, R.sup.8B, and R.sup.8C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, ORG, OC(O)R.sup.8H,
NR.sup.8IR.sup.8J, NHC(O)R.sup.8K, NHC(S)R.sup.8L, NHC(O)OR.sup.8M,
NHC(S)OR.sup.8N, NHC(O)NHR.sup.8O, NHC(S)NHR.sup.8P,
NHC(O)SR.sup.8Q, NHC(S)SR.sup.8R, NHS(O).sub.2R.sup.8S,
C(O)OR.sup.8T, and C(O)NHR.sup.5U; and each of R.sup.8G, R.sup.8H,
R.sup.8I, R.sup.8J, R.sup.8K, R.sup.8L, R.sup.8M, R.sup.8N,
R.sup.8O, R.sup.8P, R.sup.8Q, R.sup.8R, R.sup.8S, R.sup.8T, and
R.sup.8U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl,
and C.sub.1-4 heteroalkyl. In one particular embodiment, R.sup.8A
is H; each of R.sup.8B and R.sup.8C is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.8G, OC(O)R.sup.8H, NR.sup.8IR.sup.8J,
NHC(O)R.sup.8K, NHC(S)R.sup.8L, NHC(O)OR.sup.8M, NHC(S)OR.sup.8N,
NHC(O)NHR.sup.8O, NHC(S)NHR.sup.8P, NHC(O)SR.sup.8Q,
NHC(S)SR.sup.8R, NHS(O).sub.2R.sup.8S, C(O)OR.sup.8T, and
C(O)NHR.sup.8U; and each of R.sup.8G, R.sup.8H, R.sup.8I, R.sup.8J,
R.sup.8K, R.sup.8L, R.sup.8M, R.sup.8N, R.sup.8O, R.sup.8S,
R.sup.8Q, R.sup.8R, R.sup.8S, R.sup.8T, and R.sup.8U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, and C.sub.1-4 heteroalkyl.
[0016] The invention also features a pharmaceutical composition
including a compound of formula (IX), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00010##
In formula (IX) each of R.sup.9A, R.sup.9B, R.sup.9C, R.sup.9D,
R.sup.9E, and R.sup.9F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.9G, OC(O)R.sup.9H, NR.sup.9IR.sup.9J, NHC(O)R.sup.9K,
NHC(S)R.sup.9L, NHC(O)OR.sup.9M, NHC(S)OR.sup.9N, NHC(O)NHR.sup.9O,
NHC(S)NHR.sup.9P, NHC(O)SR.sup.9Q, NHC(S)SR.sup.9R,
NHS(O).sub.2R.sup.9S, C(O)OR.sup.9T, and C(O)NHR.sup.9U; X.sup.9 is
independently selected from OR.sup.9G, OC(O)R.sup.9H,
NR.sup.9IR.sup.9J, NHC(O)R.sup.9K, NHC(S)R.sup.9L, NHC(O)OR.sup.9M,
NHC(S)OR.sup.9N, NHC(O)NHR.sup.9O, NHC(S)NHR.sup.9P,
NHC(O)SR.sup.9Q, NHC(S)SR.sup.9R, and NHS(O).sub.2R.sup.9S; Ar is
selected from C.sub.2-6 heterocyclyl and C.sub.6-12 aryl; and each
of R.sup.9G, R.sup.9H, R.sup.9I, R.sup.9J, R.sup.9K, R.sup.9L,
R.sup.9M, R.sup.9N, R.sup.9O, R.sup.9P, R.sup.9Q, R.sup.9R,
R.sup.9S, R.sup.9T, and R.sup.9U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.9A, R.sup.9B, and R.sup.9C are H; each of
R.sup.9D, R.sup.9E, and R.sup.9F is, independently, selected from
H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.9G, OC(O)R.sup.9H, NR.sup.9IR.sup.9J,
NHC(O)R.sup.9K, NHC(S)R.sup.9L, NHC(O)OR.sup.9M, NHC(S)OR.sup.9N,
NHC(O)NHR.sup.9O, NHC(S)NHR.sup.9P, NHC(O)SR.sup.9Q,
NHC(S)SR.sup.9R, NHS(O).sub.2R.sup.9S, C(O)OR.sup.9T, and
C(O)NHR.sup.9U; X.sup.9 is independently OR.sup.9G; Ar is selected
from C.sub.2-6 heterocyclyl and C.sub.6-12 aryl; and each of
R.sup.9G, R.sup.9H, R.sup.9I, R.sup.9J, R.sup.9K, R.sup.9L,
R.sup.9M, R.sup.9N, R.sup.9O, R.sup.9P, R.sup.9Q, R.sup.9R,
R.sup.9S, R.sup.9T, and R.sup.9U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, and C.sub.1-4 heteroalkyl.
[0017] The invention features a pharmaceutical composition
including a compound of formula (X), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00011##
In formula (X) each of R.sup.10A, R.sup.10B, and R.sup.10C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.10G, OC(O)R.sup.10H,
NR.sup.10IR.sup.10J, NHC(O)R.sup.10K, NHC(S)R.sup.10L,
NHC(O)OR.sup.10M, NHC(S)OR.sup.10N, NHC(O)NHR.sup.10O,
NHC(S)NHR.sup.10P, NHC(O)SR.sup.10Q, NHC(S)SR.sup.10R,
NHS(O).sub.2R.sup.10S, C(O)OR.sup.10T, and C(O)NHR.sup.10U;
X.sup.10 is independently selected from OR.sup.10G, OC(O)R.sup.10H,
NR.sup.10IR.sup.10J, NHC(O)R.sup.10K, NHC(S)R.sup.10L,
NHC(O)OR.sup.10M, NHC(S)OR.sup.10N, NHC(O)NHR.sup.10O,
NHC(S)NHR.sup.10P, NHC(O)SR.sup.10Q, NHC(S)SR.sup.10R, and
NHS(O).sub.2R.sup.10S; and each of R.sup.10G, R.sup.10H, R.sup.10I,
R.sup.10J, R.sup.10K, R.sup.10L, R.sup.10M, R.sup.10N, R.sup.10O,
R.sup.10P, R.sup.10Q, R.sup.10R, R.sup.10S, R.sup.10T, and
R.sup.10U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl,
and C.sub.1-4 heteroalkyl. In one particular embodiment, R.sup.10A
is H; each of R.sup.10B and R.sup.10C is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.10G, OC(O)R.sup.10H, NR.sup.10IR.sup.10J,
NHC(O)R.sup.10K, NHC(S)R.sup.10L, NHC(O)OR.sup.10M,
NHC(S)OR.sup.10N, NHC(O)NHR.sup.10O, NHC(S)NHR.sup.10P,
NHC(O)SR.sup.10Q, NHC(S)SR.sup.10R, NHS(O).sub.2R.sup.10S,
C(O)OR.sup.10T, and C(O)NHR.sup.10U; X.sup.10 is independently
selected from NR.sup.10IR.sup.10J, NHC(O)R.sup.10K,
NHC(S)R.sup.10L, NHC(O)OR.sup.10M, NHC(S)OR.sup.10N,
NHC(O)NHR.sup.10O, NHC(S)NHR.sup.10P, NHC(O)SRO.sup.10Q,
NHC(S)SR.sup.10R, and NHS(O).sub.2R.sup.10S; and each of R.sup.10G,
R.sup.10H, R.sup.10I, R.sup.10J, R.sup.10K, R.sup.10L, R.sup.10M,
R.sup.10N, R.sup.10O, R.sup.10P, R.sup.10Q, R.sup.10R, R.sup.10S,
R.sup.10T, and R.sup.10U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.1-4 heteroalkyl.
[0018] The invention further features a pharmaceutical composition
including a compound of formula (XI), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00012##
In formula (XI) each of R.sup.11A, R.sup.11B, R.sup.11C, R.sup.11D,
R.sup.11E, and R.sup.11F is, independently, selected from H,
halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.11G, OC(O)R.sup.11H, NR.sup.11IR.sup.11J,
NHC(O).sup.11K, NHC(S)R.sup.11L, NHC(O)OR.sup.11M,
NHC(S)OR.sup.11N, NHC(O)NHR.sup.11O, NHC(S)NHR.sup.11P,
NHC(O)SR.sup.11Q, NHC(S)SR.sup.11R, NHS(O).sub.2R.sup.11S,
C(O)OR.sup.11T, and C(O)NHR.sup.11U; and each of R.sup.11G,
R.sup.11H, R.sup.11I, R.sup.11J, R.sup.11K, R.sup.11L, R.sup.11M,
R.sup.11N, R.sup.11O, R.sup.11P, R.sup.11Q, R.sup.11R, R.sup.11S,
R.sup.11T, and R.sup.11U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.11A and R.sup.11D are H; each of R.sup.11B,
R.sup.11C, R.sup.11E, and R.sup.11F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.11G, OC(O)R.sup.11H, NR.sup.11IR.sup.11J,
NHC(O)R.sup.11K, NHC(S)R.sup.11L, NHC(O)OR.sup.11M,
NHC(S)OR.sup.11N, NHC(O)NHR.sup.11O, NHC(S)NHR.sup.11P,
NHC(O)SR.sup.11Q, NHC(S)SR.sup.11R, NHS(O).sub.2R.sup.11S,
C(O)OR.sup.11T, and C(O)NHR.sup.11U; and each of R.sup.11G,
R.sup.11H, R.sup.11I, R.sup.11J, R.sup.11K, R.sup.11L, R.sup.11M,
R.sup.11N, R.sup.11O, R.sup.11P, R.sup.11Q, R.sup.11R, R.sup.11S,
R.sup.11T, and R.sup.11U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.1-4 heteroalkyl.
[0019] The invention also features a pharmaceutical composition
including a compound of formula (XII), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00013##
In formula (XII) each of R.sup.12A, R.sup.12B, R.sup.12C,
R.sup.12D, R.sup.12E, and R.sup.12F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.12G, OC(O)R.sup.12H, NR.sup.12IR.sup.12J,
NHC(O).sup.12K, NHC(S)R.sup.12L, NHC(O)OR.sup.12M,
NHC(S)OR.sup.12N, NHC(O)NHR.sup.12O, NHC(S)NHR.sup.12P,
NHC(O)SR.sup.12Q, NHC(S)SR.sup.12R, NHS(O).sub.2R.sup.12S,
C(O)OR.sup.12T, and C(O)NHR.sup.12U; and each of R.sup.12G,
R.sup.12H, R.sup.12I, R.sup.12J, R.sup.12K, R.sup.12L, R.sup.12M,
R.sup.12N, R.sup.12O, R.sup.12P, R.sup.12Q, R.sup.12R, R.sup.12S,
R.sup.12T, and R.sup.12U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.12A, R.sup.12C, and R.sup.12E are H; each of
R.sup.12B, R.sup.12D, and R.sup.12F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.12G, OC(O)R.sup.12H, NR.sup.12IR.sup.12J,
NHC(O)R.sup.12K, NHC(S)R.sup.12L, NHC(O)OR.sup.12M,
NHC(S)OR.sup.12N, NHC(O)NHR.sup.12O, NHC(S)NHR.sup.12P,
NHC(O)SR.sup.12Q, NHC(S)SR.sup.12R, NHS(O).sub.2R.sup.12S,
C(O)OR.sup.12T, and C(O)NHR.sup.12U; and each of R.sup.12G,
R.sup.12H, R.sup.12I, R.sup.12J, R.sup.12K, R.sup.12L, R.sup.12M,
R.sup.12N, R.sup.12O, R.sup.12P, R.sup.12Q, R.sup.12R, R.sup.12S,
R.sup.12T, and R.sup.12U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.1-4 heteroalkyl.
[0020] The invention features a pharmaceutical composition
including a compound of formula (XIII), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00014##
In formula (XIII) each of R.sup.13A, R.sup.13B, and R.sup.13C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.13G, OC(O)R.sup.13H,
NR.sup.13IR.sup.13J, NHC(O)R.sup.13K, NHC(S)R.sup.13L,
NHC(O)OR.sup.13M, NHC(S)OR.sup.13N, NHC(O)NHR.sup.13O,
NHC(S)NHR.sup.13P, NHC(O)SR.sup.13Q, NHC(S)SR.sup.13R,
NHS(O).sub.2R.sup.13S, C(O)OR.sup.13T, and C(O)NHR.sup.13U; and
each of R.sup.13G, R.sup.13H, R.sup.13I, R.sup.13K, R.sup.13K,
R.sup.13L, R.sup.13M, R.sup.13N, R.sup.13O, R.sup.13P, R.sup.13Q,
R.sup.13R, R.sup.13S, R.sup.13T, and R.sup.13U is, independently,
selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl, C.sub.7-14
alkaryl, C.sub.3-10 alkheterocyclyl, and C.sub.1-4 heteroalkyl. In
one particular embodiment, R.sup.13A is H; each of R.sup.13B and
R.sup.13C is, independently, selected from H, halide, nitro,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.13G,
OC(O)R.sup.13H, NR.sup.13IR.sup.13J, NHC(O)R.sup.13K,
NHC(S)R.sup.13L, NHC(O)OR.sup.13M, NHC(S)OR.sup.13N,
NHC(O)NHR.sup.13O, NHC(S)NHR.sup.13P, NHC(O)SR.sup.13Q,
NHC(S)SR.sup.13R, NHS(O).sub.2R.sup.13S, C(O)OR.sup.13T, and
C(O)NHR.sup.13U; and each of and each of R.sup.13G, R.sup.13H,
R.sup.13I, R.sup.13J, R.sup.13K, R.sup.13L, R.sup.13M, R.sup.13N,
R.sup.13O, R.sup.13P, R.sup.13Q, R.sup.13R, R.sup.13S, R.sup.13T,
and R.sup.13U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and C.sub.1-4
heteroalkyl.
[0021] The invention further features a pharmaceutical composition
including a compound of formula (XIV), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00015##
In formula (XIV) each of R.sup.14A, R.sup.14B, R.sup.14C,
R.sup.14D, R.sup.14E, and R.sup.14F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.14G, OC(O)R.sup.14H, NR.sup.14IR.sup.14J,
NHC(O)R.sup.14K, NHC(S)R.sup.14L, NHC(O)OR.sup.14M,
NHC(S)OR.sup.14N, NHC(O)NHR.sup.14O, NHC(S)NHR.sup.14P,
NHC(O)SR.sup.14Q, NHC(S)SR.sup.14R, NHS(O).sub.2R.sup.14S,
C(O)OR.sup.14T, and C(O)NHR.sup.14U; and each of R.sup.14G,
R.sup.14H, R.sup.14I, R.sup.14J, R.sup.14K, R.sup.14L, R.sup.14M,
R.sup.14N, R.sup.14O, R.sup.14P, R.sup.14Q, R.sup.14R, R.sup.14S,
R.sup.14T, and R.sup.14U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.14A and R.sup.14D are H; each of R.sup.14B,
R.sup.14C, R.sup.14E, and R.sup.14F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.14G, OC(O)R.sup.14H, NR.sup.14IR.sup.14J,
NHC(O)R.sup.14K, NHC(S)R.sup.14L, NHC(O)OR.sup.14M,
NHC(S)OR.sup.14N, NHC(O)NHR.sup.14O, NHC(S)NHR.sup.14P,
NHC(O)SR.sup.14Q, NHC(S)SR.sup.14R, NHS(O).sub.2R.sup.14S,
C(O)OR.sup.14T, and C(O)NHR.sup.14U; and each of R.sup.14G,
R.sup.14H, R.sup.14I, R.sup.14J, R.sup.14K, R.sup.14L, R.sup.14M,
R.sup.14N, R.sup.14O, R.sup.14P, R.sup.14Q, R.sup.14R, R.sup.14S,
R.sup.14T, and R.sup.14U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.1-4 heteroalkyl.
[0022] The invention also features a pharmaceutical composition
including a compound of formula (XV), or a salt thereof, and a
pharmaceutically acceptable excipient.
##STR00016##
In formula (XV) each of R.sup.15A, R.sup.15B, R.sup.15C, R.sup.15D,
R.sup.15E, and R.sup.15F is, independently, selected from H,
halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, OR.sup.15G, OC(O)R.sup.15H, NR.sup.15IR.sup.15J,
NHC(O)R.sup.15K, NHC(S)R.sup.15L, NHC(O)OR.sup.15M,
NHC(S)OR.sup.15N, NHC(O)NHR.sup.15O, NHC(S)NHR.sup.15P,
NHC(O)SR.sup.15Q, NHC(S)SR.sup.15R, NHS(O).sub.2R.sup.15S,
C(O)OR.sup.15T, and C(O)NHR.sup.15U; and each of R.sup.15G,
R.sup.15H, R.sup.15I, R.sup.15J, R.sup.15K, R.sup.15L, R.sup.15M,
R.sup.15N, R.sup.15O, R.sup.15P, R.sup.15Q, R.sup.15R, R.sup.15S,
R.sup.15T, and R.sup.15U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. In one particular
embodiment, R.sup.15A and R.sup.15D are H; each of R.sup.15B,
R.sup.15C, R.sup.15E, and R.sup.15F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.15G, OC(O)R.sup.15H, NR.sup.15IR.sup.15J,
NHC(O)R.sup.15K, NHC(S)R.sup.15L, NHC(O)OR.sup.15M,
NHC(S)OR.sup.15N, NHC(O)NHR.sup.15O, NHC(S)NHR.sup.15P,
NHC(O)SR.sup.15Q, NHC(S)SR.sup.15R, NHS(O).sub.2R.sup.15S,
C(O)OR.sup.15T, and C(O)NHR.sup.15U; and each of R.sup.15G,
R.sup.15H, R.sup.15I, R.sup.15J, R.sup.15K, R.sup.15L, R.sup.15M,
R.sup.15N, R.sup.15O, R.sup.15P, R.sup.15Q, R.sup.15R, R.sup.15S,
R.sup.15T, and R.sup.15U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, and
C.sub.1-4 heteroalkyl.
[0023] The invention further features a method of treating a
microbial or viral infection in a subject (e.g., a host animal or
plant) that includes the step of administering to the subject a
pharmaceutical composition of the invention in an amount effective
to treat the infection. Thus, the present invention can be used to
treat a range of disorders associated with infection including; but
not limited to, sepsis syndrome, cachexia, circulatory collapse and
shock resulting from acute or chronic bacterial infection, acute
and chronic parasitic and/or infectious diseases from bacterial,
viral or fungal sources, such as a HIV, AIDS (including symptoms of
cachexia, autoimmune disorders, AIDS dementia complex and
infections) can be treated, as well as Wegners Granulomatosis. In
the above method, the infection can be a microbial infection (e.g.,
a protozoan, bacterial, mycobacterium, or fungal infection) or a
viral infection.
[0024] In plants, compounds of the invention can be used to prevent
or treat a broad range of fungal infections including powdery
mildews such as Erysiphe spp. and Sphaerotheca spp., downy mildews
such as Bremia spp. and Peronospora spp., black spot including
Diplocarpon spp., blights such as Alternaria spp. and Diplodia
spp., cankers such as Penicillium spp. and Coniothyrium spp., leaf
curl such as Taphrina spp., leaf spot such as Botrytis spp. and
Rhytisma spp., rots such as Aspergillus spp., Fusarium spp.,
Rhizoctonia spp., Pythium spp., Phytophthora spp. Sclerotinia spp.,
rusts such as soybean rust (e.g., Phakopsora pachyrhizi) and
Puccinia spp., Cronartium spp., scabs such as Venturia spp., and
smuts such as Urocystis spp. The compounds of the invention may
also be used to prevent or treat various bacterial infections in
plants including infections by Erwinia spp., Agrobacterium spp.,
Pseudomonas spp., and Xanthomonas spp.
[0025] In one particular embodiment, the infection to be treated is
a bacterial infection selected from bacterial pathogens that cause
community-acquired pneumonia, upper and lower respiratory tract
infections, skin and soft tissue infections, hospital-acquired lung
infections, bone and joint infections, respiratory tract
infections, acute bacterial otitis media, bacterial pneumonia,
urinary tract infections, complicated infections, noncomplicated
infections, pyelonephritis, intra-abdominal infections, deep-seated
abcesses, bacterial sepsis, central nervous system infections,
bacteremia, wound infections, peritonitis, meningitis, infections
after burn, urogenital tract infections, gastro-intestinal tract
infections, pelvic inflammatory disease, endocarditis, and other
intravascular infections. The methods of treating bacterial
infections described herein are useful in treating an infection by
a Gram-positive bacterium. Desirably, the methods are used to treat
infection by a Gram-positive coccus, or by a drug-resistant
Gram-positive coccus. Desirably, the Gram-positive coccus is
selected from S. aureus, S. epidermidis, S. pneumoniae, S.
pyogenes, M. catarrhalis, H. influenzae, and Enterococcus spp.
Alternatively, the bacterial infection to be treated is by
Chlamydia pneumoniae or Chlamydia trachomatis. The methods of
treating bacterial infections described herein can also be useful
in treating an infection is by a Gram-negative bacterium,
including, without limitation, Pseudonomas aeruginosa, Klebsiella
pneumoniae, Escherichia coli, Haemophilus influenzae, Citrobacter
freundii and Enterobacter spp. Further, the methods of treating
bacterial infections described herein can also be useful for
treating agriculturally important bacterial infections such as, for
example, Erwinia spp., Agrobacterium tumefaciens, Agrobacterium
rhizogenes, Pseudomonas syringae, or Xanthomonas spp. infections in
plants.
[0026] The methods of the invention can be used to reduce or
eliminate the incidence of postoperative infections in subjects
undergoing surgical procedures or implantation of prosthetic
devices.
[0027] The invention further features a method of treating an
infection by multi-drug resistant bacteria in a subject (e.g., a
host animal or plant). The method includes administering to the
subject a pharmaceutical composition of the invention, wherein the
compound is administered in an amount effective to treat the
multi-drug resistant infection. Resistant strains of bacteria
include penicillin-resistant, methicillin-resistant,
quinolone-resistant, macrolide-resistant, and/or
vancomycin-resistant bacterial strains. The multi-drug resistant
bacterial infections to be treated using the methods of the
invention include, for example, infections by penicillin-,
methicillin-, macrolide-, vancomycin-, and/or quinolone-resistant
Streptococcus pneumoniae; penicillin-, methicillin-, macrolide-,
vancomycin-, and/or quinolone-resistant Staphylococcus aureus;
penicillin-, methicillin-, macrolide-, vancomycin-, and/or
quinolone-resistant Streptococcus pyogenes; and penicillin-,
methicillin-macrolide-, vancomycin-, and/or quinolone-resistant
enterococci.
[0028] In the above method, the infection can be caused by a fungus
selected from, without limitation, Absidia corymbifera, Acremonium
falciforme, A. kiliense, A. recifei, Ajellomyces dermatitidis, A.
capsulata, Aspergillus spp., (e.g., A. flavus, A. fumigatus, A.
nidulans, A. niger, A. terreus), Candida spp. (e.g., C. albicans,
C. glabrata, C. guillermondii, C. krusei, C. parapsilosis, C.
kefyr, C. tropicalis), Crytococcus spp. (e.g., C. neoformans, C.
gattii, C. grubii), Cunninghamella elegans, Emmonsia parva,
Epidermophyton floccosum, Exophialia dermitidis, E. werneckii, E.
jeanselmei, E. spinifera, E. richardsiae, Filobasidiella
neoformans, Fonsecaea compacta, F. pedrosoi, Histoplasma
capsulatum, Leptoshaeria senegarlensis, Madurella mycetomatis, M.
grisea, Malassezia furfur, Microsporum spp, Neotestudina rosatii,
Paracoccidioides brasiliensis, Penicillium marneffei, Phialophora
verrucosa, Piedraia hortae, Pneumocystis spp., Pseudallescheria
boydii, Pyrenochaeta romeroi, Rhizomucor pusillus, Sporothrix
schenckii, Trichophyton spp, Trichosporon beigelii, Wangiella
dermatitidis and Xylohypha bantiana. In addition, in plants, the
infection can be caused by a fungus selected from, without
limitation, Botrytis spp., Fusarium spp., Alternaria spp., Erysiphe
spp., Rhytisma spp., Thielaviopsis spp., Verticillium spp.,
Aspergillus spp., Magnaporthe grisea, Rhizoctonia spp., Phakospora
pachyrhizi, Puccinia spp., Diplocarpon spp., Sphaerotheca spp.,
Phytophthora spp., Venturia spp., Taphrina spp., Phythium spp.,
Penicillium spp., Urocystis spp., and Coniothyrium spp.
[0029] Fungal infections that can be treated using the methods of
the invention include tinea capitis, tinea corporis, tinea pedis,
tinea barbae, tinea cruris, tinea versicolor, onychomycosis,
perionychomycosis, pityriasis versicolor, tinea unguium, oral
thrush, vaginal candidosis, respiratory tract candidosis, biliary
candidosis, eosophageal candidosis, urinary tract candidosis,
systemic candidosis, mucocutaneous candidosis, mycetoma,
cryptococcosis, aspergillosis, mucormycosis, chromoblastomycosis,
paracoccidioidomycosis, North American blastomycosis,
histoplasmosis, coccidioidomycosis, or sporotrichosis.
[0030] In the above method, the infection can be a viral infection,
such as, vesicular stomatitis virus, influenza, HIV, or
mononucleosis. In plants, the viral infection may be a nepovirus,
poytvirus, tymovirus, ilarvirus, potexvirus, caulimovirus,
tobravirus, closterovirus, carlavirus, cucumovirus, tobamovirus,
comovirus, carmovirus, necrovirus, nucleorhabdovirus, tospovirus,
luteovirus, fijivirus, or tenuivirus infection. In particular, the
viral infection may be a tobacco mosaic virus infection or a
leafroll virus infection.
[0031] In a related aspect, the invention features a kit including
a pharmaceutical composition of the invention and instructions for
administering the composition to a subject (e.g., a host organism
or a plant) for the treatment of a microbial or viral infection.
The specific infection to be treated and recited in the kit
instructions can be any described herein.
[0032] Pharmaceutical formulations containing a compound of the
invention can include isomers such as diastereomers and
enantiomers, mixtures of isomers, including racemic mixtures,
salts, solvates, and polymorphs thereof.
[0033] In another aspect, the invention features a method for
identifying a compound that is capable of inhibiting a pathogen in
an invertebrate animal host organism. This method involves (a)
exposing the invertebrate host organism to a pathogen; (b)
incubating the exposed invertebrate host organism in liquid medium
in the presence of at least one candidate compound; and (c)
identifying a compound that inhibits the pathogen in the
invertebrate host organism. In desirable embodiments, step (b) is
performed before step (a). In other desirable embodiments, prior to
step (a), the invertebrate animal host organism is exposed to the
candidate compound. These desirable embodiments can be used to
identify a prophylactic compound.
[0034] In other desirable embodiments, identifying of step (c)
involves using an automated microscope and identifying desirably
involves determining whether the host organism is alive or dead. In
further desirable embodiments, the exposed invertebrate host
organism is transferred to the liquid medium by a robot. In yet
other desirable embodiments the identifying step involves using a
fluorescent marker (e.g., green fluorescent protein, Nile Red, or
MitoTracker). Desirably, the fluorescent marker selectively stains
dead or dying cells and, desirably, is Sytox.RTM. green or
Sytox.RTM. orange.
[0035] In additional desirable embodiments of this aspect of the
invention, inhibiting kills the pathogen. Desirably, the compound
is a low molecular weight chemical compound.
[0036] In further desirable embodiment, the invertebrate host
organism is a nematode (e.g., a Caenorhabditis elegans nematode).
The Caenorhabditis elegans nematode desirably contains a mutation
in the mitogen-activated protein kinase (MAPK) pathway. A desirable
example of such a mutation in the MAPK pathway is sek-1. In other
desirable embodiments, the Caenorhabditis elegans nematode is a
temperature sensitive sterile mutant Caenorhabditis elegans
nematode. Desirably, the temperature sensitive sterile mutant
Caenorhabditis elegans nematode is a glp-4 mutant.
[0037] In additional desirable embodiments, the invertebrate host
organism is a Drosophila larva (e.g., a Drosophila melanogaster
larva). In yet other desirable embodiments, the invertebrate host
organism is a Plutella xylostella larva or a Galleria mellonella
larva.
[0038] In another desirable embodiment of this aspect of the
invention, the pathogen is a bacterial pathogen (e.g.,
Enterococcus, Pseudomonas, Salmonella, or Staphylococcus).
Desirably, the Enterococcus is Enterococcus faecalis, the
Pseudomonas is Pseudomonas aeruginosa, the Salmonella is Salmonella
typhimurium, or the Staphylococcus is Staphylococcus aureus,
Staphylococcus epidermidis, or Staphylococcus saprophyticus.
[0039] In yet other desirable embodiments of this aspect of the
invention, the pathogen is a fungal pathogen (e.g., Candida,
Cryptococcus, Fusarium, Rhodotorula, Aspergillus, or
Saccharomyces). Desirably, the Candida is Candida albicans, Candida
glabrata, or Candida parapsilosis, the Cryptococcus is Cryptococcus
neoformans, Crytococcus gattii, or Crytococcus grubii, the
Rhodotorula is Rhodotorula mucilaginosa, the Saccharomyces is
Saccharomyces cerevisiae, or the Aspergillus is Aspergillus flavus,
Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, or
Aspergillus terreus.
[0040] In further desirable embodiments of this aspect of the
invention, the pathogen is a viral pathogen (e.g., vesicular
stomatitis virus (VSV)). In another desirable embodiment of this
aspect of the invention, the pathogen is a protozoan. In yet
another desirable embodiment of this aspect of the invention, the
pathogen is a Mycobacterium (e.g., Mycobacterium tuberculosis).
[0041] In another aspect, the invention features a container
including (i) an invertebrate animal host organism infected with a
pathogen, (ii) liquid media, and (iii) a candidate compound.
Desirably, the container is a 24-well plate, a 48-well plate, a
96-well plate, a 384-well plate, a 1536-well plate, or a 3456-well
plate. In desirable embodiments, each well of the plate contains a
different candidate compound.
[0042] Desirably, the liquid medium contains a fluorescent marker,
such as a fluorescent marker that selectively stains dead or dying
cells. In desirable embodiments, the fluorescent marker is
Sytox.RTM. green or Sytox.RTM. orange.
[0043] In other desirable embodiments of this aspect of the
invention, the compound is a low molecular weight chemical
compound.
[0044] In additional desirable embodiments of this aspect of the
invention, the invertebrate host organism is a nematode (e.g., a
Caenorhabditis elegans nematode). Desirably, the Caenorhabditis
elegans nematode contains a mutation in the mitogen-activated
protein kinase (MAPK) pathway. In a desirable embodiment, the
mutation in the MAPK pathway is sek-1. In another desirable
embodiment of this aspect of the invention, the Caenorhabditis
elegans nematode is a temperature sensitive sterile mutant
Caenorhabditis elegans nematode. The temperature sensitive sterile
mutant Caenorhabditis elegans nematode desirably is a glp-4
mutant.
[0045] In another desirable embodiment of this aspect of the
invention, the invertebrate host organism is a Drosophila larva
(e.g., a Drosophila melanogaster larva). In other desirable
embodiments, the invertebrate host organism is a Plutella
xylostella larva or a Galleria mellonella larva.
[0046] In further desirable embodiments of this aspect of the
invention, the pathogen is a bacterial pathogen (e.g.,
Enterococcus, Pseudomonas, Salmonella, or Staphylococcus).
Desirably, the Enterococcus is Enterococcus faecalis, the
Pseudomonas is Pseudomonas aeruginosa, the Salmonella is Salmonella
typhimurium, or the Staphylococcus is Staphylococcus aureus,
Staphylococcus epidermidis, or Staphylococcus saprophyticus.
[0047] In other desirable embodiments of this aspect of the
invention, the pathogen is a fungal pathogen (e.g., Candida,
Cryptococcus, Fusarium, Rhodotorula, Aspergillus, or
Saccharomyces). Desirably, the Candida is Candida albicans, Candida
glabrata, or Candida parapsilosis, the Cryptococcus is Cryptococcus
neoformans, Crytococcus gattii, or Crytococcus grubii, the
Rhodotorula is Rhodotorula mucilaginosa, the Saccharomyces is
Saccharomyces cerevisiae, or the Aspergillus is Aspergillus flavus,
Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, or
Aspergillus terreus.
[0048] In yet another desirable embodiment of this aspect of the
invention, the pathogen is a viral pathogen (e.g., vesicular
stomatitis virus (VSV)). In a further desirable embodiment, the
pathogen is a protozoan. In yet another desirable embodiment of
this aspect of the invention, the pathogen is a Mycobacterium
(e.g., Mycobacterium tuberculosis).
[0049] In another aspect, the invention features a method for
identifying a compound that is capable of inhibiting a pathogen in
a plant host organism. This method involves (a) exposing the plant
host organism to a pathogen; (b) incubating the exposed plant host
organism in liquid medium in the presence of at least one candidate
compound; and (c) identifying a compound that inhibits the pathogen
in the plant host organism. In desirable embodiments, step (b) is
performed before step (a). In other desirable embodiments, the
plant is exposed to the candidate compound prior to step (a). In
additional desirable embodiments of this aspect of the invention,
the identifying step (c) involves using an automated microscope.
Desirably, the identifying step (c) involves determining whether
the host organism is alive or dead.
[0050] In a desirable embodiment, the plant host organism is an
Arabidopsis thaliana seedling.
[0051] In yet another desirable embodiment, the exposed plant host
organism is transferred to the liquid medium by a robot. In yet
other desirable embodiments of this aspect of the invention, the
identifying step (c) involves using a fluorescent or luminescent
marker. Desirably, the fluorescent marker is green fluorescent
protein, Nile Red, or MitoTracker and the luminescent marker is
luciferase.
[0052] In other desirable embodiments of this aspect of the
invention, the fluorescent marker selectively stains dead or dying
cells. Such a fluorescent marker desirably is Sytox.RTM. green or
Sytox.RTM. orange. In additional desirable embodiments of this
aspect of the invention, the inhibiting kills the pathogen. In
further desirable embodiments, the compound is a low molecular
weight chemical compound.
[0053] In yet another desirable embodiment of this aspect of the
invention, the pathogen is a bacterial pathogen (e.g., Pseudomonas,
Xanthomonas, Erwinia, or Agrobacterium). Desirably, the Pseudomonas
is Pseudomonas aeruginosa or Pseudomonas syringae.
[0054] In another desirable embodiment of this aspect of the
invention, the pathogen is a fungal pathogen. Desirably, the fungal
pathogen is Candida, Cryptococcus, Fusarium, Rhodotorula,
Aspergillus, Saccharomyces, Botrytis, Erysiphe, Alternaria,
Rhytisma, Thielaviopsis, Verticillium, Aspergillus, Magnaporthe
grisea, Rhizoctonia, Phakospora pachyrhizi, Puccinia, Diplocarpon,
Sphaerotheca, Phytophthora, Venturia, Taphrina, Phythium,
Penicillium, Urocystis, or Coniothyrium. In particularly desirable
embodiments, the Candida is Candida albicans, Candida glabrata, or
Candida parapsilosis, the Cryptococcus is Cryptococcus neoformans,
Crytococcus gattii, or Crytococcus grubii, the Rhodotorula is
Rhodotorula mucilaginosa, the Saccharomyces is Saccharomyces
cerevisiae, or the Aspergillus is Aspergillus flavus, Aspergillus
fumigatus, Aspergillus nidulans, Aspergillus niger, or Aspergillus
terreus, the Botrytis is Botrytis cinera, or the Fusarium is
Fusarium oxysporum.
[0055] In further desirable embodiments of this aspect of the
invention, the pathogen is a viral pathogen. Desirably, the viral
pathogen is a nepovirus, poytvirus, tymovirus, ilarvirus,
potexvirus, caulimovirus, tobravirus, closterovirus, carlavirus,
cucumovirus, tobamovirus, comovirus, carmovirus, necrovirus,
nucleorhabdovirus, tospovirus, luteovirus, fijivirus, or
tenuivirus. In particularly desirable embodiments, the viral
pathogen is tobacco mosaic virus, tobacco necrosis virus, potato
leaf roll virus, potato virus X, potato virus Y, tomato spotted
wilt virus, or tomato ring spot virus. In yet other desirable
embodiments, the pathogen is a protozoan or a Mycobacterium.
[0056] In yet another aspect, the invention features a container
including (i) a plant host organism infected with a pathogen, (ii)
liquid media, and (iii) a candidate compound. Desirably, the
container is a 24-well plate, a 48-well plate, a 96-well plate, a
384-well plate, a 1536-well plate, or a 3456-well plate. Desirably,
each well of the plate contains a different candidate compound. The
plant host organism desirably is an Arabidopsis thaliana
seedling.
[0057] In desirable embodiments of this aspect of the invention,
the liquid medium contains a fluorescent or luminescent marker.
Desirably, the luminescent marker is luciferase. In another
desirable embodiment, the fluorescent marker selectively stains
dead or dying cells. Desirable examples of such fluorescent markers
are Sytox.RTM. green and Sytox.RTM. orange.
[0058] In an additional desirable embodiment, the compound is a low
molecular weight chemical compound.
[0059] In further desirable embodiments, the pathogen is a
bacterial pathogen (e.g., Pseudomonas, Xanthomonas, Erwinia, or
Agrobacterium). Desirably, the Pseudomonas is Pseudomonas
aeruginosa or Pseudomonas syringae.
[0060] In other desirable embodiments, the pathogen is a fungal
pathogen. Desirably, the fungal pathogen is Candida, Cryptococcus,
Fusarium, Rhodotorula, Aspergillus, Saccharomyces, Botrytis,
Erysiphe, Alternaria, Rhytisma, Thielaviopsis, Verticillium,
Aspergillus, Magnaporthe grisea, Rhizoctonia, Phakospora
pachyrhizi, Puccinia, Diplocarpon, Sphaerotheca, Phytophthora,
Venturia, Taphrina, Phythium, Penicillium, Urocystis, or
Coniothyrium. In particularly desirable embodiments, the Candida is
Candida albicans, Candida glabrata, or Candida parapsilosis, the
Cryptococcus is Cryptococcus neoformans, Crytococcus gattii,
Crytococcus grubii, the Rhodotorula is Rhodotorula mucilaginosa,
the Saccharomyces is Saccharomyces cerevisiae, or the Aspergillus
is Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans,
Aspergillus niger, or Aspergillus terreus, the Botrytis is Botrytis
cinera, or the Fusarium is Fusarium oxysporum.
[0061] In yet other desirable embodiments, the pathogen is a viral
pathogen. Desirably, the viral pathogen is a nepovirus, poytvirus,
tymovirus, ilarvirus, potexvirus, caulimovirus, tobravirus,
closterovirus, carlavirus, cucumovirus, tobamovirus, comovirus,
carmovirus, necrovirus, nucleorhabdovirus, tospovirus, luteovirus,
fijivirus, or tenuivirus. In particularly desirable embodiments,
the viral pathogen is tobacco mosaic virus, tobacco necrosis virus,
potato leaf roll virus, potato virus X, potato virus Y, tomato
spotted wilt virus, or tomato ring spot virus.
[0062] In further desirable embodiments, the pathogen is a
protozoan or a Mycobacterium.
[0063] In yet another aspect, the invention features a method for
identifying a compound that increases the lifespan of an
invertebrate organism. This method involves (a) incubating an
invertebrate organism in liquid medium in the presence of at least
one candidate compound; and (b) identifying a compound that
increases the lifespan of the invertebrate organism relative to a
control organism not contacted with the candidate compound.
[0064] In desirable embodiments of this aspect of the invention,
the identifying of step (c) involves using an automated microscope.
In other desirable embodiments, the exposed invertebrate organism
is transferred to the liquid medium by a robot.
[0065] In yet another desirable embodiment of this aspect of the
invention, the identifying of step (c) involves using a fluorescent
marker. Desirable, the fluorescent marker is green fluorescent
protein, Nile Red, or MitoTracker. In other desirable embodiments,
the fluorescent marker selectively stains dead or dying cells. Such
a fluorescent marker desirably is Sytox.RTM. green or Sytox.RTM.
orange.
[0066] In additional desirable embodiments, the identifying of step
(c) involves determining whether the host organism is alive or
dead. In yet another desirable embodiment of this aspect of the
invention, the compound is a low molecular weight chemical
compound.
[0067] In further desirable embodiments, the invertebrate host
organism is a nematode (e.g., a Caenorhabditis elegans nematode).
In yet further desirable embodiments of this aspect of the
invention, the invertebrate host organism is a Drosophila larva
(e.g., a Drosophila melanogaster larva). In other desirable
embodiments the invertebrate host organism is a Plutella xylostella
larva or a Galleria mellonella larva.
[0068] As used herein, the term "pharmaceutical composition" refers
to a composition containing a compound of the invention (i.e., a
compound of any of formulas (I)-(XV)), formulated with a
pharmaceutically acceptable excipient, and manufactured or sold
with the approval of a governmental regulatory agency as part of a
therapeutic regimen for the treatment or prevention of disease in a
mammal. Pharmaceutical compositions can be formulated, for example,
for oral administration in unit dosage form (e.g., a tablet,
capsule, caplet, gelcap, or syrup), for topical administration
(e.g., as a cream, gel, lotion, or ointment), for intravenous
administration (e.g., as a sterile solution free of particulate
emboli and in a solvent system suitable for intravenous use), or
any other formulation described herein.
[0069] As used herein, the term "treating" refers to administering
a pharmaceutical composition for prophylactic and/or therapeutic
purposes. To "prevent disease" refers to prophylactic treatment of
a subject (e.g., a host organism or a plant) who is not yet ill or
diseased, but who is susceptible to, or otherwise at risk of, a
particular disease. To "treat disease" or use for "therapeutic
treatment" refers to administering treatment to a subject already
suffering from a disease to improve or stabilize the subject's
condition. Thus, in the claims and embodiments, treating is the
administration to a subject either for therapeutic or prophylactic
purposes.
[0070] By "effective" amount is meant the amount of a
pharmaceutical composition of the invention required to treat or
prevent an infection or a disease associated with an infection,
such as peripheral artery disease. The effective amount of a
pharmaceutical composition of the invention used to practice the
invention for therapeutic or prophylactic treatment of conditions
caused by or contributed to by an infection varies depending upon
the manner of administration, the age, body weight, and general
health of the subject. Ultimately, the attending physician or
veterinarian will decide the appropriate amount and dosage regimen.
Such amount is referred to as an "effective" amount.
[0071] By "infection" is meant the invasion of a host by a pathogen
(e.g., bacteria, fungi, protozoa, mycobacteria, or viruses). For
example, the infection may include the excessive growth of microbes
or viruses that are normally present in or on the body of a mammal
or growth of microbes or viruses that are not normally present in
or on a mammal or plant. More generally, a microbial or viral
infection can be any situation in which the presence of a microbial
or viral population(s) is damaging to a host body. Thus, a mammal
is "suffering" from an infection when an excessive amount of a
microbial or viral population is present in or on the mammal's
body, or when the presence of a microbial or viral population(s) is
damaging the cells or other tissue of the mammal.
By a "pathogen" is meant a bacterium, Mycobacterium, protozoan,
fungus, or virus that can cause an infection of a host organism.
Examples of pathogenic bacteria include, without limitation,
Aerobacter, Aeromonas, Acinetobacter, Agrobacterium, Bacillus,
Bacteroides, Bartonella, Bortella, Brucella, Calymmatobacterium,
Campylobacter, Citrobacter, Clostridium, Cornyebacterium,
Enterobacter, Erwinia, Escherichia, Francisella, Haemophilus,
Hafnia, Helicobacter, Klebsiella, Legionella, Listeria, Morganella,
Moraxella, Proteus, Providencia, Pseudomonas, Salmonella, Serratia,
Shigella, Staphylococcus, Streptococcus, Treponema, Xanthomonas,
Vibrio, and Yersinia. In desirable embodiments, the bacterial
pathogen is at Gram-positive coccus such as Staphylococcus aureus,
Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus
pyogenes, Moraxella catarrhalis, Haemophilus influenzae, or
Enterococcus spp. In other desirable embodiments the bacterial
pathogen is a Gram-negative bacterium, such as, without limitation,
Pseudonomas aeruginosa, Klebsiella pneumoniae, Escherichia coli,
Haemophilus influenzae, Citrobacter freundii, or Enterobacter spp.
In other desirable embodiments the bacterial pathogen is Chlamydia
pneumoniae or Chlamydia trachomatis. In particularly desirable
embodiments, the bacterial pathogen is Enterococcus faecalis,
Pseudomonas aeruginosa, Pseudomonas syringae, Salmonella
typhimurium, Staphylococcus aureus, Staphylococcus epidermidis, or
Staphylococcus saprophyticus. In other particularly desirable
embodiments, the bacterial pathogen is Rickettsia prowazekii,
Rickettsia rickettsii, Vibrio alginolyticus, Yersinia pestis,
Bacillus anthracis, Clostridium botulinum, Brucella abortus,
Brucella melitensis, Brucella suis, Burkholderia mallei,
Burkholderia pseudomallei, Clostridium perfringens, Coxiella
burnetii, Francisella tularensis, Shigella dysenteriae,
Staphylococcus lugdunensis, Staphylococcus schleiferi,
Staphylococcus caprae, Cowdria ruminantium, Mycoplasma capricolum,
Mycoplasma mycoides mycoides, Candidatus Liberobacter africanus,
Candidatus Liberobacter asiaticus, Ralstonia solanacearum,
Xanthomonas oryzae pv. oryzicola, or Xylella fastidiosa.
[0072] In desirable embodiments, a fungal pathogen is, without
limitation, Absidia corymbifera, Acremonium falciforme, A.
kiliense, A. recifei, Ajellomyces dermatitidis, A. capsulata,
Aspergillus spp., (e.g., A. flavus, A. fumigatus, A. nidulans, A.
niger, A. terreus), Candida spp. (e.g., C. albicans, C. glabrata,
C. guillermondii, C. krusei, C. parapsilosis, C. kefyr, C.
tropicalis), Crytococcus spp. (e.g., C. neoformans, C. gattii, C.
grubii), Cunninghamella elegans, Emmonsia parva, Epidermophyton
floccosum, Exophialia dermitidis, E. werneckii, E. jeanselmei, E.
spinifera, E. richardsiae, Filobasidiella neoformans, Fonsecaea
compacta, F. pedrosoi, Histoplasma capsulatum, Leptoshaeria
senegarlensis, Madurella mycetomatis, M. grisea, Malassezia furfur,
Microsporum spp, Neotestudina rosatii, Paracoccidioides
brasiliensis, Penicillium marneffei, Phialophora verrucosa,
Piedraia hortae, Pneumocystis spp., Pseudallescheria boydii,
Pyrenochaeta romeroi, Rhizomucor pusillus, Sporothrix schenckii,
Trichophyton spp, Trichosporon beigelii, Wangiella dermatitidis, or
Xylohypha bantiana. In particularly desirable embodiments, the
fungal pathogen is Coccidioides posadasii, Coccidioides immitis,
Fusarium sporotrichioides, Peronosclerospora philippinensis,
Schlerophthora rayssiae var zeae, or Synchytrium endobioticum.
[0073] In plants, in desirable embodiments, a fungal pathogen is,
without limitation Alternaria spp. (for example, A. brassicola and
A. solani), Ascochyta spp. (for example, A. pisi), Aspergillus
spp., Botrytis spp. (for example, B. cinerea), Cercospora spp. (for
example, C. kikuchii and C. zaea-maydis), Colletotrichum spp. (for
example, C. lindemuthianum), Coniothyrium spp., Diplocarpon spp.,
Diplodia spp. (for example, D. maydis), Erysiphe spp. (for example,
E. graminis f. sp. graminis and E. graminis f. sp. hordei),
Fusarium spp. (for example, F. nivale and F. oxysporum, F.
graminearum, F. solani, F. monilforme, and F. roseum),
Gaeumanomyces spp. (for example, G. graminis f. sp. tritici),
Helminthosporium spp. (for example, H. turcicum, H. carbonum, and
H. maydis), Macrophomina spp. (for example, M. phaseolina and
Maganaporthe grisea), Nectria spp. (for example, N. heamatocacca),
Penicillium spp., Phakospora pachyrhizi, Peronospora spp. (for
example, P. manshurica, P. tabacina), Phoma spp. (for example, P.
betae), Phymatotrichum spp. (for example, P. omnivorum),
Phytophthora spp. (for example, P. cinnamomi, P. cactorum, P.
phaseoli, P. parasitica, P. citrophthora, P. megasperma f. sp.
sojae, and P. infestans), Plasmopara (for example, P. viticola),
Podosphaera spp. (for example, P. leucotricha), Puccinia spp. (for
example, P. sorghi, P. striiformis, P. graminis f. sp. tritici, P.
asparagi, P. recondita, and P. arachidis), Puthium spp. (for
example, P. aphanidermatum), Pyrenophora spp. (for example, P.
tritici-repentens), Pyricularia spp. (for example, P. oryzea),
Pythium spp. (for example, P. ultimum), Rhizoctonia spp. (for
example, R. solani and R. cerealis), Rhytisma spp., Scerotium spp.
(for example, S. rolfsii), Sclerotinia spp. (for example, S.
sclerotiorum), Septoria spp. (for example, S. lycopersici, S.
glycines, S. nodorum and S. tritici), Sphaerotheca spp., Taphrina
spp., Thielaviopsis spp. (for example, T. basicola), Uncinula spp.
(for example, U. necator), Urocystis spp., Venturia spp. (for
example, V. inaequalis), or Verticillium spp. (for example, V.
dahliae and V. albo-atrum).
[0074] In desirable embodiments, the viral pathogen is vesicular
stomatitis virus (VSV). In particularly desirable embodiments, the
viral pathogen is cercopithecine herpesvirus 1 (herpes B virus),
Crimean-Congo haemorrhagic fever virus, Ebola virus, Lassa fever
virus, Marburg virus, monkeypox virus, reconstructed 1918 influenza
virus, South American haemorrhagic fever viruses (e.g., Flexal,
Guanarito, Junin, Machupo, and Sabia), tick-borne encephalitis
(e.g., Central European tick-borne encephalitis, Far Eastern
tick-borne encephalitis, Kyasanur Forest disease, Omsk hemorrhagic
fever, and Russian spring-summer encephalitis), variola major virus
(e.g., smallpox virus), variola minor virus (e.g., alastrim),
Eastern equine encephalitis virus, Hendra virus, Nipah virus, Rift
Valley fever virus, Venezuelan equine encephalitis virus, African
horse sickness virus, African swine fever virus, Akabane virus,
avian influenza virus, Bluetongue virus, bovine spongiform
encephalitis, camelpox virus, classical swine fever virus,
foot-and-mouth disease virus, goat pox virus, Japanese encephalitis
virus, lumpy skin disease virus, malignant catarrhal fever virus
(alcelaphine herpesvirus type 1), Menangle virus, Newcastle disease
virus, Peste des petits ruminants virus, rinderpest virus, sheep
pox virus, or swine vesicular disease virus.
[0075] In plants, the viral pathogen desirably is a nepovirus,
poytvirus, tymovirus, ilarvirus, potexvirus, caulimovirus,
tobravirus, closterovirus, carlavirus, cucumovirus, tobamovirus,
comovirus, carmovirus, necrovirus, nucleorhabdovirus, tospovirus,
luteovirus, fijivirus, tenuivirus, tobacco mosaic virus, or
leafroll virus. In particular desirable embodiments, the viral
pathogen is tobacco mosaic virus, tobacco necrosis virus, potato
leaf roll virus, potato virus X, potato virus Y, tomato spotted
wilt virus, or tomato ring spot virus.
[0076] In other desirable embodiments, the Mycobacterium is
Mycobacterium tuberculosis.
[0077] By a "compound," "candidate compound," or "factor" is meant
a chemical, be it naturally-occurring or artificially-derived.
Compounds may include, for example, peptides, polypeptides,
synthetic organic molecules, naturally-occurring organic molecules,
nucleic acid molecules, and components or combinations thereof.
Desirably, a candidate compound is a low molecular weight chemical
compound.
[0078] By "increases the lifespan of an organism" is meant an
increase of at least 10% in the average age attained at the time of
death of a population of organisms relative to a control population
of the same organism. In desirable embodiments, the increase in age
is at least 20%, 50%, 75%, 100%, or even 200%.
[0079] By "inhibiting a pathogen" is meant the ability of a
candidate compound to decrease, suppress, attenuate, diminish, or
arrest the development or progression of a pathogen-mediated
disease or an infection in a eukaryotic host organism. Preferably,
such inhibition decreases pathogenicity by at least 5%, more
preferably by at least 25%, and most preferably by at least 50%, as
compared to symptoms in the absence of candidate compound in any
appropriate pathogenicity assay (for example, those assays
described herein). In one particular example, inhibition may be
measured by monitoring pathogenic symptoms in a host organism
exposed to a test compound or extract, a decrease in the level of
symptoms relative to the level of pathogenic symptoms in a host
organism not exposed to the compound indicating compound-mediated
inhibition of the pathogen. In other desirable embodiments,
inhibiting a pathogen kills the pathogen.
[0080] By a "low molecular weight chemical compound" as used herein
is meant a molecule of less than about 10 kDa molecular weight. The
compound desirably is a synthetic organic or inorganic compound or
a natural organic compound. Desirably, the molecular weight is less
than about 1000 Da, more desirably less than about 750 Da, and most
desirably less than about 500 Da. Nucleic acid molecules are
specifically excluded from the definition of a low molecular weight
chemical compound.
[0081] The invention features 15 lead compounds and, in some
instances, their structural homologues for use as antimicrobials,
antifungals, and antivirals. A compound is identified as a
structural homologue of a lead compound if it has a significant
number of substructures in common with that lead compound, such
that the Tanimoto coefficient measuring structural similarity
between those two compounds has a value of 0.85 or greater. The
Tanimoto coefficient between two compounds is determined by
calculating the ratio of the number of substructures held in common
by the two compounds to the number of total substructures present
in at least one of the two compounds. Tanimoto coefficients can
also be calculated using hashed binary fingerprints wherein each
fingerprint encodes the substructure composition of one compound
and the Tanimoto coefficient is the ratio of the number of bits set
to "1" in both compounds' fingerprints divided by the number of
bits set to "1" in either compound's fingerprint using methods
known in the art, e.g., such as Patterson et al. 1996 J. Med. Chem.
39:3049-3059; Matter et al. 1997 J. Med. Chem. 40:1219-1229; Potter
et al. J. Med. Chem. 1998 41:478-488; Taylor et al. 1995 J. Chem
Inf. Comp. Sci. 35:59-67; Delaney et al., 1996 Mol. Diversity
1:217-222. In the present invention, one such method using Daylight
fingerprints is used to calculate Tanimoto coefficients and thus
identify compounds present in publicly available chemical
databases, e.g., such as PubChem and Chembank, that are structural
homologues of a lead compound (Martin et al., 2002 J. Med. Chem.
45:4350-4358; Daylight Chemical Information Systems Inc.: Irvine,
Calif.). A lead compound and structurally homologous compound
having a Tanimoto coefficient of 0.85 or greater as calculated
using Daylight fingerprints are known to have a probability of 30%
or greater, e.g., such as 50% or 75%, of having the same biological
activity, depending in part on the potency of the lead compound,
e.g., having an IC.sub.50 less than 10 micromolar or having an
IC.sub.50 between 10 micromolar and 100 micromolar, and in part on
the biological activity under consideration, as known in the art,
e.g., as described in Martin et al., 2002 J. Med. Chem.
45:4350-4358 and Klekota et al. 2005 J. Chem. Info. Model.
45:1824-1836.
[0082] In the generic descriptions of compounds of this invention,
the number of atoms of a particular type in a substituent group is
generally given as a range, e.g., an alkyl group containing from 1
to 4 carbon atoms or C.sub.1-4 alkyl. Reference to such a range is
intended to include specific references to groups having each of
the integer number of atoms within the specified range. For
example, an alkyl group from 1 to 4 carbon atoms includes each of
C.sub.1, C.sub.2, C.sub.3, and C.sub.4. A C.sub.1-4 heteroalkyl,
for example, includes from 1 to 3 carbon atoms in addition to one
or more heteroatoms. Other numbers of atoms and other types of
atoms may be indicated in a similar manner.
[0083] As used herein, the terms "alkyl" and the prefix "alk-" are
inclusive of both straight chain and branched chain groups and of
cyclic groups, i.e., cycloalkyl. Exemplary cyclic groups include
cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl groups. The
C.sub.1-4 alkyl group may be substituted or unsubstituted.
Exemplary substituents include alkoxy, aryloxy, sulfhydryl,
alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl,
amino, aminoalkyl, disubstituted amino, quaternary amino,
hydroxyalkyl, carboxyalkyl, and carboxyl groups. C.sub.1-4 alkyls
include, without limitation, methyl; ethyl; n-propyl; isopropyl;
cyclopropyl; cyclopropylmethyl; cyclopropylethyl; n-butyl;
iso-butyl; sec-butyl; tert-butyl; and cyclobutyl.
[0084] By "C.sub.2-4 alkenyl" is meant a branched or unbranched
hydrocarbon group containing one or more double bonds and having
from 2 to 4 carbon atoms. The C.sub.2-4 alkenyl group may be
substituted or unsubstituted. Exemplary substituents include
alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxyl,
fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino,
quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
C.sub.2-4 alkenyls include, without limitation, vinyl; allyl;
2-cyclopropyl-1-ethenyl; 1-propenyl; 1-butenyl; 2-butenyl;
3-butenyl; 2-methyl-1-propenyl; and 2-methyl-2-propenyl.
[0085] By "C.sub.2-4 alkynyl" is meant a branched or unbranched
hydrocarbon group containing one or more triple bonds and having
from 2 to 4 carbon atoms. The C.sub.2-4 alkynyl group may be
substituted or unsubstituted. Exemplary substituents include
alkoxy, aryloxy, sulfhydryl, alkylthio, arylthio, halide, hydroxy,
fluoroalkyl, perfluoralkyl, amino, aminoalkyl, disubstituted amino,
quaternary amino, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
C.sub.2-4 alkynyls include, without limitation, ethynyl,
1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, and 3-butynyl.
[0086] By "C.sub.2-6 heterocyclyl" is meant a stable 5- to
7-membered monocyclic or 7- to 14-membered bicyclic heterocyclic
ring which is saturated partially unsaturated or unsaturated
(aromatic), and which consists of 2 to 6 carbon atoms and 1, 2, 3
or 4 heteroatoms independently selected from the group consisting
of N, O, and S and including any bicyclic group in which any of the
above-defined heterocyclic rings is fused to a benzene ring. The
heterocyclyl group may be substituted or unsubstituted. Exemplary
substituents include alkoxy, aryloxy, sulfhydryl, alkylthio,
arylthio, halide, hydroxy, fluoroalkyl, perfluoralkyl, amino,
aminoalkyl, disubstituted amino, quaternary amino, hydroxyalkyl,
carboxyalkyl, and carboxyl groups. The nitrogen and sulfur
heteroatoms may optionally be oxidized. The heterocyclic ring may
be covalently attached via any heteroatom or carbon atom which
results in a stable structure, e.g., an imidazolinyl ring may be
linked at either of the ring-carbon atom positions or at the
nitrogen atom. A nitrogen atom in the heterocycle may optionally be
quaternized. Preferably when the total number of S and O atoms in
the heterocycle exceeds 1, then these heteroatoms are not adjacent
to one another. Heterocycles include, without limitation,
1H-indazole, 2-pyrrolidonyl, 2H,6H-1,5,2-dithiazinyl, 2H-pyrrolyl,
3H-indolyl, 4-piperidonyl, 4aH-carbazole, 4H-quinolizinyl,
6H-1,2,5-thiadiazinyl, acridinyl, azocinyl, benzimidazolyl,
benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl,
benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl,
benzisothiazolyl, benzimidazalonyl, carbazolyl, 4aH-carbazolyl,
b-carbolinyl, chromanyl, chromenyl, cinnolinyl,
decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,
dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl,
imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl,
indolinyl, indolizinyl, indolyl, isobenzofuranyl, isochromanyl,
isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,
isothiazolyl, isoxazolyl, morpholinyl, naphthyridinyl,
octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,
1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,
oxazolidinyl, oxazolyl, oxazolidinylperimidinyl, phenanthridinyl,
phenanthrolinyl, phenarsazinyl, phenazinyl, phenothiazinyl,
phenoxathiinyl, phenoxazinyl, phthalazinyl, piperazinyl,
piperidinyl, pteridinyl, piperidonyl, 4-piperidonyl, pteridinyl,
purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl,
pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole,
pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl,
pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl,
quinuclidinyl, carbolinyl, tetrahydrofuranyl,
tetrahydroisoquinolinyl, tetrahydroquinolinyl,
6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl,
1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl,
thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl,
thiophenyl, triazinyl, 1,2,3-triazolyl, 1,2,4-triazolyl,
1,2,5-triazolyl, 1,3,4-triazolyl, xanthenyl. Preferred 5 to 10
membered heterocycles include, but are not limited to, pyridinyl,
pyrimidinyl, triazinyl, furanyl, thienyl, thiazolyl, pyrrolyl,
pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, tetrazolyl,
benzofuranyl, benzothiofuranyl, indolyl, benzimidazolyl,
1H-indazolyl, oxazolidinyl, isoxazolidinyl, benzotriazolyl,
benzisoxazolyl, oxindolyl, benzoxazolinyl, quinolinyl, and
isoquinolinyl. Preferred 5 to 6 membered heterocycles include,
without limitation, pyridinyl, pyrimidinyl, triazinyl, furanyl,
thienyl, thiazolyl, pyrrolyl, piperazinyl, piperidinyl, pyrazolyl,
imidazolyl, oxazolyl, isoxazolyl, and tetrazolyl.
[0087] By "C.sub.6-12 aryl" is meant an aromatic group having a
ring system comprised of carbon atoms with conjugated .pi.
electrons (e.g., phenyl). The aryl group has from 6 to 12 carbon
atoms. Aryl groups may optionally include monocyclic, bicyclic, or
tricyclic rings, in which each ring desirably has five or six
members. The aryl group may be substituted or unsubstituted.
Exemplary substituents include alkyl, hydroxy, alkoxy, aryloxy,
sulfhydryl, alkylthio, arylthio, halide, fluoroalkyl, carboxyl,
hydroxyalkyl, carboxyalkyl, amino, aminoalkyl, monosubstituted
amino, disubstituted amino, and quaternary amino groups.
[0088] By "C.sub.7-14 alkaryl" is meant an alkyl substituted by an
aryl group (e.g., benzyl, phenethyl, or 3,4-dichlorophenethyl)
having from 7 to 14 carbon atoms.
[0089] By "C.sub.3-10 alkheterocyclyl" is meant an alkyl
substituted heterocyclic group having from 7 to 14 carbon atoms in
addition to one or more heteroatoms (e.g., 3-furanylmethyl,
2-furanylmethyl, 3-tetrahydrofuranylmethyl, or
2-tetrahydrofuranylmethyl).
[0090] By "C.sub.1-4 heteroalkyl" is meant a branched or unbranched
alkyl, alkenyl, or alkynyl group having from 1 to 4 carbon atoms in
addition to 1, 2, or 3 heteroatoms independently selected from the
group consisting of N, O, S, and P. Heteroalkyls include, without
limitation, tertiary amines, secondary amines, ethers, thioethers,
amides, thioamides, carbamates, thiocarbamates, hydrazones, imines,
phosphodiesters, phosphoramidates, sulfonamides, and disulfides. A
heteroalkyl may optionally include monocyclic, bicyclic, or
tricyclic rings, in which each ring desirably has three to six
members. The heteroalkyl group may be substituted or unsubstituted.
Exemplary substituents include alkoxy, aryloxy, sulfhydryl,
alkylthio, arylthio, halide, hydroxyl, fluoroalkyl, perfluoralkyl,
amino, aminoalkyl, disubstituted amino, quaternary amino,
hydroxyalkyl, hydroxyalkyl, carboxyalkyl, and carboxyl groups.
[0091] By "acyl" is meant a chemical moiety with the formula
R--C(O)--, wherein R is selected from C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12
aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or C.sub.1-4
heteroalkyl.
[0092] By "halide" is meant bromine, chlorine, iodine, or
fluorine.
[0093] By "fluoroalkyl" is meant an alkyl group that is substituted
with a fluorine.
[0094] By "perfluoroalkyl" is meant an alkyl group consisting of
only carbon and fluorine atoms.
[0095] By "carboxyalkyl" is meant a chemical moiety with the
formula --(R)--COOH, wherein R is selected from C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or
C.sub.1-4 heteroalkyl.
[0096] By "hydroxyalkyl" is meant a chemical moiety with the
formula --(R)--OH, wherein R is selected from C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or
C.sub.1-4 heteroalkyl.
[0097] By "alkoxy" is meant a chemical substituent of the formula
--OR, wherein R is selected from C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12
aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or C.sub.1-4
heteroalkyl.
[0098] By "aryloxy" is meant a chemical substituent of the formula
--OR, wherein R is a C.sub.6-12 aryl group.
[0099] By "alkylthio" is meant a chemical substituent of the
formula --SR, wherein R is selected from C.sub.1-4 alkyl, C.sub.2-4
alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12
aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, or C.sub.1-4
heteroalkyl.
[0100] By "arylthio" is meant a chemical substituent of the formula
--SR, wherein R is a C.sub.6-12 aryl group.
[0101] By "quaternary amino" is meant a chemical substituent of the
formula --(R)--N(R')(R'')(R''').sup.+, wherein R, R', R'', and R'''
are each independently an alkyl, alkenyl, alkynyl, or aryl group. R
may be an alkyl group linking the quaternary amino nitrogen atom,
as a substituent, to another moiety. The nitrogen atom, N, is
covalently attached to four carbon atoms of alkyl and/or aryl
groups, resulting in a positive charge at the nitrogen atom.
[0102] Other features and advantages of the invention will be
apparent from the following Detailed Description, the Drawings, and
the Claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0103] FIG. 1 depicts the structure of compound A1 and compounds
A2-A9, which are homologues of A1.
[0104] FIG. 2 depicts the structure of compound B1 and compound B2,
which is a homologue of B1.
[0105] FIG. 3 depicts the structure of compound C1 and compounds
C2-C7, which are homologues of C1.
[0106] FIG. 4 depicts the structure of compound D1 and compounds
D2-D4, which are homologues of D1.
[0107] FIGS. 5A and 5B depict the structure of compound E1 and
compounds E2-E25, which are homologues of E1.
[0108] FIGS. 6A and 6B depict the structure of compound F1 and
compounds F2-F7, which are homologues of F1.
[0109] FIG. 7 depicts the structure of compound G1 and compound G2,
which is a homologue of G1.
[0110] FIG. 8 depicts the structure of compound H1 and compounds
H2-H8, which are homologues of H1.
[0111] FIG. 9 depicts the structure of compound I1 and compound I2,
which is a homologue of I1.
[0112] FIG. 10 depicts the structure of compound J1 and compounds
J2-J6, which are homologues of J1.
[0113] FIGS. 11A-11D show the curing of a nematode E. faecalis
infection on solid medium by antibiotic treatment. FIG. 11A is a
graph depicting the Kaplan-Meier survival curves of wild-type N2 C.
elegans feeding continuously on lawns of E. faecalis (Efs) V583
(.tangle-solidup.) or E. faecium (Efm) DO (). Nematodes feeding for
24 hours on E. faecalis and subsequently transferred to lawns of E.
faecium (.box-solid.) die with an LT50 of 14.9 days. Error bars
equal SEM. FIG. 11B is a graph depicting the Kaplan-Meier survival
curves of E. faecalis infected N2 C. elegans transferred to lawns
of E. faecium on media containing tetracycline at 10 .mu.g/ml (A,
p<0.0001), 2 .mu.g/ml (V, p<0.0014), 0.4 .mu.g/ml (*,
p<0.06), 0.08 .mu.g/ml (*, p=0.56), or no tetracycline (m). FIG.
11C is a graph showing the antibiotic concentrations required to
promote rescue of E. faecalis infected N2 nematodes. Survival was
measured 13 days post-infection after treatment with tetracycline,
vancomycin, or ciprofloxacin. Error bars are standard deviations.
FIG. 11D is a graph depicting the Kaplan-Meier survival curve of
glp-4; sek-1 nematodes infected for 12 hours on E. faecalis OG1RF
and transferred to BHI media containing 20 .mu.g/ml tetracycline
(.tangle-solidup.), 40 .mu.g/ml vancomycin (), or no additional
antibiotic (.box-solid.). Error bars equal SEM.
[0114] FIGS. 12A-12C show the liquid infection assay gauges
differences in nematode killing due to E. faecalis strains with
varying degrees of pathogenicity or due to antibiotic treatment.
FIG. 12A is a graph depicting the Kaplan-Meier survival curves of
glp-4; sek-1 nematodes that were infected with E. faecalis OG1RF
(.box-solid.,.tangle-solidup.) or the two component quorum sensing
regulator mutant OG1RF .DELTA.fsrB () or with the E. faecium strain
11M12 ( ). OG1RF infected nematodes were treated with 0
(.box-solid.) or 20 .mu.g/ml (.tangle-solidup.) tetracycline. Error
bars equal SEM. FIG. 12B is a graph depicting the Kaplan-Meier
survival curves of glp-4; sek-1 nematodes that were infected with
the cytolysin (Cyl) producing E. faecalis strain MMH594
(.tangle-solidup.,.diamond-solid.) or the E. faecium strain 11M12 (
). Cyl infected nematodes were treated with 0 (.tangle-solidup.) or
20 .mu.g/ml (.diamond-solid.) tetracycline. FIG. 12C is a bar chart
showing the bacterial load in the nematode intestinal tract after
antibiotic treatment. E. faecalis infected nematodes were treated
in liquid media containing 20 .mu.g/ml ampicillin or tetracycline
and the number of CFU per worm was determined. Error bars equal
standard deviations.
[0115] FIG. 13 is a series of pictures depicting the scoring
live/dead worms in the liquid killing assay. Living nematodes in
the liquid infection assay maintain a sinusoidal shape, whereas
dead nematodes in the liquid infection assay appear as straight,
rigid rods as the corpse becomes filled with bacteria.
[0116] FIG. 14 is a graph depicting the curing kinetics of selected
hit compounds. The Kaplan-Meier survival curves of infected
nematodes treated with 25 .mu.g/ml of compound C1
(.diamond-solid.), 50 .mu.g/ml of compound G1 ( ), 25 .mu.g/ml of
compound N1 (.quadrature.), 2 .mu.g/ml tetracycline (), 20 .mu.g/ml
tetracycline (.tangle-solidup.), or mock treatment (.box-solid.).
In pairwise comparisons to mock treatment using log-rank tests, the
difference for all of the treatments was significant with
p<0.0001.
[0117] FIGS. 15A-15C show the structures of the lead compounds A1,
B1, C1, D1, E1, E30, F1, G1, H1, I1, J1, K1, L1, M1, N1, and
O1.
[0118] FIGS. 16A and 16B are a series of graphs showing that
killing of C. elegans nematodes after exposure to Candida spp.
depends on the nematode MAPK-pathway and on the Candida strain.
FIG. 16A shows that killing of 60-70 C. elegans N2 wild type
nematodes takes place mostly on approximately day 3 of the assay,
and in over one third of the nematodes, killing is associated with
matricidal effect involving the premature hatching of eggs in the
C. elegans uterus. Survival of 60-70 C. elegans glp-4; sek-1
nematodes in liquid pathogen-free media, after feeding on lawns of
C. albicans strain DAY185 is significantly shorter compared to the
strain glp-4 (P<0.001). 30-40 control N2 nematodes were exposed
to E. coli OP50. FIG. 16B shows survival of C. elegans glp-4; sek-1
feeding on lawns of C. albicans ATCC#90028, C. parapsilosis
ATCC#20019 or C. krusei ATCC#6258. P<0.001 for each of the yeast
strains compared to control nematodes that were exposed to E. coli
OP50. 60-70 worms were exposed to each pathogen or E. coli.
[0119] FIGS. 17A through 17D are a series of graphs showing that
established antifungals prolong the survival of C. elegans glp-4;
sek-1 nematodes infected by Candida spp. and evaluation of
antifungals in the C. elegans assay allows the concomitant
evaluation of toxicity. FIG. 17A shows that addition of caspofungin
(8 .mu.g/ml), amphotericin B (16 .mu.g/ml) and fluconazole (32
.mu.g/ml) were effective against the fluconazole-susceptible C.
albicans strain MLR62. FIG. 17B shows that only caspofungin and
amphotericin B were effective against C. krusei strain ATCC#6258,
the most fluconazole-resistant strain tested (P<0.001). FIG. 17C
shows that caspofungin (8 .mu.g/ml), amphotericin B (16 .mu.g/ml),
and fluconazole (32 .mu.g/ml) prolonged the survival of C. elegans
glp-4; sek-1 nematodes (P<0.001) against C. parasilosis
ATCC#20019. FIG. 17D shows that the life span of nematodes was
significantly longer in 4 .mu.g/mL and 32 .mu.g/mL fluconazole
compared to those in no antifungal (P=0.0001 and P<0.0001,
respectively) against C. parasilosis ATCC#20019. However, at very
high concentrations (100 g/mL) nematodes died faster, probably
because of toxicity (P=0.01, compared to those in no antifungal).
60-70 nematodes were used for each condition.
[0120] FIGS. 18A through 18C are a series of images showing the
phenotype of nematodes exposed to compounds with (FIG. 18A) and
without antifungal efficacy (FIGS. 18B and 18C). After exposure to
strain C. albicans MLR62, C. elegans glp-4; sek-1 nematodes were
pipetted into 96-well plates that contained compounds from chemical
libraries. Nematodes exposed to compounds that had antifungal
efficacy (in this case caffeic acid phenethyl ester, CAPE) had no
fluorescence within the intestine (FIG. 18A), while nematodes
exposed to compounds without antifungal efficacy did not
demonstrate any movement (FIG. 18B) and developed filaments outside
the nematodes (FIG. 18C). Roughly 25 worms were used per well.
[0121] FIG. 19 is a series of images showing that the Sytox.RTM.
orange fluorophore stains dead worms in the liquid infection assay.
At the end point of the infection assay, worms in the 384-well
plate were washed to remove the infection media and bacteria. Worms
were the incubated with the Sytox.RTM. orange, which specifically
stains dead and dying worms. Fluorescent and transmitted light
images were captured for each well. The images displayed on the
left column are from an untreated well in which most of the worms
are dead and fluorescent. The images displayed on the right column
are from an ampicillin-treated well in which most of the worms are
alive and not fluorescently-labeled.
[0122] FIG. 20 is a series of images and a schematic diagram
showing worm survival quantification using CellProfiler software.
After drug treatment (e.g., vancomycin), worms in 384-well plates
are incubated with Sytox.RTM. orange, which specifically stains
dead worms. The top row shows raw fluorescent Sytox.RTM. orange and
bright-field images captured using a 2.times. objective. The images
were analyzed using CellProfiler software through a pipeline of 22
processing steps. The results of two of the processing steps are
shown. Images in the middle row show the results of well area
selection and light correction of the bright-field images. Images
in the bottom row show the result of the worm identification
function after inverting and cropping the well area. Finally, the
total object area of the fluorescent and the bright-field images
are measured. Assuming that there are exactly the same number of
worms per well (.+-.5% sorting error) and that all worms have
roughly the same size, the Sytox.RTM. orange objects area and
bright-field objects area are used to approximate the number of
dead worms and the total number of worms, respectively. Worm
survival=1-(Sytox.RTM. orange area/total bright-field worm
area).
[0123] FIG. 21 is an image depicting near complete killing of
seedlings in a 96-well plate assay of Arabidopsis (Col ecotype)
seedlings by Pseudomonas aeruginosa (strain PA14) compared to much
healthier (and greener) control seedlings five days post
infection.
[0124] FIG. 22 is an image depicting damage to Arabidopsis (Col
ecotype) seedlings by a plant pathogen Pseudomonas syringae pv.
tomato (DC3000) and much healthier seedlings (compared to control
untreated seedlings) by the typeIII secretion mutant DC3000(hrcC)
five days post infection. The treated seedlings are boxed and
labeled.
[0125] FIG. 23 is a graphical representation of relative
luminescent units (RLU) of Arabidopsis seedlings ectopically
expressing a luciferase reporter from a CaMV 35S promoter.
Indicated are RLU just prior to treatment with P. aeruginosa
(strain PA14) or a mutant impaired in production of some virulence
factors--PA14(lasR) or control untreated seedlings.
DETAILED DESCRIPTION
[0126] To discover novel antimicrobial, antifungal, and antiviral
compounds we devised screens to identify compounds that promote the
survival of the model laboratory nematode Caenorhabditis elegans
infected with the human opportunistic pathogen Enterococcus
faecalis and with Candida strains. The screening methods can also
be used to identify compounds that increase the lifespan of an
organism.
[0127] E. faecalis colonizes the nematode intestinal tract forming
a persistent lethal infection. Death of infected nematodes was
prevented by antibiotic treatment in a dose-dependent manner and
antibiotic treatment markedly reduced the number of bacteria
colonizing the nematode intestine. To facilitate high throughput
screening of compound libraries, we adapted the traditional
agar-based C. elegans-E. faecalis infection assay so that it could
be carried out in liquid medium in standard microtiter plates. An
important feature of the assay is that the first assay endpoint is
nematode survival, which automatically eliminates highly toxic
compounds that affect nematode viability. We used this simple
infection system to screen 6000 synthetic compounds. We identified
16 compounds that promoted nematode survival. Some of the compounds
inhibited E. faecalis growth in vitro, but in contrast to
traditional antibiotics, the in vivo effective dose of many of
these compounds was significantly lower than the minimum inhibitory
concentration needed to prevent the growth of E. faecalis in vitro.
Moreover, many of the compounds had little or no affect on in vitro
bacterial growth. Our findings indicate that the whole-animal C.
elegans screen not only identifies traditional antibiotics, but
also compounds that target bacterial virulence or stimulate host
defense.
[0128] We have also found that Candida albicans, as well as other
Candida strains, form persistant infections in the C. elegans
intestine and can kill C. elegans. We used these findings to
develop a C. elegans-based infection model that can be performed in
standard 96-well plates in liquid media, thereby enabling automated
addition of compounds. An important feature of the assay is that
the first assay endpoint is nematode survival, which automatically
eliminates highly toxic compounds that affect nematode viability.
Moreover, important components of Candida pathogenesis in mammals,
such as filament formation, are involved in nematode killing and
filamentation becomes apparent as the nematode dies, providing a
second clinically relevant endpoint. As described herein, we
screened a total of 1,266 compounds and identified 15 that
prolonged nematode survival and completely or almost completely
inhibited filamentation. An additional 52 of the compounds
prolonged nematode survival but had no or minimal effect on
filamentation.
High-Throughput Screening
[0129] Whole-organism (e.g., C. elegans or plant seedling) screens
have several advantages compared to in vitro screens that use
planktonic cells. Some virulence traits are induced only in the
host and, therefore, the identification of compounds that are
effective against these virulence traits may require detection in
vivo. Also, the whole-organism approach provides relatively
unambiguous assay endpoints (e.g., survival or death of the animal
or plant), allows the use of liquid handling robots for filling
assay plates and for pin transfer of compounds from library stock
plates to assay plates, and permits automated or semi-automated
readout using plate readers or automated imaging microscopes. The
whole-organism assays using, e.g., invertebrates, also have many
advantages compared to screens using mammalian models. The study of
pathogenesis in mammals is complicated by difficulty of handling,
long reproductive cycles, small brood sizes, complexity of
mammalian hosts, high cost, and ethical considerations.
[0130] Despite the potential value of live animals (e.g., C.
elegans or Drosophila or moth larvae) or plant seedlings for gene,
drug, or drug-target discovery, screens using whole animals, prior
to the present invention, were generally carried out manually and,
therefore, were extremely labor intensive. In the laboratory, for
example, C. elegans assays are typically carried out by
transferring nematodes from lawns of Escherichia coli strain OP50
(their normal laboratory food) to lawns of pathogenic bacteria or
yeast grown on solid agar media. However, screening chemical
libraries using an agar-based C. elegans killing assay is not
readily compatible with the use of robots for filling assay plates
and pin transfer of compounds, homogeneous distribution of
chemicals in the medium, or the use of automated plate readers or
automated screening microscopes to monitor host (e.g., nematode)
survival. The previously available liquid assays also had their
deficiencies, as these assays used volumes that were incompatible
with high-throughput screens of chemical libraries. The methods for
identifying candidate compounds described herein are automated,
performed in a small volume of liquid (e.g., 20-100 .mu.l), and the
scoring of the compounds is automated and quantitative.
[0131] The methods for identifying a candidate compound described
herein feature exposing a host organism to a pathogen and
incubating the exposed invertebrate animal or plant host organism
in a liquid medium in the presence of at least one candidate
compound. In desirable embodiments, the invertebrate animal or
plant host is contacted with the candidate compound prior to being
exposed to the pathogen. A candidate compound that inhibits the
pathogen in the host organism may be identified depending on the
survival or death of the host organism. The host organism may be,
e.g., a nematode (e.g., C. elegans). The nematode may have a
mutation in the mitogen-activated protein kinase (MAPK) pathway
(e.g., a sek-1 mutation) or may have a temperature-sensitive
sterile mutation (e.g., a glp-4 mutation). The invertebrate host
organisms may also be, e.g., Drosophila larvae, Plutella xylostella
larvae, Galleria mellonella larvae, or a plant seedling (e.g., a
Arabidopsis thaliana seedling). The pathogen that infects the host
organism may be, e.g., a bacterial pathogen (e.g., Enterococcus
faecalis, Pseudomonas aeruginosa, Pseudomonas syringae, Salmonella
typhimurium, or Staphylococcus aureus or epidermidis), a fungal
pathogen (e.g., Candida albicans, Candida glabrata, Candida
parapsilosis, Crytococcus spp. (e.g., C. neoformans, C. gattii, C.
grubii), Rhodotorula mucilaginosa, Fusarium oxysporum, Botrytis
cinerea, or Saccharomyces cerevisiae), a viral pathogen (e.g.,
vesicular stomatitis virus), a protozoan, or a mycobacterium. The
exposed invertebrate host organism may be incubated with at least
one candidate compound in a liquid medium. The liquid medium may
include, e.g., a buffer (e.g., M9 buffer), brain heart infusion
media, MS (Murashige and Skoog) medium, cholesterol, and an
antibiotic (e.g., kanamycin).
[0132] The invention also features a container that includes an
invertebrate animal or a plant host organism that is infected with
a pathogen, liquid media, and a candidate compound. The container
may be, e.g., a 24-well, 48-well, 96-well plate, a 384-well plate,
a 1536-well plate, a 3456-well plate, or any other suitable
container. Each well of the container may contain a different
candidate compound.
[0133] The methods of screening described herein may also be used
to enable quantitative analysis of a wide range of biological
processes such as the response to different types of biotic (e.g.
pathogens) or abiotic (e.g., exposure to heavy metals, ultraviolet
radiation, or heat) stresses that affect viability. Traditional
longevity studies may be performed using the methods described
herein, which would allow for the automated screening of any
phenotypic read-out based on fluorescent markers (e.g., green
fluorescent protein, Nile Red, MitoTracker (Invitrogen), or
Sytox.RTM. green or orange). Automated screening of ectopic
fluorescent and luminescent markers such as GFP and luciferase
could facilitate the finding of genes that affect, e.g.,
reproduction, cell proliferation, cell death, fat accumulation,
insulin signaling, pathogen resistance, and neurotransmission. In
addition, the study of chemical or genetic perturbations that
affect growth rate or body size could be performed using the
methods described herein.
[0134] Using these screens, we have identified compounds that are
useful for the treatment of infections (compounds of formulas
(I)-(XV), below).
Test Extracts and Candidate Compounds
[0135] In general, novel antimicrobial, antifungal, or antiviral
drugs are identified from large libraries of both natural-product
or synthetic (or semi-synthetic) extracts or chemical libraries
according to methods known in the art. Those skilled in the field
of drug discovery and development will understand that the precise
source of test extracts or compounds is not critical to the
screening procedure(s) of the invention. Accordingly, virtually any
number of chemical extracts or compounds can be screened using the
methods described herein. Examples of such extracts or compounds
include, but are not limited to, plant-, fungal-, prokaryotic- or
animal-based extracts, fermentation broths, and synthetic
compounds, as well as modification of existing compounds. Numerous
methods are also available for generating random or directed
synthesis (e.g., semi-synthesis or total synthesis) of any number
of chemical compounds, including, but not limited to, saccharide-,
lipid-, peptide-, and nucleic acid-based compounds. Synthetic
compound libraries are commercially available from Brandon
Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee,
Wis.). Alternatively, libraries of natural compounds in the form of
bacterial, fungal, plant, and animal extracts are commercially
available from a number of sources, including Biotics (Sussex, UK),
Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft.
Pierce, Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In
addition, natural and synthetically produced libraries are
produced, if desired, according to methods known in the art, e.g.,
by standard extraction and fractionation methods. Furthermore, if
desired, any library or compound is readily modified using standard
chemical, physical, or biochemical methods.
[0136] In addition, those skilled in the art of drug discovery and
development readily understand that methods for dereplication
(e.g., taxonomic dereplication, biological dereplication, and
chemical dereplication, or any combination thereof) or the
elimination of replicates or repeats of materials already known for
their antimicrobial, antifungal, or antiviral activity should be
employed whenever possible.
[0137] When a crude extract is found to have antimicrobial,
antifungal, or antiviral activity, further fractionation of the
positive lead extract is necessary to isolate chemical constituents
responsible for the observed effect. Thus, the goal of the
extraction, fractionation, and purification process is the careful
characterization and identification of a chemical entity within the
crude extract having antimicrobial, antifungal, or antiviral
activity. Methods of fractionation and purification of such
heterogenous extracts are known in the art. If desired, compounds
shown to be useful agents for the treatment of pathogenicity are
chemically modified according to methods known in the art.
Synthesis of Compounds
[0138] The synthesis of the compounds of the invention may involve
selective protection and deprotection of alcohols, amines,
sulfhydryls and carboxylic acid functional groups in one or more
reactants. For example, commonly used protecting groups for amines
include carbamates, such as tert-butyl, benzyl,
2,2,2-trichloroethyl, 2-trimethylsilylethyl, 9-fluorenylmethyl,
allyl, and m-nitrophenyl. Other commonly used protecting groups for
amines include amides, such as formamides, acetamides,
trifluoroacetamides, sulfonamides, trifluoromethanesulfonyl amides,
trimethylsilylethanesulfonamides, and tert-butylsulfonyl amides.
Examples of commonly used protecting groups for carboxylic acids
include esters, such as methyl, ethyl, tert-butyl,
9-fluorenylmethyl, 2-(trimethylsilyl)ethoxy methyl, benzyl,
diphenylmethyl, O-nitrobenzyl, ortho-esters, and halo-esters.
Examples of commonly used protecting groups for alcohols include
ethers, such as methyl, methoxymethyl, methoxyethoxymethyl,
methylthiomethyl, benzyloxymethyl, tetrahydropyranyl, ethoxyethyl,
benzyl, 2-napthylmethyl, O-nitrobenzyl, P-nitrobenzyl,
P-methoxybenzyl, 9-phenylxanthyl, trityl (including
methoxy-trityls), and silyl ethers. Examples of commonly used
protecting groups for sulfhydryls include many of the same
protecting groups used for hydroxyls. In addition, sulfhydryls can
be protected in a reduced form (e.g., as disulfides) or an oxidized
form (e.g., as sulfonic acids, sulfonic esters, or sulfonic
amides). Protecting groups can be chosen such that selective
conditions (e.g., acidic conditions, basic conditions, catalysis by
a nucleophile, catalysis by a lewis acid, or hydrogenation) are
required to remove each, exclusive of other protecting groups in a
molecule. The conditions required for the addition of protecting
groups to amine, alcohol, sulfhydryl, and carboxylic acid
functionalities and the conditions required for their removal are
provided in detail in "T.W. Green and P.G.M. Wuts: Protective
Groups in Organic Synthesis" (2.sup.nd ed., 1991, John Wiley &
Sons) and "P. J. Kocienski: Protecting Groups" (1994 Georg Thieme
Verlag); each of which is hereby incorporated by reference.
[0139] In the synthetic schemes provided herein, the use of
protecting groups is indicated in a structure by the letter P,
where P for any amine, aldehyde, carboxylic acid, sulfhydryl, or
alcohol may be any of the protecting groups listed above.
Compounds of Formula I
[0140] Compounds of the invention include compounds of formula
(I).
##STR00017##
In formula (I) each of R.sup.1A, R.sup.1B, R.sup.1C, R.sup.1D,
R.sup.1E, and R.sup.1F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.1G, OC(O)R.sup.1H, NR.sup.1IR.sup.1J, NHC(O)R.sup.1K,
NHC(S)R.sup.1L, NHC(O)OR.sup.1M, NHC(S)OR.sup.1N, NHC(O)NHR.sup.1O,
NHC(S)NHR.sup.1P, NHC(O)SR.sup.1Q, NHC(S)SR.sup.1R,
NHS(O).sub.2R.sup.1S, C(O)OR.sup.1T, and C(O)NHR.sup.1U; and each
of R.sup.1G, R.sup.1H, R.sup.1I, R.sup.1J, R.sup.1K, R.sup.1L,
R.sup.1M, R.sup.1N, R.sup.1O, R.sup.1P, R.sup.1Q, R.sup.1R,
R.sup.1S, R.sup.1T, and R.sup.1U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Compounds of formula
(I) include compounds A1-A9 (see FIG. 1).
[0141] Compounds of formula (I) can by prepared by hydrazinolysis
of an activated benzoic acid derivative with hydrazine hydrate,
followed by condensation with a benzaldehyde derivative to give the
desired hydrazide (see Scheme 1).
##STR00018##
Compounds of Formula II
[0142] Compounds of the invention include compounds of formula
(II).
##STR00019##
In formula (II) each of R.sup.2A, R.sup.2B, R.sup.2C, R.sup.2D,
R.sup.2E, and R.sup.2F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.2G, OC(O)R.sup.2H, NR.sup.2IR.sup.2J, NHC(O)R.sup.2K,
NHC(S)R.sup.2L, NHC(O)OR.sup.2M, NHC(S)OR.sup.2N, NHC(O)NHR.sup.2O,
NHC(S)NHR.sup.2P, NHC(O)SR.sup.2Q, NHC(S)SR.sup.2R,
NHS(O).sub.2R.sup.2S, C(O)OR.sup.2T, and C(O)NHR.sup.2U; X.sup.2 is
independently selected from OR.sup.2G, OC(O)R.sup.2H,
NR.sup.2IR.sup.2J, NHC(O)R.sup.2K, NHC(S)R.sup.2L, NHC(O)OR.sup.2M,
NHC(S)OR.sup.2N, NHC(O)NHR.sup.2O, NHC(S)NHR.sup.2P,
NHC(O)SR.sup.2Q, NHC(S)SR.sup.2R, and NHS(O).sub.2R.sup.2S; and
each of R.sup.2G, R.sup.2H, R.sup.2I, R.sup.2J, R.sup.2K, R.sup.2L,
R.sup.2M, R.sup.2N, R.sup.2O, R.sup.2P, R.sup.2Q, R.sup.2R,
R.sup.2S, R.sup.2T, and R.sup.2U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Compounds of formula
(II) include compounds B and B2 (see FIG. 2).
[0143] Compounds of formula (II) can be prepared by condensation of
a phenylglyoxylic acid derivative with a phenylhydrazine derivative
to form the desired hydrazone (see Scheme 2).
##STR00020##
Phenylglyoxylic acid derivatives can be prepared by oxidation of
the corresponding mandelic acid derivative or styrene derivative
(see, for example, Hurd et al., J. Am. Chem. Soc. 61:2979
(1939)).
Compounds of Formula III
[0144] Compounds of the invention include compounds of formula
(III).
##STR00021##
In formula (II) each of R.sup.3A, R.sup.3B, and R.sup.3C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.3G, OC(O)R.sup.3H,
NR.sup.3IR.sup.3J, NHC(O)R.sup.3K, NHC(S)R.sup.3L, NHC(O)OR.sup.3M,
NHC(S)OR.sup.3N, NHC(O)NHR.sup.3O, NHC(S)NHR.sup.3P,
NHC(O)SR.sup.3Q, NHC(S)SR.sup.3R, NHS(O).sub.2R.sup.3S,
C(O)OR.sup.3T, and C(O)NHR.sup.3U; X.sup.3 is independently
selected from OR.sup.3G, OC(O)R.sup.3H, NR.sup.3IR.sup.3J,
NHC(O)R.sup.3K, NHC(S)R.sup.3L, NHC(O)OR.sup.3M, NHC(S)OR.sup.3N,
NHC(O)NHR.sup.3O, NHC(S)NHR.sup.3P, NHC(O)SR.sup.3Q,
NHC(S)SR.sup.3R, and NHS(O).sub.2R.sup.3S; and each of R.sup.3G,
R.sup.3H, R.sup.3I, R.sup.3J, R.sup.3K, R.sup.3L, R.sup.3M,
R.sup.3N, R.sup.3O, R.sup.3P, R.sup.3Q, R.sup.3R, R.sup.3S,
R.sup.3T, and R.sup.3U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Formula (III) includes
compound C1-C7 (see FIG. 3).
[0145] Compounds of formula III can be synthesized using methods
analogous to those described by Kopiichuk I. I., Med. Inst., Lvov,
Farmatsevtichnii Zhurnal (Kiev) 21:7-10 (1966). First,
3-(1-Carboxy-2-methylpropyl)rhodanine can be prepared by mixing 0.3
mole valine in 1 portion of KOH solution (3 moles in 80 ml
H.sub.2O) with 0.3 mole CS.sub.2 in the same amount of KOH
solution. After 3 hours of mixing, 0.3 mole ClCH.sub.2CO.sub.2H
neutralized by K.sub.2CO.sub.3 is added to the mixture and the
reaction is stirred for 20-30 minutes, followed by neutralization
with concentration HCl. The reaction is heated at 900 for 20-30
minutes and the 3-(1-Carboxy-2-methylpropyl)rhodanine can be
isolated as a crystalline solid. Subsequent condensation with
aromatic aldehydes can yield compounds of formula (III).
Compounds of Formula IV
[0146] Compounds of the invention include compounds of formula
(IV).
##STR00022##
In formula (IV) each of R.sup.4A, R.sup.4B, R.sup.4C, R.sup.4D,
R.sup.4E, and R.sup.4F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.4G, OC(O)R.sup.4H, NR.sup.4IR.sup.4J, NHC(O)R.sup.4K,
NHC(S)R.sup.4L, NHC(O)OR.sup.4M, NHC(S)OR.sup.4N, NHC(O)NHR.sup.4O,
NHC(S)NHR.sup.4P, NHC(O)SR.sup.4Q, NHC(S)SR.sup.4R,
NHS(O).sub.2R.sup.4S, C(O)OR.sup.4T, and C(O)NHR.sup.4U; X.sup.4 is
--S(O)-- or --S(O).sub.2--; and each of R.sup.4G, R.sup.4H,
R.sup.4I, R.sup.4J, R.sup.4K, R.sup.4L, R.sup.4M, R.sup.4N,
R.sup.4O, R.sup.4P, R.sup.4Q, R.sup.4R, R.sup.4S, R.sup.4T, and
R.sup.4U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl,
and C.sub.1-4 heteroalkyl. Compounds of formula (IV) include
compounds D1-D4 (see FIG. 4).
[0147] Compounds of formula (IV) can be synthesized using existing
methodology. For example, a simple, unsymmetrical diaryl sulfones
can be prepared from aryl boronic acids and arylsulfonyl chlorides
(see Bandgar et al., Org. Lett., 6:2105-2108 (2004)) and from
sulfinic acid salts and aryl halides or triflates (see Cacchi et
al., J. Org. Chem. 69:5608-5614 (2004)). These approaches are
depicted in Schemes 4a and 4b, respectively.
##STR00023##
##STR00024##
[0148] Sulfoxides of formula (IV) can be synthesized from their
corresponding sulfide by hydrogen peroxide monooxidation (see, for
example, Matteucci et al., Org. Lett., 5:235-237 (2003) and Scheme
4c, below).
##STR00025##
Compounds of Formula V
[0149] Compounds of the invention include compounds of formula
(V).
##STR00026##
wherein Ar is described by any of formulas (Va), (Vb), or (Vc).
##STR00027##
In formula (V), each of R.sup.5A, R.sup.5B, R.sup.5C, R.sup.5D,
R.sup.5E, and R.sup.5F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.5G, OC(O)R.sup.5H, NR.sup.5IR.sup.5J, NHC(O)R.sup.5K,
NHC(S)R.sup.5L, NHC(O)OR.sup.5M, NHC(S)OR.sup.5N, NHC(O)NHR.sup.5O,
NHC(S)NHR.sup.5P, NHC(O)SR.sup.5Q, NHC(S)SR.sup.5R,
NHS(O).sub.2R.sup.5S, C(O)OR.sup.5T, and C(O)NHR.sup.5U; each of
X.sup.5A and X.sup.5B is, independently, selected from O and S;
X.sup.5C is selected from C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl,
C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, and C.sub.1-4
heteroalkyl; and each of R.sup.5G, R.sup.5H, R.sup.5I, R.sup.5J,
R.sup.5K, R.sup.5L, R.sup.5M, R.sup.5N, R.sup.5O, R.sup.5P,
R.sup.5Q, R.sup.5R, R.sup.5S, R.sup.5T, and R.sup.5U is,
independently, selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl,
C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl, and C.sub.1-4
heteroalkyl. Compounds of formula (V) include compounds E1-E25 (see
FIGS. 5A and 5B) and compound E30 (see FIG. 15).
[0150] Compounds of formula (V) can be synthesized using the
approach outlined in Scheme 5 for compound E1. Substituted
arylureas and arylthioureas can be employed (e.g., to produce
compounds described by formulas Va-Vc and to produce compounds in
which X.sup.5A is O or S). The synthesis can be completed by amide
coupling with any desired benzoic acid derivative.
##STR00028##
Compounds of Formula VI
[0151] Compounds of the invention include compounds of formula
(VI).
##STR00029##
In formula (VI), each of R.sup.6A, R.sup.6B, R.sup.6C, R.sup.6D,
R.sup.6E, and R.sup.6F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.6G, OC(O)R.sup.6H, NR.sup.6IR.sup.6, NHC(O)R.sup.6K,
NHC(S)R.sup.6L, NHC(O)OR.sup.6M, NHC(S)OR.sup.6N, NHC(O)NHR.sup.6O,
NHC(S)NHR.sup.6P, NHC(O)SR.sup.6Q, NHC(S)SR.sup.6R,
NHS(O).sub.2R.sup.6S, C(O)OR.sup.6T, and C(O)NHR.sup.6U; each of
X.sup.6A and X.sup.6B is, independently, selected from O and S; and
each of R.sup.6G, R.sup.6H, R.sup.6I, R.sup.6J, R.sup.6K, R.sup.6L,
R.sup.6M, R.sup.6N, R.sup.6O, R.sup.6P, R.sup.6Q, R.sup.6R,
R.sup.6S, R.sup.6T, and R.sup.6U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Compounds of formula
(VI) include compounds F1-F7 (see FIG. 6).
[0152] Compounds of formula (VI) can be synthesized using the
approach outlined in Scheme 5 for compound F1.
##STR00030##
Compounds of Formula VII
[0153] Compounds of the invention include compounds of formula
(VII).
##STR00031##
In formula (VII), each of R.sup.7A, R.sup.7B, R.sup.7C, R.sup.7D,
R.sup.7E, and R.sup.7F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.7G, OC(O)R.sup.7H, NR.sup.7IR.sup.7J, NHC(O)R.sup.7K,
NHC(S)R.sup.7L, NHC(O)OR.sup.7M, NHC(S)OR.sup.7N, NHC(O)NHR.sup.7O,
NHC(S)NHR.sup.7P, NHC(O)SR.sup.7Q, NHC(S)SR.sup.7R,
NHS(O).sub.2R.sup.7S, C(O)OR.sup.7T, and C(O)NHR.sup.7U; X.sup.7 is
independently selected from OR.sup.7G, OC(O)R.sup.7H,
NR.sup.7IR.sup.7J, NHC(O)R.sup.7K, NHC(S)R.sup.7L, NHC(O)OR.sup.7M,
NHC(S)OR.sup.7N, NHC(O)NHR.sup.7O, NHC(S)NHR.sup.7P,
NHC(O)SR.sup.7Q, NHC(S)SR.sup.7R, and NHS(O).sub.2R.sup.7S; and
each of R.sup.7G, R.sup.7H, R.sup.7I, R.sup.7J, R.sup.7K, R.sup.7L,
R.sup.7M, R.sup.7T, R.sup.7O, R.sup.7P, R.sup.7Q, R.sup.7R,
R.sup.7S, R.sup.7T, and R.sup.7U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Compounds of formula
(VII) include compounds G1 and G2 (see FIG. 7).
[0154] Compounds of formula (VII) can be synthesized using the
approach outlined in Scheme 7 for compound G1.
##STR00032##
Compounds of Formula VIII
[0155] Compounds of the invention include compounds of formula
(VIII).
##STR00033##
In formula (VIII), each of R.sup.8A, R.sup.8B, and R.sup.8C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.8G, OC(O)R.sup.8H,
NR.sup.8IR.sup.8J, NHC(O)R.sup.8K, NHC(S)R.sup.8L, NHC(O)OR.sup.8M,
NHC(S)OR.sup.8N, NHC(O)NHR.sup.8O, NHC(S)NHR.sup.8P,
NHC(O)SR.sup.8Q, NHC(S)SR.sup.8R, NHS(O).sub.2R.sup.8S,
C(O)OR.sup.8T, and C(O)NHR.sup.8U; and each of R.sup.8G, R.sup.8H,
R.sup.8I, R.sup.8J, R.sup.8K, R.sup.8L, R.sup.8M, R.sup.8N,
R.sup.8O, R.sup.8P, R.sup.8Q, R.sup.8R, R.sup.8S, R.sup.8T, and
R.sup.8U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl,
and C.sub.1-4 heteroalkyl. Compounds of formula (VIII) include
compounds H1-H8 (see FIG. 8).
[0156] Compounds of formula (VIII) can by prepared by condensation
of 5-amino-2-benzimidazolinone (aka 5-Amino-2-hydroxybenzimidazole,
CAS 95-23-8; Pfaltz&Bauer Cat. No. A17950) with a benzaldehyde
derivative (see Scheme 8a). Compound H1 can be prepared as
described in Scheme 8b.
##STR00034##
##STR00035##
Compounds of Formula IX
[0157] Compounds of the invention include compounds of formula
(IX).
##STR00036##
In formula (IX), each of R.sup.9A, R.sup.9B, R.sup.9C, R.sup.9D,
R.sup.9E, and R.sup.9F is, independently, selected from H, halide,
nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl,
OR.sup.9G, OC(O)R.sup.9H, NR.sup.9IR.sup.9J, NHC(O)R.sup.9K,
NHC(S)R.sup.9L, NHC(O)OR.sup.9M, NHC(S)OR.sup.9N, NHC(O)NHR.sup.9O,
NHC(S)NHR.sup.9P, NHC(O)SR.sup.9Q, NHC(S)SR.sup.9R,
NHS(O).sub.2R.sup.9S, C(O)OR.sup.9T, and C(O)NHR.sup.9U; X.sup.9 is
independently selected from OR.sup.9G, OC(O)R.sup.9H,
NR.sup.9IR.sup.9J, NHC(O)R.sup.9K, NHC(S)R.sup.9L, NHC(O)OR.sup.9M,
NHC(S)OR.sup.9N, NHC(O)NHR.sup.9O, NHC(S)NHR.sup.9P,
NHC(O)SR.sup.9Q, NHC(S)SR.sup.9R, and NHS(O).sub.2R.sup.9S; Ar is
selected from C.sub.2-6 heterocyclyl and C.sub.6-12 aryl; and each
of R.sup.9G, R.sup.9H, R.sup.9I, R.sup.9J, R.sup.9K, R.sup.9L,
R.sup.9M, R.sup.9N, R.sup.9O, R.sup.9P, R.sup.9Q, R.sup.9R,
R.sup.9S, R.sup.9T, and R.sup.9U is, independently, selected from
H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Compounds of formula
(IX) include compounds I1 and I2 (see FIG. 9).
[0158] Compounds of formula (IX) can be prepared by boiling
2-Hydroxy-1,4-naphthoquinone (Aldrich Cat. No. H46805) in sulfuric
acid with alcohols of the formula Aryl-CH(OH)-Aryl' as described by
Zalukaev et al., Voronezh. Gos. Univ., Voronezh, USSR. Izvestiya
Vysshikh Uchebnykh Zavedenii, Khimiya i Khimicheskaya Tekhnologiya
16:1599-600 (1973).
Compounds of Formula X
[0159] Compounds of the invention include compounds of formula
(X).
##STR00037##
In formula (X), each of R.sup.10A, R.sup.10B, R.sup.10C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.10G, OC(O)R.sup.10H,
NR.sup.10IR.sup.10J, NHC(O)R.sup.10K, NHC(S)R.sup.10L,
NHC(O)OR.sup.10M, NHC(S)OR.sup.10N, NHC(O)NHR.sup.10O,
NHC(S)NHR.sup.10P, NHC(O)SR.sup.10Q, NHC(S)SR.sup.10R,
NHS(O).sub.2R.sup.10S, C(O)OR.sup.10T, and C(O)NHR.sup.10U;
X.sup.10 is independently selected from OR.sup.10G, OC(O)R.sup.10H,
NR.sup.10IR.sup.10J, NHC(O)R.sup.10K, NHC(S)R.sup.10L,
NHC(O)OR.sup.10M, NHC(S)OR.sup.10N, NHC(O)NHR.sup.10O,
NHC(S)NHR.sup.10P, NHC(O)SR.sup.10Q, NHC(S)SR.sup.10R, and
NHS(O).sub.2R.sup.10S; and each of R.sup.10G, R.sup.10H, R.sup.10I,
R.sup.10J, R.sup.10K, R.sup.10L, R.sup.10M, R.sup.10N, R.sup.10O,
R.sup.10P, R.sup.10Q, R.sup.10R, R.sup.10S, R.sup.10T, and
R.sup.10U is, independently, selected from H, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6 heterocyclyl,
C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10 alkheterocyclyl,
and C.sub.1-4 heteroalkyl. Compounds of formula (X) include
compounds J1-J6 (see FIG. 10).
[0160] Compounds of formula (X) can be synthesized using the
approach outlined in Scheme 10 for compound J1.
##STR00038##
Compounds of Formula XI
[0161] Compounds of the invention include compounds of formula
(XI).
##STR00039##
In formula (XI), each of R.sup.11A, R.sup.11B, R.sup.11C,
R.sup.11D, R.sup.11E, and R.sup.11F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.11G, OC(O)R.sup.11H, NR.sup.11IR.sup.11J,
NHC(O).sup.11K, NHC(S)R.sup.11L, NHC(O)OR.sup.11M,
NHC(S)OR.sup.11N, NHC(O)NHR.sup.11O, NHC(S)NHR.sup.11P,
NHC(O)SR.sup.11Q, NHC(S)SR.sup.11R, NHS(O).sub.2R.sup.11S,
C(O)OR.sup.11T, and C(O)NHR.sup.11U; and each of R.sup.11G,
R.sup.11H, R.sup.11I, R.sup.11J, R.sup.11K, R.sup.11L, R.sup.11M,
R.sup.11N, R.sup.11O, R.sup.11P, R.sup.11Q, R.sup.11R, R.sup.11S,
R.sup.11T, and R.sup.11U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Formula (XI) includes
compound K1 (see FIG. 15).
[0162] Compounds of formula (XI) can be synthesized using the
approach outlined in Scheme 11 for compound K1.
##STR00040##
Compounds of Formula XII
[0163] Compounds of the invention include compounds of formula
(XII).
##STR00041##
In formula (XII), each of R.sup.12A, R.sup.12B, R.sup.12C,
R.sup.12D, R.sup.12E, and R.sup.12F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.12G, OC(O)R.sup.12H, NR.sup.12IR.sup.12J,
NHC(O)R.sup.12K, NHC(S)R.sup.12L, NHC(O)OR.sup.12M,
NHC(S)OR.sup.12N, NHC(O)NHR.sup.12O, NHC(S)NHR.sup.12P,
NHC(O)SR.sup.12Q, NHC(S)SR.sup.12R, NHS(O).sub.2R.sup.12S,
C(O)OR.sup.12T, and C(O)NHR.sup.12U; and each of R.sup.12G,
R.sup.12H, R.sup.12I, R.sup.12J, R.sup.12K, R.sup.12L, R.sup.12M,
R.sup.12N, R.sup.12O, R.sup.12P, R.sup.12Q, R.sup.12R, R.sup.12S,
R.sup.12T, and R.sup.12U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Formula (XII) includes
compound L1 (see FIG. 15).
[0164] Compounds of formula (XII) can by prepared by the sequential
alkylation of piperazine (see Scheme 12a below, X is a leaving
group (e.g., a halide) and P is a protecting group). Compound L1
can be synthesized as described in Scheme 12b.
##STR00042##
##STR00043##
[0165] Alternatively, piperazines of formula (XII) can be prepared
by condensation of piperazine, or a derivative thereof, with the
desired biphenyl aldehyde, followed by reduction of the resulting
imine as described by Forsee et al., J. Am. Chem. Soc. 57:1788
(1935).
Compounds of Formula XIII
[0166] Compounds of the invention include compounds of formula
(XIII).
##STR00044##
In formula (XIII), each of R.sup.13A, R.sup.13B, and R.sup.13C is,
independently, selected from H, halide, nitro, C.sub.1-4 alkyl,
C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, OR.sup.13G, OC(O)R.sup.13H,
NR.sup.13IR.sup.13J, NHC(O)R.sup.13K, NHC(S)R.sup.13L,
NHC(O)OR.sup.13M, NHC(S)OR.sup.13N, NHC(O)NHR.sup.15O,
NHC(S)NHR.sup.13P, NHC(O)SR.sup.13Q, NHC(S)SR.sup.13R,
NHS(O).sub.2R.sup.13S, C(O)OR.sup.13T, and C(O)NHR.sup.13U; and
each of R.sup.13G, R.sup.13H, R.sup.13I, R.sup.13J, R.sup.13K,
R.sup.13L, R.sup.13M, R.sup.13N, R.sup.13O, R.sup.13P, R.sup.13Q,
R.sup.13R, R.sup.13S, R.sup.13T, and R.sup.13U is, independently,
selected from H, C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4
alkynyl, C.sub.2-6 heterocyclyl, C.sub.6-12 aryl, C.sub.7-14
alkaryl, C.sub.3-10 alkheterocyclyl, and C.sub.1-4 heteroalkyl.
Formula (XIII) includes compound M1 (see FIG. 15).
[0167] Compounds of formula (XIII) can by prepared by activation of
3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (CAS
55701-05-8) and reaction with 3-amino-1-propanol (Aldrich Cat. No.
23,984-4), followed by reaction with the appropriately substituted
phenyl isocyanate to form the carbamate of formula (XII) (see
Scheme 13a). Compound M1 can be prepared as described in Scheme
13b.
##STR00045##
##STR00046##
Compounds of Formula XIV
[0168] Compounds of the invention include compounds of formula
(XIV).
##STR00047##
In formula (XIV), each of R.sup.14A, R.sup.14B, R.sup.14C,
R.sup.14D, R.sup.14E, and R.sup.14F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.14G, OC(O)R.sup.14H, NR.sup.14IR.sup.14J,
NHC(O)R.sup.14K, NHC(S)R.sup.14L, NHC(O)OR.sup.14M,
NHC(S)OR.sup.14N, NHC(O)NHR.sup.14O, NHC(S)NHR.sup.14P,
NHC(O)SR.sup.14Q, NHC(S)SR.sup.14R, NHS(O).sub.2R.sup.14S,
C(O)OR.sup.14T, and C(O)NHR.sup.14U; and each of R.sup.14G,
R.sup.14H, R.sup.14I, R.sup.14J, R.sup.14K, R.sup.14L, R.sup.14M,
R.sup.14N, R.sup.14O, R.sup.14P, R.sup.14Q, R.sup.14R, R.sup.14S,
R.sup.14T, and R.sup.14U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Formula (XIV) includes
compound N1 (see FIG. 15).
[0169] Compounds of formula (XIV) can by prepared, for example, by
coupling an aryl iodide derivative with carboxy-protected lactic
acid. Hydrazinolysis of the resulting product, followed by
condensation with a benzaldehyde derivative can yield the desired
hydrazide (see Scheme 14a).
##STR00048##
Conditions for coupling aryl iodides and alcohols are described by
Manbeck et al., J. Org. Chem. 70:244 (2005) and Wolter et al., Org.
Lett. 4:973 (2002), schemes 14b and 14c, respectively.
##STR00049##
##STR00050##
Compound N1 can be prepared as described in Scheme 14d.
##STR00051##
Compounds of Formula XV
[0170] Compounds of the invention include compounds of formula
(XV).
##STR00052##
In formula (XV), each of R.sup.15A, R.sup.15B, R.sup.15C,
R.sup.15D, R.sup.15E, and R.sup.15F is, independently, selected
from H, halide, nitro, C.sub.1-4 alkyl, C.sub.2-4 alkenyl,
C.sub.2-4 alkynyl, OR.sup.15G, OC(O)R.sup.15H, NR.sup.15IR.sup.15J,
NHC(O)R.sup.15K, NHC(S)R.sup.15L, NHC(O)OR.sup.15M,
NHC(S)OR.sup.15N, NHC(O)NHR.sup.15O, NHC(S)NHR.sup.15P,
NHC(O)SR.sup.15Q, NHC(S)SR.sup.15R, NHS(O).sub.2R.sup.15S,
C(O)OR.sup.15T, and C(O)NHR.sup.15U; and each of R.sup.15G,
R.sup.15H, R.sup.15I, R.sup.15J, R.sup.15K, R.sup.15L, R.sup.15M,
R.sup.15N, R.sup.15O, R.sup.15P, R.sup.15Q, R.sup.15R, R.sup.15S,
R.sup.15T, and R.sup.15U is, independently, selected from H,
C.sub.1-4 alkyl, C.sub.2-4 alkenyl, C.sub.2-4 alkynyl, C.sub.2-6
heterocyclyl, C.sub.6-12 aryl, C.sub.7-14 alkaryl, C.sub.3-10
alkheterocyclyl, and C.sub.1-4 heteroalkyl. Formula (XV) includes
compound O1 (see FIG. 15).
[0171] Compounds of formula (XV) can by prepared, for example, by
sequential condensation of morpholine, a benzyl amine derivative,
and hydrazine with cyanuric acid (Aldrich Cat. No. C9,550-1).
Subsequent condensation with a benzaldehyde derivative can yield
the desired hydrazone (see Scheme 15a). Compound O1 can be prepared
as described in Scheme 15b.
##STR00053##
##STR00054##
Biological Activity
[0172] Our screen of a synthetic compound library for substances
that cure C. elegans of a persistent E. faecalis infection suggests
that in contrast to a traditional antibiotic screen, the E.
faecalis assay not only identifies compounds that block pathogen
replication in vitro but also identifies compounds that may be
prodrugs, that affect the virulence of the pathogen, that suppress
pathogen survival, or that enhance the immune response of the host.
Because some of the identified compounds only have significant
activity in vivo in the whole-animal assay, this provides
proof-of-principle for using a whole-animal screen in a drug
discovery program to identify novel antimicrobial, antifungal, and
antiviral compounds.
[0173] One of the most interesting features of many of the
identified compounds is their unusual ability to promote nematode
survival at concentrations that were much lower than their MIC
value in vitro. In contrast, the effective dose in the C. elegans
curing assay of all of the known antibiotics that we tested was
several fold higher than the in vitro MIC. The effective dosage in
the nematode model is similar to the therapeutic concentration of
most antibiotics in human blood serum, which is typically 5-20 fold
higher than the MIC (Schulz et al., Pharmazie 58:447-474 (2003)). A
plausible explanation for this observation is that the C. elegans
curing assay specifically identifies compounds that target
functions only important for in vivo survival or virulence, or
activators of innate immunity and that compounds with these
activities may be more common than traditional antibiotics.
[0174] Compounds that inhibit E. faecalis virulence could
potentially target expression or function of cytolysin, serine
protease, gelatinase; the quorum sensing pathway, or colonization
of the bacteria in the worm gut. A possible mode of action for
these compounds is the inhibition of bacterial colonization, but
this activity is difficult to distinguish from antibiotic activity,
which also reduces bacterial colonization.
[0175] Compounds that function as immune enhancers may activate the
C. elegans immune pathway downstream of a conserved p38 MAPK
cascade. A cascade consisting of the C. elegans PMK-1, SEK-1, and
NSY-1 proteins, corresponding to the p38 MAP Kinase (MAPK), and
upstream MAPKK and MAPKKK, is required for the response to a
variety of bacterial and fungal pathogens and loss of any of these
signaling components results in nematodes that have enhanced
susceptibility to the pathogens (Kim et al., Science 297:623-626
(2002)). The C. elegans-E. faecalis curing assay utilized a sek-1
mutant worm strain that dies more quickly when exposed to E.
faecalis and a potential activity of a hit would be an activator of
the downstream p38 MAP kinase. Restoring the activity of the p38
MAP kinase in the sek-1; glp-4 strain that we used would be
detectable in the killing assay since this results in the doubling
of the survival time from the E. faecalis infection, as illustrated
by comparing the killing kinetics of the glp-4; sek-1 and glp-4
mutants.
[0176] Natural or synthetic compounds that block the virulence of
pathogens ("virulence anti-infectives") are a largely unexplored
class of antimicrobial, antifungal, and antiviral agents. The
successful targeting of a virulence product is demonstrated in a
recent paper by Hung et al. (Science 310:670-674 (2005)), in which
a high throughput screen was used to identify compounds that
inhibit the activity of the Vibrio cholerae transcriptional
regulator ToxT that is required for expression of cholera toxin and
the toxin coregulated pilus. A small molecule identified in this
screen, 4-[N-(1,8-naphthalimide)]-n-butyric acid, protected infant
mice from intestinal colonization by V. cholerae. Although cholera
is effectively treated with a simple fluid-replacement regime,
targeting a key toxin or particularly potent virulence factor may
be an effective therapeutic in the case of other pathogens.
Therapy
[0177] The invention features compositions and methods for treating
or preventing a disease or condition in an animal or a plant
associated with a microbial or viral infection by administering a
compound of formula I-XV.
[0178] Compounds of the present invention may be administered by
any appropriate route for treatment or prevention of a disease or
condition associated with a microbial or viral infection. These may
be administered to humans, domestic pets, livestock, or other
animals with a pharmaceutically acceptable diluent, carrier, or
excipient, in unit dosage form. Administration may be topical,
parenteral, intravenous, intra-arterial, subcutaneous,
intramuscular, intracranial, intraorbital, ophthalmic,
intraventricular, intracapsular, intraspinal, intracisternal,
intraperitoneal, intranasal, aerosol, by suppositories, or oral
administration.
[0179] Therapeutic formulations may be in the form of liquid
solutions or suspensions; for oral administration, formulations may
be in the form of tablets or capsules; and for intranasal
formulations, in the form of powders, nasal drops, ear drops, or
aerosols.
[0180] Methods well known in the art for making formulations are
found, for example, in "Remington: The Science and Practice of
Pharmacy" (20th ed., ed. A. R. Gennaro, 2000, Lippincott Williams
& Wilkins). Formulations for parenteral administration may, for
example, contain excipients, sterile water, or saline, polyalkylene
glycols such as polyethylene glycol, oils of vegetable origin, or
hydrogenated napthalenes. Biocompatible, biodegradable lactide
polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Nanoparticulate formulations (e.g.,
biodegradable nanoparticles, solid lipid nanoparticles, liposomes)
may be used to control the biodistribution of the compounds. Other
potentially useful parenteral delivery systems include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for
inhalation may contain excipients, for example, lactose, or may be
aqueous solutions containing, for example, polyoxyethylene-9-lauryl
ether, glycholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel. The
concentration of the compound in the formulation will vary
depending upon a number of factors, including the dosage of the
drug to be administered, and the route of administration.
[0181] The compound may be optionally administered as a
pharmaceutically acceptable salt, such as a non-toxic acid addition
salts or metal complexes that are commonly used in the
pharmaceutical industry. Examples of acid addition salts include
organic acids such as acetic, lactic, pamoic, maleic, citric,
malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic,
tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic
acids or the like; polymeric acids such as tannic acid,
carboxymethyl cellulose, or the like; and inorganic acid such as
hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid,
or the like. Metal complexes include zinc, iron, and the like.
[0182] Administration of compounds in controlled release
formulations is useful where the compound of formula I has (i) a
narrow therapeutic index (e.g., the difference between the plasma
concentration leading to harmful side effects or toxic reactions
and the plasma concentration leading to a therapeutic effect is
small; generally, the therapeutic index, TI, is defined as the
ratio of median lethal dose (LD.sub.50) to median effective dose
(ED.sub.50)); (ii) a narrow absorption window in the
gastro-intestinal tract; or (iii) a short biological half-life, so
that frequent dosing during a day is required in order to sustain
the plasma level at a therapeutic level.
[0183] Many strategies can be pursued to obtain controlled release
in which the rate of release outweighs the rate of metabolism of
the therapeutic compound. For example, controlled release can be
obtained by the appropriate selection of formulation parameters and
ingredients, including, e.g., appropriate controlled release
compositions and coatings. Examples include single or multiple unit
tablet or capsule compositions, oil solutions, suspensions,
emulsions, microcapsules, microspheres, nanoparticles, patches, and
liposomes.
[0184] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose and sorbitol), lubricating
agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc
stearate, stearic acid, silicas, hydrogenated vegetable oils, or
talc).
[0185] Formulations for oral use may also be provided in unit
dosage form as chewable tablets, tablets, caplets, or capsules
(i.e., as hard gelatin capsules wherein the active ingredient is
mixed with an inert solid diluent, or as soft gelatin capsules
wherein the active ingredient is mixed with water or an oil
medium).
[0186] The formulations can be administered to human subjects in
therapeutically effective amounts. Typical dose ranges are from
about 0.01 .mu.g/kg to about 2 mg/kg of body weight per day. The
preferred dosage of drug to be administered is likely to depend on
such variables as the type and extent of the disorder, the overall
health status of the particular subject, the specific compound
being administered, the excipients used to formulate the compound,
and its route of administration. Standard clinical trials maybe
used to optimize the dose and dosing frequency for any particular
compound.
[0187] For agricultural uses, the compounds described herein and
additional compounds identified using the methods disclosed herein
may be used as chemicals applied as sprays or dusts on the foliage
of plants to treat, for example, cankers, rots (e.g., stalk or root
rot), rusts, blights (e.g., potato blight), downey or powdery
mildew, scabs, smuts, leaf spot, black spot, vascular wilt disease,
crown gall, leaf curl, or hairy root. Typically, such agents are to
be administered on the surface of the plant in advance of the
pathogen in order to prevent infection. Seeds, bulbs, roots,
tubers, and corms are also treated to prevent pathogenic attack
after planting by controlling pathogens carried on them or existing
in the soil at the planting site. Soil to be planted with
vegetables, ornamentals, shrubs, or trees can also be treated with
chemical fumigants for control of a variety of microbial pathogens.
Treatment is preferably done several days or weeks before planting.
The chemicals can be applied by either a mechanized route, e.g., a
tractor or with hand applications. In addition, chemicals
identified using the methods of the assay can be used as
disinfectants.
[0188] The formulation of the compounds described herein for
agricultural chemical compositions may be an emulsifiable
concentrate, a wettable powder, granule, a dust formulation, a
suspension concentrate or plowable, as well as a liquid
formulation. Accordingly, other additives such as an emulsifier, a
dispersing agent, and a carrier may be optionally contained
depending on the formulation. Such agricultural chemical
compositions may be prepared using standard methods in the art.
[0189] The compounds described herein and additional compounds
identified using the methods of the invention may also be used to
increase growth rate or feed conversion in an animal (e.g., a cow,
pig, sheep, goat, duck, chicken, goose, or turkey). For example,
the compounds may be administered to the animal by means of an
injection, in the feed, or in the drinking water.
[0190] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the methods and compounds claimed herein are
performed, made, and evaluated, and are intended to be purely
exemplary of the invention and are not intended to limit the scope
of what the inventors regard as their invention.
EXAMPLES
Example 1
High-Throughput C. elegans Chemical Screens
[0191] We developed protocols to perform automated, high-throughput
(HT) whole-animal chemical screens. The described methods enable
quantitative analyses of a wide range of biological processes such
as the response to different types of biotic (pathogens) or abiotic
(heavy metals, ultraviolet radiation, heat) stresses that affect
viability, as well as traditional longevity studies. We optimized
methods to perform screens in liquid media. In particular, for 96-
and 384-well format, small perturbations such as salt
concentration, amount of food, and the number of animals has a
greater effect than in an assay using a larger format.
[0192] Exemplary assays described herein were designed to screen
for compounds (e.g., low molecular weight compounds) that prevent
the lethal effect of the infection of C. elegans by a pathogen such
as the bacterial pathogen Enterococcus faecalis. C. elegans were
first grown to the young adult stage and then infected on lawns of
Enterococcus faecalis. The infected worms were washed and
transferred to 384-well plates, each well containing liquid media
and the compound or compounds to be tested. The plates were
incubated until the infection killed untreated worms. The worms
were washed and stained with a fluorescent dye such as Nile Red or
MitoTracker or a fluorescent dye such as Sytox.RTM. orange that
specifically stains dead worms. Images of the wells were captured
with an automated microscope and analyzed to quantify worm
survival.
[0193] In particular, the below protocol allows the screening of
twenty 384-well plates per experiment. The rate limiting steps are
worm dispensing and imaging. Each of these steps takes
approximately 15 minutes per plate.
[0194] Amplify Worm Stocks
[0195] (1) Inoculate one colony of E. coli HB101 into a 2 Liter
flask containing 500 ml LB supplemented with 200 .mu.g/ml
streptomycin sulfate. Incubate for 16 hours at 37.degree. C. and
250 rpm. Centrifuge the saturated culture at 5,000.times.g for 10
minutes and resuspend in LB (Luria Broth; 10 g Bacto-tryptone, 5 g
yeast extract, 10 g NaCl, water to 1 liter, pH to 7.5 with NaOH) to
concentrate the bacteria 20 fold. Spread 100 .mu.l of the
concentrated bacteria onto 90 mm plates of NGM (3 g NaCl, 2.5 g
peptone, 17 g agar, 975 ml water, autoclaved, cooled to 55.degree.
C. and supplemented with sterile solutions of (i) 1 ml of 5 mg/ml
cholesterol dissolved in ethanol (ii) 1 ml 1M CaCl2, (iii) 1 ml 1M
MgSO.sub.4, and (iv) 25 ml 1M potassium phosphate pH 6.0 (108.3 g
KH.sub.2PO.sub.4, 35.6 g K.sub.2HPO.sub.4, water to 1 liter)), or
SK-NS agar (3 g NaCl, 3.5 g peptone, 20 g agar, 975 ml water,
autoclaved, cooled to 55.degree. C. and supplemented with sterile
solutions of (i) 1 ml of 5 mg/ml cholesterol dissolved in ethanol
(ii) 1 ml 1M CaCl2, (iii) 1 ml 1M MgSO.sub.4, (iv) 25 ml 1M
potassium phosphate pH 6.0, (v) 1 ml 100 mg/ml streptomycin
sulfate, and (vi) 1 ml 62.5 U/.mu.l nystatin). (Nystatin and
streptomycin are added to inhibit fungal and bacterial
contamination, respectively.) Incubate the plates at RT (room
temperature) for 1 day to produce the lawns of E. coli that will
serve as the food source for the worms. Plates can be stored at RT
for up to 1 week.
[0196] (2) Grow approximately 1,000 glp-4(bn2ts); sek-1(km4) L1
worms on each 90 mm plate of HB101 on NGM at 15.degree. C. for 5
days until the worms become gravid adults.
[0197] (i) Harvest the gravid adults by washing the plates with 10
ml of M9 buffer (6 g Na.sub.2HPO.sub.4, 3 g KH.sub.2PO.sub.4, 5 g
NaCl, 0.25 g MgSO.sub.4-7H.sub.20, water to 1 liter) and transfer
them into 15 ml conical tubes. Centrifuge the tubes at
1,500.times.g for 30 seconds to pellet the worms. Remove the
supernatant leaving behind 0.5 ml of liquid and the sedimented
worms. Immediately before use, prepare a solution consisting of 0.1
ml 5 M NaOH and 0.4 ml bleach, and add the 0.5 ml solution to the
tube. Continuously agitate and vortex the tube for 3-5 minutes,
until most of the adult worms have ruptured. Do not overexpose to
the bleach solution because this will result in damaged embryos. An
average often to fifteen progeny per glp-4(bn2ts); sek-1(km4) adult
can be obtained.
[0198] (ii) Wash the eggs 3 times with 14 ml M9 buffer.
[0199] (iii) Resuspend the eggs in 5 ml M9 buffer and let them
hatch at RT for 20 hours with gentle rocking. The hatched worms
arrest at the L1 larval stage in the M9 buffer. Seed approximately
4,000 L1s onto each HB101 on SK-NS plate and grow the worms at
25.degree. C. for approximately 54 hours until the worms become
young adults.
[0200] Prepare E. faecalis Lawns
[0201] To prepare lawns for the pathogenic E. faecalis bacteria as
the source of the infection, inoculate one colony of E. faecalis
strain MMH594 in BHI (Brain-Heart Infusion; BD, Sparks, Md.) liquid
media and incubate at 37.degree. C. for 6 hours. Spread 100 .mu.l
of the culture over the entire surface of 90 mm plates containing
BHI agar. Incubate the plates at 25.degree. C. overnight to grow
the lawns and then cool the plates at 15.degree. C.
[0202] Infect Adult Worms
[0203] (1) To each plate of sterile, adult worms, add 15 ml of M9
buffer and resuspend the worms by gently shaking the plate for 10
seconds. Transfer the worms into sterile 50 ml tubes by pouring.
Allow the worms to settle to the bottom of the tubes and remove the
supernatant. Wash worms twice with M9 buffer to remove the E.
coli.
[0204] (2) Using large orifice pipette tips, seed the worms onto
the lawns of E. faecalis MMH594 on BHI agar. Transfer up to 8,000
animals per plate. Incubate the plates for 15 hours at 15.degree.
C. to allow the infection to become established. The worms are
infected on lawns of pathogen for a period of time that allows
persistent intestinal colonization but at which symptoms of the
infection are not yet observed.
[0205] Seed Infected Worms in Screening Plates
[0206] (1) Using a multiplate dispenser, dispense 20 .mu.l of
1.75.times. media to each well of the 384-well plate. 1.75.times.
media consists of 35% BHI, 63.4% M9 buffer, 1.25% DMSO, 109 U/ml
nystatin, and 175 .mu.g/ml kanamycin sulfate.
[0207] (2) Pin transfer 100 nl of a 5 mg/ml compound stock
dissolved in DMSO into each well of a 384-well plate (final DMSO
concentration is 1% and compound concentration is 14 g/ml).
[0208] (3) Sterilize and prepare COPAS Biosort. In particular, the
COPAS Biosort is prepared by sterilizing the worm sorter tubing
system by running 200 ml of 10% bleach through the system. The
bleach is washed out by running 200 ml of sterile ddH.sub.20
through the system. The system is further sterilized by running 200
ml of 70% ethanol through the system and the ethanol is washed out
by running 200 ml of sterile ddH.sub.20 through the system. The
system is equilibrated with sample buffer, by running 50 ml of
S-buffer through the system.
[0209] Following the sterilization and preparation procedure, 200
ml of S-buffer and 2 ml of Sytox.RTM.-stained L1 suspension are
added to the sample cup. An acquisition cycle of 500 objects is
performed. For accurate dispensing of L1s, the flow rate should be
approximately 10 events/sec. The L1 suspension is diluted and added
accordingly. Based on size and intensity of the fluorescent signal,
the gate of interest is defined. A test is performed by dispensing
5 non-Sytox.RTM.-stained objects (live L1s) per well in the cover
of a 96-well plate. The accuracy is checked under the dissecting
scope.
[0210] (4) Resuspend the infected worms in M9 buffer. Dispense 15
young adult worms into each well of the 384-well plates (black
walled, clear bottom; Corning Cat. #3712, Lowell, Mass.) using the
COPAS Biosort (Harvard Bioscience, Holliston, Mass.). In
particular, the worm synchronization method described results in a
population of worms in which 95% are young adults. Based on time or
flight or length (TOF), the COPAS Biosort differentiates young
adults from younger animals. The remaining 5% of the worm
population corresponds to slower growing animals that are
discarded. The COPAS Biosort transfers each worm in a volume of
.about.1 .mu.l. The final volume of transferred worms per well is
35 .mu.l. Final concentrations are as follows: 20% BHI, 36% M9
buffer, 1% DMSO, 100 .mu.g/ml kanamycin sulfate, 62.5 U/ml
nystatin, and the remaining liquid consists of sheath fluid (worm
sorter specific fluid; Pulak, Methods Mol. Biol. 351:275-286, 2006)
and M9 buffer.
[0211] (5) Dry the top of the 384-well plates with laboratory
tissue to allow adhesion of membranes and seal the plates with gas
permeable membranes (Diversified Biotech #BEM-1, Boston,
Mass.).
[0212] (6) Incubate plates at 26.5.degree. C. with 85% relative
humidity for 7 days. The plates are placed in a single layer on top
of the shelves and incubated without agitation. Roughly 90% of the
untreated worms die from the infection during the 7-day incubation
period. In contrast, less than 15% of the worms die when treated
with antibiotics such as ampicillin or tetracycline. Humidity is
set at the maximum of the incubator capacity to reduce evaporation.
Alternatively, the microtiter plates could be placed into
containers that are lined with wet paper towels.
[0213] Prepare Samples to Score
[0214] (1) Resuspend the worms and bacteria by vortexing the plates
for 5 seconds at a high setting. Centrifuge the plates at
1,000.times.g for 10 seconds to remove the liquid from the
membranes. Remove the membrane seals. Using a plate washer,
dispense 65 .mu.l of M9 buffer per well using the maximum dispense
speed to facilitate washing (e.g., using a thermo multidrop combi
(Milford, Mass.) or equivalent liquid dispenser for 384-well
microtiter plates). Let the worms settle to the bottom of the wells
for at least 3 minutes. Remove three-fourths of the liquid from the
top of the plate using the aspirating head of the plate washer
(Bio-Tek Elx405, Winooski, Vt.; or equivalent plate washer with an
adjustable-height aspiration manifold). Wash the plates a total of
4 times. After the final wash, aspirate enough liquid to leave
.about.25 .mu.l/well. In addition to staining dead worms,
Sytox.RTM. Orange also stains the bacteria in the assay wells and
therefore the bacteria need to be removed to allow quantification
of the fraction of dead worms.
[0215] (2) Using the multiplate dispenser, dispense 50 .mu.l of 1
.mu.M Sytox.RTM. Orange (Invitrogen Cat. #S11368, Carlsbad, Calif.)
diluted in M9 buffer per well resulting in a stain concentration of
0.7 .mu.M.
[0216] (3) Seal the plates with gas permeable membranes and
incubate at 20.degree. C. for 20 hours at 85% relative humidity
(RH). Arrange the plates in a single layer on the incubator shelves
and incubate without agitation.
[0217] Image Capture
[0218] Wells are imaged using a Molecular Devices Discovery-1
automated microscope (Sunnyvale, Calif.) or equivalent. Fluorescent
TRITC (535 nm excitation, 610 nm emission) and transmitted light
images are captured. Hardware capture conditions are as described
in Table 1, except that the focal plane is set at the bottom of the
well (laser based scanning). The use of a 2.times. objective allows
capturing the entire area of a well in a 384 well plate.
[0219] Settings used to capture images of worms growing on
half-area 96-well agar media plates with a Discovery-1 automated
microscope are listed (Table 1). Images are taken in 3 wavelengths:
bright-field, GFP, and Nile Red.
TABLE-US-00001 TABLE 1 Parameter Setting Plate Reference Point (a)
14337.5 Reference Objective (b) 5 Parfocality Offset (c) 1445 Plate
Height (d) 14.2 Well Depth (e) 10500 Find 2nd Maximum (f) FALSE
Start z position (g) 19982.5 Full range (.mu.m) (h) 1000 Full max
step (.mu.m) (i) 590 Plate bottom exposure (j) 3 Wide range (.mu.m)
(k) 50 Wide max step (.mu.m) (l) 10 Accuracy (.mu.m) (m) 59
Magnification 2x Camera binning 2 Gain 2(4x) Transmitted light
exposure 10 ms Image based range (.mu.m) 500 Max. step (.mu.m) 100
Nile Red: filter set 572/630 exposure time 100 ms GFP: filter set
470/530 exposure time 250 ms (a) Reference point of flat sheet in
plate holder. Value is distance from application Z origin. (b)
Objective position used for setting reference point (c) Offset
distance between current objective to reference point objective (d)
Height defined for current plate (e) Depth of well for current
plate (f) TRUE = 2 peak search, FALSE = single peak search (g)
Start z position of search in units (h) Total range covered in
.mu.m (I) Incremental steps in .mu.m (j) Image exposure (ms) (k)
Search range at bottom of well in .mu.m (I) Incremental steps in
.mu.m (m) Accuracy to which focus will be found (.mu.m)
Image Analysis
[0220] Analysis is performed with CellProfiler software which is
publicly available at cellprofiler.org. An optimized pipeline to
quantify worm survival (1-(Sytox.RTM. Orange worm area/bright-field
worm area)=1-(dead worm area/total worm area)) is also available at
cellprofiler.org. An example of the quantification of worm survival
using the described methodology is shown in FIGS. 19 and 20.
Survival can be determined by measuring areas (number of pixels)
instead of counting worms. CellProfiler cannot distinguish
overlapping worms as independent objects. Also, the intensity of
fluorescent Sytox.RTM. orange staining in dead worms varied
greatly. This variation, in addition to the inhomogeneous nature of
the worm suspensions, also prevented the use of a fluorescent plate
reader.
Example 2
High-Throughput C. elegans Screen for Identifying Chemical
Compounds with Antimicrobial Activity
Materials and Methods
[0221] Bacterial and Nematode Strains.
[0222] Wild-type Bristol N2 (Brenner S., Genetics 77: 71-94 (1974))
and glp-4(bn2ts); sek-1(km4) (Beanan et al., Development
116:755-766 (1992); and Tanaka-Hino et al., EMBO Rep 3:56-62
(2002)) C. elegans strains were maintained using standard practices
(see Lewis et al., Basic Culture Methods. In: Epstein H F, Shakes D
C, editors. Caenorhabditis elegans Modern Biological Analysis of an
Organism. San Diego, Calif.: Academic Press. pp. 3-29 (1995)). E.
faecalis strains MMH594 (Huycke et al., Antimicrob. Agents
Chemother. 35:1626-1634 (1991)), OG1RF (Murray et al., J.
Bacteriol. 175:5216-5223 (1993)), OG1RF .DELTA.fsrB (Qin et al.,
Infect. Immun. 68:2579-2586 (2000)), V583 (Sahm et al., Antimicrob.
Agents Chemother. 33:1588-1591 (1989)), VS583 (Moy et al., Infect.
Immun. 72:4512-4520 (1989)) and E. faecium strains DO (Arduino et
al., Infect. Imnmun. 62:5587-5594 (1994)) and 11M12 (Moy et al.,
Infect. Immun. 72:4512-4520 (1989)) were grown on brain heart
infusion (BHI) media (Difco Becton Dickinson, Sparks, Md.) at
37.degree. C.
[0223] Nematode Killing and Rescue.
[0224] N2 or glp-4(bn2ts); sek-1(km4) worms were synchronized by
isolating eggs from gravid adults, hatching the eggs overnight in
M9 buffer, and plating L1-stage worms onto lawns of E. coli on NGM
agar media. Worms were grown to sterile, young adults by incubation
at 25.degree. C. for 48 to 52 hours, washed off the plates with M9
buffer, resuspended and washed in M9 buffer, deposited onto lawns
of E. faecalis grown on BHI agar plates containing kanamycin at 80
.mu.g/ml to inhibit E. coli growth, incubated for 8 to 12 hours at
25.degree. C., and resuspended in M9 buffer. For assays using agar
media, approximately 35 infected worms were washed and then
deposited onto 35 mm plates containing the appropriate antibiotics.
Plates were incubated at 25.degree. C. and scored for worm survival
at regular intervals. Worms were considered dead if they were
unresponsive to touch with a platinum wire pick. For the initial
assays using liquid media, approximately 80 infected worms were
transferred to wells of a 6 well plate containing 2 ml media
consisting of 10% BHI 80 .mu.g/ml kanamycin, 90% M9 buffer, and the
appropriate antibiotics. The plates were incubated without
agitation at 25.degree. C. and 80-85% relative humidity. To score
for worm survival, the 6 well plates were shaken by hand, and the
worms were considered to be dead if they did not move or exhibit
muscle tone.
[0225] Bacterial Colonization.
[0226] Infected worms were washed three times with M9 buffer
containing 1 mM sodium azide. Approximately 10 worms were
transferred to a 2 ml microcentrifuge tube and the volume was
brought to 250 .mu.l. Fifty .mu.l of buffer was removed and plated
to determine the number of external CFU. Approximately 400 mg of
1.0 mm silicon carbide particles (BioSpec Products, Catalog
#11079110sc) were added to each tube, the tubes were vortexed at
maximum speed for one minute, which disrupts the worms but does not
affect bacterial survival, and the resulting suspension was diluted
and plated onto selective media to determine CFU.
[0227] Screening for Anti-Infectives.
[0228] Synchronized L4 stage to young adult glp-4(bn2ts);
sek-1(km4) worms were infected for 8 hours on lawns of MMH594 as
described above. The worms were resuspended in media composed of
20% BHI, 80 .mu.g/ml kanamycin, and 80% M9 buffer. Approximately 25
worms in a volume of 50 .mu.l were transferred into 0.3 ml wells of
96 well plates. An equal volume of liquid media containing 125
units/ml nystatin and the compounds to be tested were mixed into
the wells. Each compound was tested in individual wells and the
screen was performed using duplicate 96 well plates. The final
concentration of compounds from ChemBridge Diverset E was 25
.mu.g/ml with dimethyl sulfoxide (DMSO) at 1%. The plates were
sealed with gas permeable membranes (Breatheasy, Diversified
Biotech, Boston Mass.) and incubated without agitation at
26.degree. C. and 80-85% relative humidity.
[0229] Each 96 well plate contained 80 test compounds that were
tested with worms infected with the cytolysin-positive E. faecalis
strain MMH594. The remaining 16 wells contained positive and
negative controls to determine whether the assay yielded
predictable and reproducible responses to antibiotics or avirulent
E. faecalis mutants and a clear threshold between positive and
negative responses. Each plate contained 8 negative control wells
that did not contain any antinfective compound and 4 positive
control wells that contained 20 .mu.g/ml ampicillin (2 wells) or 20
.mu.g/ml tetracycline (2 wells). These control wells were seeded
with glp-4; sek-1 infected with E. faecalis MMH594. In addition,
each plate contained 4 positive control wells that were seeded with
glp-4; sek-1 worms infected with the E. faecalis .DELTA.fsrB
mutant.
[0230] In total, there were 1,312 control wells in the ChemBridge
and NCI screens. The false positive rate was 2.7% and the false
negative rate was 1.9%. False positives were defined as more than
50% survival of infected worms in wells that did not contain an
antibiotic. False negatives were defined as less than 50% survival
of worms treated with an antibiotic or infected with the
.DELTA.fsrB mutant.
[0231] Worm survival was scored manually after 6 days of
incubation. A total of 6,000 compounds were tested from a
ChemBridge library Diverset E. Compounds that increased worm
survival by two- to three-fold were retested for activity. Ninety
ChemBridge compounds were retested in the worm infection model.
Eighteen of the compounds promoted worm survival upon retesting.
Additional quantities of the 18 ChemBridge hits were ordered from
ChemBridge, Inc. for further analysis and use in antibacterial
susceptibility testing. MICs were determined against E. faecalis
strain MMH594 using 2-fold dilution in BHI media according to the
broth microdilution protocol of the NCCLS (CLSI) (National
Committee for Clinical Laboratory Standards, Approved Standard
M7-A5; Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria that Grow Aerobically, Villanova, Pa.: NCCLS (2000)). The
compounds were also tested by their ability to hemolyze sheep
erythrocytes based on the protocol of Ciornei (Ciornei et al.,
Antimicrob. Agents Chemother. 49:2845-2850 (2005)) with the
following modifications: sheep erythrocytes (Rockland
Immunochemicals Inc., Gilbertsville, Pa.) were treated with 100
.mu.g/ml of the compounds in phosphate-buffered saline with DMSO at
2% for 2 hours and the supernatants were monitored at OD.sub.540.
The results are provided in Table 2 (below).
TABLE-US-00002 TABLE 2 Fold Therapeutic Amt. Survival Concentration
Colonization Compound vs untreated.sup.2 (.mu.g/ml) vs
untreated.sup.3 MIC (.mu.g/ml) A1 3.0 25 22% >125 B1 2.6 12.5
13% >125 C1 2.7 50 7% >125 D1 2.3 12.5 1% >125 E1 3.0 25
1% 31.3 E30 1.6 6.3 2% .sup. >30.sup.1 F1 3.1 25 3% >125 G1
3.3 25 6% 31.3 H1 3.0 50 1% 15.6 I1 2.7 6.3 9% 7.8 J1 2.1 6.3 11%
2.0 K1 1.9 100 61% >125 L1 1.8 25 101% >125 M1 2.2 100 8%
>125 N1 2.6 25 5% 3.9 O1 2.6 25 8% 15.6 Tetracycline.sup.4 4.0
1.6 5% 0.24 .sup.1aqueous solubility limit. .sup.2untreated = 24%
survival. .sup.3100% = 3.28 .times. 10.sup.4 CFU/worm.
.sup.4control.
[0232] Toxicity Testing.
[0233] Fifteen out of 16 compounds identified in this study did not
show any signs of toxicity against C. elegans or mammalian
erythrocytes indicating that the screen was able to select against
toxic compounds. These results indicate that the worm infection
model will be able to select against at least some compounds that
exhibit toxicity. In other studies, C. elegans has been used as an
indicator of toxicity from heavy metals, environmental pollutants,
organic solvents, and neurotoxins (Sochova et al., Environ. Int.
32:374-83 (2005)). Toxicity against nematodes has been quantified
based on nematode survival, growth, reproduction, expression of
stress response proteins, feeding behavior, and movement. The
utility of C. elegans in toxicology testing greatly depends on how
it correlates to toxicity in mammalian models. Williams and
Dusenbery (Williams et al., Toxicol. Ind. Health 4:469-478 (1988))
determined that toxicity of heavy metals against C. elegans as
measured by the LC50 values (concentration resulting in 50% death)
correlates well with toxicity against mice or rats in rank order
tests. Additionally, Cole et al. reported a significant correlation
from rank order toxicity tests from organophosphates between C.
elegans and rodents (Cole et al., Toxicol. Appl. Pharmacol.
194:248-256 (2004)).
Example 3
High-Throughput C. elegans Screen for Identifying Chemical
Compounds with Antifungal Activity
[0234] Our objective was to develop a semi-automated, high
throughput, whole animal C. elegans assay useful for identifying
chemical compounds with antifungal activity. Key features of the C.
elegans assay are that it allows concurrent evaluation of toxicity
and antifungal activity and that it identifies compounds that
target important pathways associated with human Candida infection,
including filament formation. Moreover, the assay allows the study
of Candida cells that are in non-planktonic form and identification
of antifungal compounds in a system where both the pathogen and the
host can be genetically manipulated.
Materials and Methods.
[0235] Strains and Media.
[0236] The Candida strains used in these experiments are summarized
in Table 3 or described in the text. Yeast cultures were maintained
on Yeast Peptone Dextrose (YPD, Difco) agar or as frozen stocks.
The C. elegans strains were propagated on E. coli strain OP50 or E.
coli strain HB101 using established procedures (Moy et al., Proc.
Natl. Acad Sci. USA 103:10414 (2006) and Brenner, S. Genetics 77:71
(1974)).
TABLE-US-00003 TABLE 3 Candida Strain Description Relevant
Characteristics or Phenotype LT.sub.50.sup.a DAY185.sup.b Arg.sup.+
Ura.sup.+ His.sup.+ Forms meshwork comprised almost 3 days
Reference exclusively of long hyphae MIC: strain 0.5 .mu.g/ml for
amphotericin B, 2 .mu.g/ml for fluconazole and caspofungin
MLR28.sup.b KEM1 Restored the ability to form normal 3 days
reconstituted hyphae (no strain difference) GKO443.sup.b suv3
Biofilm-defective mutant that is 6 days defective in hypha
production (P < 0.0001) MLR3.sup.b SUV3 Produced thick biofilms
containing 4 days reconstituted hyphae and excluded calcofluor from
the (no strain basal cell layers difference) MLR74.sup.b kem1
deletion Biofilm-defective mutant that is 5 days mutant defective
in hypha production (P = 0.0008) MLR62.sup.c GFP-expressing GFP
gene linked to the constitutively 4.5 days WT active TEF1 promoter
C. albicans WT Isolated from blood, Iowa 3.5 days ATCC#90028.sup.d
MIC: 2 .mu.g/ml for amphotericin B and 1 .mu.g/ml for caspofungin
C. krusei WT Isolated from sputum of patient with 4 days
ATCC#6258.sup.d bronchomycosis, Sri Lanka MIC: 2 .mu.g/ml for
amphotericin B.sup.d, 32 .mu.g/ml for fluconazole.sup.d and 0.5-2
.mu.g/ml caspofungin C. parapsilosis WT Isolated from case of
sprue, Puerto Rico 3 days ATCC#22019.sup.d MIC: 2 .mu.g/ml for
amphotericin B.sup.d, 2 .mu.g/ml for fluconazole.sup.d and 0.5-2
.mu.g/ml caspofungin C. albicans Transformed Expresses GFP in a
hyphae-specific ND HGFP3.sup.e with manner pHWP1GFP3 .sup.aC.
elegans strain glp-4; sek-1 killing (P value compared to parent
strain, when relevant); .sup.bRichard et al., Eukaryot Cell4: 1493
(2005); .sup.cNobile et al., Curr. Biol. 15: 1150 (2005);
.sup.dSTANDARDS., N. C. F. C. L. Methods for antifungal disk
diffusion susceptibility testing of yeasts; approved guideline
M44-A. (National Committee for Clinical Laboratory Standards,
Wayne, Pa., 2004.); .sup.eStaab et al., Microbiology 149: 2977
(2003) Abbreviations: ATCC: American Type Culture Collection, GFP:
Green fluorescent protein, LT: Lethal Time to mortality, MIC:
Minimal Inhibitory Concentration, ND: Not done, WT: Wild type
[0237] C. elegans Liquid Medium Killing Assays.
[0238] Candida strains were inoculated in 2 ml of YPD and grown at
30.degree. C. for 24 hours. Lawns were prepared by spreading 10
.mu.l of each culture on 35 mm tissue-culture plates (Falcon)
containing solid brain heart infusion (BHI) media (Difco) with
kanamycin (45 .mu.g/ml), ampicillin (100 .mu.g/ml), and
streptomycin (100 .mu.g/ml). The plates were incubated at 30'C for
24 hours followed by 25.degree. C. for 24 hours. glp-4; sek-1 and
glp-4 worms were grown at 15.degree. C. until they reached the L2
stage, and then they were transferred to 25.degree. C. for 24 h. N2
wild-type worms were grown at 15.degree. C. for 72 h, until they
reached the L4 stage. Approximately 100 worms were picked onto each
lawn and allowed to feed for 30 minutes to 2 hours. The worms were
washed off the plates with M9 buffer and allowed to crawl on
unseeded BHI plates to remove yeast cells from their cuticles.
Roughly 70-80 worms were then picked to wells in a six-well
microtiter dish that contained 1.5 ml liquid medium of 79% M9
buffer, 20% BHI, 2 .mu.M cholesterol and 90 .mu.g/ml kanamycin. The
plates were incubated at 25.degree. C. overnight and then examined
at 24-hour intervals for survival. Worms were considered dead when
they did not respond to being touched with a platinum wire
pick.
[0239] C. elegans Filamentation Staining.
[0240] C. albicans strains DAY185 and HGFP3 lawns were prepared and
C. elegans glp-4; sek-1 worms exposed to C. albicans as described
above for the killing assays. The worms were then transferred to
the liquid media described above in both the presence and absence
of 32 .mu.g/ml fluconazole. At 24, 48, 144, and 192 hours, 20-40
worms exposed to DAY185 were stained in 200 .mu.l of 10 .mu.M and
25 .mu.g/ml Concavalin A-Alexafluor for 45 minutes (Li et al.,
Microbiology 149:353 (2003)). Pictures were taken with a confocal
laser microscope (TCS NT, Leica Microsystems). FUN-1 is a
fluorescent yellow dye that is absorbed by metabolically active
fungal cells and fluoresces red when illuminated with 480 nm
(fluorescence emission). Concanavalin A-Alexafluor (fluorescence
emission at 519 nm) is a fluorescent green dye that binds to
polysaccharides and stains filaments.
[0241] Evaluation of Fungal Burden within C. elegans.
[0242] The number of C. albicans colony forming units in C. elegans
was quantified based on the protocol detailed by Tang et al.,
Infect. Immun. 73:8219 (2005) and Garsin et al., Proc. Natl. Acad
Sci. USA 98:10892 (2001) with a few modifications. C. albicans
strain MLR62 lawns were prepared on BHI as described above. C.
elegans glp-4; sek-1 worms were exposed to the lawns for 30 minutes
and then moved to liquid media and incubated at 25.degree. C. in
the presence or absence of antifungal agents. At appropriate time
points, 3 groups of 10 worms each were washed twice in 8 .mu.l
drops of M9 buffer on BHI agar plates, in order to remove surface
Candida. Each group of 10 worms was then disrupted using a Pellet
Pestle Motor (Kontes) and plated on YPD agar containing kanamycin
(45 .mu.g/ml), ampicillin (100 .mu.g/ml), and streptomycin (100
.mu.g/ml). The plates were incubated for 48 hours at 30'C and
colonies were counted.
[0243] Screen of Compound Library in C. elegans
[0244] For testing the efficacy of chemical compounds, C. albicans
strain MLR62 was inoculated into 2 ml of YPD and grown at
30.degree. C. for 24 hours; 10 .mu.l of the culture was spread on
35-mm tissue-culture plates (Falcon) containing brain heart
infusion (BHI) agar (Difco) with kanamycin (45 .mu.g/ml),
ampicillin (100 .mu.g/ml), and streptomycin (100 .mu.g/ml). The
plates were incubated at 30.degree. C. for 24 hours. C. elegans
animals at the L4 developmental stage (grown as detailed above
under C. elegans liquid medium killing assays) were transferred
from a lawn of E. coli HB101 onto C. albicans lawns on BHI medium,
incubated at 25.degree. C. for 30 minutes, and then pipetted in 50
.mu.l into 96-well plate wells that contained 100 .mu.l liquid
media (79% of M9 buffer, 20% BHI media, 2 .mu.M cholesterol and 90
.mu.g/ml kanamycin). C. elegans glp-4; sek-1 nematodes were
pipetted into 96-well plates that contained compounds from chemical
libraries. Library 1361 was assembled by Biomol and contains
well-characterized compounds that affect many different aspects of
cellular pathways. The NINDS custom collection (plates 501-503) was
put together by MicroSource Discovery Systems for the National
Institute of Neurological Disorders and Stroke (NINDS), the
Huntington's Disease Society of America (HDSA), the Amyotrophic
Lateral Sclerosis (ALS) Association, and the Hereditary Disease
Foundation (HDF). It mostly contains FDA approved drugs. The
Prestwick library (plate 1569) contains compounds that are known to
be safe and bioactive in humans. The majority of the compounds are
marketed drugs.
[0245] On day 6, nematodes were evaluated for survival, filament
formation and optical density. The plates were incubated at
25.degree. C. and examined on day 6 for survival with a Nikon
SMZ645 dissecting microscope. In addition, images were obtained
using a CellWorx High Content Cell Analysis System (Applied
Precision) at 4.times. magnification. Dead worms were counted but
not removed. Worms were considered dead if they had developed
filamentation, were rod shaped, or did not respond to the well
being tapped. Each compound was also scored by the number of dead
worms in the well that developed hyphea. Compounds were considered
to have completely or almost completely inhibited filament
formation if fewer than 25% of the dead worms in the well developed
hyphea. Compounds were categorized as having a minimal effect on
filament formation if 25-75% of the dead worms developed hyphea.
The compounds that had no effect of filament formation allowed over
75% of the dead worms to form hyphea.
[0246] Approximately 25 nematodes were used to analyze each
chemical compound tested. Two controls were used in all plates. The
positive control was the antifungal caspofungin (that allows
>75% survival at day 6). PBS was the negative control (<25%
of nematodes in PBS are alive at Day 6). In this pilot screen, we
considered a compound as a "hit" when survival in the compound well
with was 50% and above of the median survival of the control wells.
Depending on the compound library, the final concentration of the
compounds was 33.33 .mu.g/ml, 13.33 .mu.g/ml, or 6.67 mM.
[0247] Results.
[0248] Killing of C. elegans by Candida Spp.
[0249] We sought to develop a C. elegans-Candida spp. killing assay
that could be performed in liquid media. We found that when
wild-type L4 stage N2 nematodes were fed Candida spp. on solid
brain heart infusion (BHI) medium for 2 hours and then transferred
to a liquid medium consisting of 20% BHI and 80% M9 buffer, the
longevity of the worms was significantly reduced compared to worms
not infected with Candida (FIG. 16a). Notably, because of brood
production, we removed progeny nematodes that survived beyond the
L1 or early L2 developmental stage. We tested different times of
exposure to Candida spp. on solid BHI medium (from 5 minutes to 24
hours) and in all cases the longevity of the worms was
significantly reduced compared to un-infected worms.
[0250] Although Candida spp. kill wild-type C. elegans in liquid
medium, the killing does not occur rapidly enough to prevent the
production of progeny produced by 3- and 4-day old hermaphroditic
nematodes. The presence of a brood in the assay mix makes it
difficult to score the viability of the infected parents. Moreover,
Candida-infected wild type nematodes exhibit significant matricidal
death (over 30%) involving the premature hatching of eggs in the C.
elegans uterus (which is not directly associated with an
infectious-like process) around day 3 of the experiment. To avoid
these problems associated with progeny production, we substituted
C. elegans glp-4 mutants for wild-type in the Candida spp. killing
assay. C. elegans glp-4 mutant animals have normal morphology and
brood sizes at 15.degree. C., but do not make gonads and are unable
to produce eggs at 25.degree. C. (see Moy et al., Proc. Natl. Acad.
Sci. U SA 103:10414 (2006); Mylonakis et al., Proc. Natl. Acad.
Sci. USA 99:15675 (2002); Beanan et al., Development 116:755
(1992); and Roussell et al., Proc. Natl. Acad. Sci. USA 90:9300
(1993)). As shown in FIG. 16a, Candida spp. also killed glp-4
nematodes, but the rate of killing was significantly slower than
for wild-type worms, similar to results reported previously for the
killing of glp-4 and other sterile mutants by bacterial
pathogens.
[0251] Although glp-4 mutant worms could have potentially been used
in a anti-fungal screening assay, we sought to increase the rate of
killing of the glp-4 mutant by utilizing glp-4; sek-1 double mutant
worms. SEK-1 encodes a C. elegans homologue of the mammalian p38
mitogen activated protein kinase (MAPK) that was shown previously
to be an important component of the C. elegans defense response to
pathogens (see Moy et al., Proc. Natl. Acad. Sci. USA 103:10414
(2006) and Kim et al., Science 297:623 (2002)). As shown in FIG.
16a, the C. elegans glp-4; sek-1 double mutant is significantly
more susceptible to Candida-than the glp-4 mutant (P<0.001),
suggesting that the C. elegans homologue of the mammalian p38 MAPK
is involved in the C. elegans immune response to Candida, similar
to it role in candidiasis in mammals (Deva et al., J. Immunol.
171:3047 (2003)) and consistent with the observation that sek-1
mutants are more susceptible to a variety of pathogens (see Moy et
al., Proc. Natl. Acad Sci. USA 103:10414 (2006) and Kim et al.,
Science 297:623 (2002)). Because the glp-4; sek-1 mutant was
similarly susceptible to a variety of C. albicans and non-albicans
strains (FIG. 16b), because it did not produce progeny, and because
we found it compelling to study Candida pathogenesis in
immunocompromised nematodes (because of the analogy with
candidiasis in immunocompromised humans), we utilized the glp-4;
sek-1 strain in all of the further studies described below.
[0252] Antifungals Prolong Nematode Survival
[0253] To develop a system that allows high throughput screening
for antifungal compounds, we first evaluated whether established
antifungals prolong the survival of nematodes infected with Candida
spp. For this, we used different C. albicans isolates as well as
non-albicans strains, including those used as reference strains by
clinical microbiology laboratories (STANDARDS, N. C. F. C. L.
Methods for antifungal disk diffusion susceptibility testing of
yeasts; approved guideline M44-A. (National Committee for Clinical
Laboratory Standards, Wayne, Pa., 2004)). Notably, all strains
tested are susceptible to amphotericin B and caspofungin, but have
different susceptibilities to fluconazole (Richard et al., Eukaryot
Cell 4:1493 (2005); Staab et al., Microbiology 149:2977 (2003);
Nobile et al., Curr. Biol. 15:1150 (2005); and Espinel-Ingroff et
al., J. Clin. Microbiol. 36:2950 (1998)). We found that antifungals
that are active against a particular Candida strain resulted in
prolongation of C. elegans survival when the nematodes were
infected with the relevant Candida strain (FIGS. 17a-d).
Caspofungin, a fungicidal agent that is active against Candida
biofilm (Bachmann et al., Antimicrob. Agents Chemother. 46:3591
(2002) and Kuhn et al., Antimicrob. Agents Chemother. 46:1773
(2002)) and the also fungicidal agent amphotericin B were the most
active agents tested in prolonging nematode survival to C. albicans
strain MLR62 (FIG. 17a), C. krusei ATCC#6258 (FIG. 17b), C.
parapsilosis ATCC#20019 (FIGS. 17c and 17d) and C. albicans
ATCC#90028. In the case of C. krusei ATCC#6258, the most resistant
to fluconazole among the strains we tested, the difference in
survival between fluconazole and caspofungin treated worms was
highly significant (P<0.0001), as was the difference between
amphotericin B and caspofungin treatments (P=0.0005; FIG. 17b). In
the case of C. parapsilosis ATCC#20019, caspofungin was
significantly more effective than fluconazole (P=0.01), but the
difference between amphotericin B and caspofungin was not
significant (FIG. 17c). Caspofungin was effective at concentrations
as low as 1 .mu.g/ml in the case of C. albicans strain MLR62
(P<0.0001). Since filament formation is only seen in dead
nematodes, there were significantly fewer nematodes with filaments
in the wells that contained antifungal compounds.
[0254] Interestingly, at high concentrations, the beneficial effect
of fluconazole was lost and the nematodes died faster than
non-treated infected worms. Fluconazole up to 32 .mu.g/ml was
effective in prolonging survival of nematodes exposed to the
fluconazole-susceptible strain C. parapsilosis ATCC#20019, but at
higher concentrations (100 jg/ml) nematode survival was diminished,
even compared to the nematodes in the control group with no
antifungals (P=0.01; FIG. 17d). Probably this toxicity is present
at even lower concentrations, but the beneficial effect from the
antifungal activity outweighs the toxic effect. For example, when
nematodes were exposed to the fluconazole-resistant strain C.
krusei ATCC#6258, fluconazole at 32 .mu.g/ml not only had no effect
on nematode survival, but killing in the fluconazole group was
significantly faster than untreated worms (P=0.02; FIG. 17b).
Similarly, fluconazole at concentrations as low as XYZ .mu.g/ml
also decreased the longevity of nematodes feeding only on E. coli.
Taken together, these observations suggest that the model can be
used for the concurrent screen for antifungal activity and host
toxicity.
[0255] We also evaluated the effect of antifungal agents in
reducing fungal burden in the nematode intestine. For example, in
one particular experiment, untreated C. elegans infected with C.
albicans MLR62 had an average of 170.8.+-.63.5 colony-forming units
(cfu) per worm, whereas caspofungin treated animals (at 8 .mu.g/ml)
had almost cleared the infection with an average of 0.3.+-.0.2 cfu
per worm (P<0.001). Fluconazole and amphotericin B also
significantly decreased the concentration of Candida cfu in the
nematodes and microscopic examination of the worms confirmed that
treatment with antifungals was effective at clearing the worm
intestine of visible Candida cells (data not shown).
[0256] Antifungal Compounds Identified Using the C. elegans-Candida
Killing Assay
[0257] To facilitate adaptation of the C. elegans-Candida killing
assay to screening chemical libraries for antifungal compounds, we
used C. albicans strain MLR62 that expresses GFP (linked to the
constitutively active TEF1 promoter) and exhibits similar killing
kinetics to the parent strain DAY185 in the C. elegans assay. Using
this strain, nematodes exposed to compounds that have significant
antifungal efficacy exhibit sinusoidal movement and no green
fluorescence in the intestine at the endpoint of the assay (FIG.
18a), whereas nematodes exposed to compounds without antifungal
efficacy do not demonstrate any movement, are rod-shaped, exhibit
high levels of intestinal fluorescence (FIG. 18b), and developed
filaments. (FIG. 18c).
[0258] As an initial test of the C. elegans-Candida screening
assay, we utilized libraries of compounds made available through
the Institute of Chemistry and Cell Biology (ICCB) at Harvard
Medical School that includes known compounds that affect diverse
cellular pathways as well as US Food and Drug Administration
approved drugs that are known to be safe and bioactive in humans.
We screened a total of 1,266 compounds (Table 4) and identified 15
(.about.1.2%) that prolonged nematode survival and completely or
almost completely inhibited filamentation. An additional 52
(.about.4.1%) of the compounds prolonged nematode survival but had
no or minimal effect on filamentation (Table 4). To the best of our
knowledge, the C. elegans screen identified all of the compounds
among the 1,266 tested with established, clinically documented
antifungal activity against Candida. Among the known antifungals,
ketoconazole and butoconazole were among the most effective
compounds identified (Table 4).
TABLE-US-00004 TABLE 4 Compound Action/Use Killing Compounds that
completely or almost completely inhibited filament formation.
Caffeic acid phenethyl NFkappaB inhibitor which plays important
role in 0 ester transcription factors pertinent to interleukin 1
action Ketoconazole Antifungal 0.056, 0.167 AG-370 Belongs to
tyrphostin family of tyrosine kinase 0.111 inhibitors. Inhibits
mitogenesis in human fibroblast mediated by platelet derived growth
factor Butoconazole nitrate Antifungal 0.128 Methscopolamine
Anticholinergic; blocks M1 muscarinic receptors 0.167 bromide who
are present in Saccharomyces cerevisiae Lapachol
Antibacterial/Antiparasitic 0.182 Diethylstilbestrol Synthetic
estrogen; estrogens stimulate 0.235 colonization of vagina with
Candida albicans Probucol Lipid-lowering agent/Antioxidant 0.278
Sulfabenzamide Antibiotic 0.286 Prochlorperazine
Antiemetic/Antipsychotic; antibacterial activity in 0.300 edisylate
vitro Budesonide Glucocorticoid 0.385 Cephaloridine Antibiotic
0.385 Dyclonine Local anesthetic; inhibits ergosterol synthesis
0.444 hydrochloride Flutrimazole Antifungal 0.444 Hydroxytacrine
Metabolite of tacrine; tacrine is an 0.467 maleate
acetylcholinesterase inhibitor that is used against Alzheimer's
disease; reduces apoptosis in neuronal cells Compounds that had
minimal effect on filament formation 5 (S) HPETE Ca.sup.2+ channel
activation resulting in B-lymphocyte 0.261 activation Ketorolac
Anti-inflammatory 0.267 tromethamine Nimodipine Ca.sup.2+ channel
blocker (L-type)/Probable antioxidant 0.273 action (see
methoxyverapamil) Lipoxin A4 Product of interaction of 15-HPETE
with human 0.273 leukocytes; anti-inflammatory mediator, suppresses
inflammation; inhibits NK cell cytotoxicity N- Conjugate of lipids
and amino acids; suppresses 0.275 Arachidonoylglycine tonic
inflammatory pain in bovine and rat brain; produces
endocannabinoid-induced inhibition of T-cell proliferation
Lidocaine Na.sup.+ channel blocker; local anesthetic; 0.286
antiarrhythmic; has antibacterial activity against E. coli, P.
aeruginosa, S. aureus in vitro and antifungal activity against C.
albicans L-cis-Diltiazem Ca.sup.2+ channel blocker; antioxidant
0.296 AG-1478 Epidermal growth factor inhibitor; antioxidant 0.3
7,7- Probable antioxidant activity 0.304 Dimethyleicosadienoic acid
MBCQ Selective inhibitor of cGMP-specific 0.32 phosphodiesterase
1-Stearoyl-2- Probably enhances the effect of TNF alpha 0.333
linoleoyl-glycerol Bafilomycin A1 Endosomal H(+)-ATPase inhibitor
in yeasts; 0.333 Inhibits TNF-.alpha. and expression of
metalloproteinases; reduces killing of Aspergillus fumigatus by
alveolar macrophages Eicosatrienoic acid Product of arachidonic
acid; induces mitogenesis 0.333 (20:3 n-3) Farnesylthioacetic acid
Farnesyl derivative; Ras antagonist 0.343 Niflumic acid NSAID;
inhibits growth of C. albicans via 0.355 cytosolic acidification
and inhibition of glycolysis Siguazodan Phosphodiesterase III
inhibitor; inhibits generation 0.375 of interleukins-4 and-13
Aminobenzamide (3- Tx+ poly(ADP-ribose) synthetase inhibitor; 0.389
ABA) [3- inhibits apoptosis in rat intestinal cells; suppresses
aminobenzamide (3- inflammatory response and brain injury in ABA)]
experimental E. coli meningitis Acetyl (N) s farnesyl 1 Inhibitor
of isoprenylated protein endoprotease 0.4 cys resulting in
inhibition of function of ras protein Cefmetazole sodium
Chemotactic agent for neutrophils 0.4 Glipizide Sulfonylouria;
increases oxidative stress in red 0.4 cells in vitro
Phenylpropanolamine Catecholamine; inotropic 0.407 hydrochloride 15
(S) HPETE Enhances TNF/cycloheximide induced apoptosis, 0.412
activates caspases-3 and-9 Ascorbic acid Vitamin C; antioxidant;
appears to enhance growth 0.417 of Cryptococcus neoformans and
Candida tropicalis in vitro; enhances neutrophil motility and
lymphocyte transformation in vitro and in vivo after infection with
Candida albicans FPL-64176 Induces apoptotic cell death via
activation of L- 0.429 type Ca.sup.2+ activation Ikarugamycin
Antiprotozoan/Inhibits degradation of CD4 0.429 SDZ-201106 Na.sup.+
channel modulator 0.432 Cytochalasin B Actin polymerization
inhibitor 0.438 Docosatrienoic acid Fatty acid; reduces GABA
response in rat 0.439 (22:3 n-3) substantia nigra neurons; is
metabolized by fungal lipases to a high-laurate canola oil 3,4-
NF-kappaB inhibitor; decreases TNF alpha effect 0.444
Dichloroisocoumarin on migration of lymphocytes ALA-ALA-PHE-CMK
Catalytic residue; probably antioxidant 0.448 Eicosapentaenoic acid
Fatty acid; is metabolized by fungal lipases to a 0.448 (20:5 n-3)
high-laurate canola oil Rolipram Phosphodiesterase 4A inhibitor;
anti- 0.455 inflammatory; inhibits IL-2R AG-490 Blocks cell growth
and induces apoptosis in human 0.462 acute lymphocytic leukemia
cells; blocks the activity of interleukin-7 in T-lymphocytes
Misoprostol, free acid Prostaglandin; reverses suppression of
TGF-.beta. 0.464 caused by acetyl salicylic acid; activates
protein- kinase A resulting in inhibition of HIV-replication into
macrophages Betulinic acid Inhibits secreted aspartic proteinases
of C. albicans 0.483 Leukotriene B4 Stimulates monocytes,
neutrophils and dendritic 0.483 cell chemotaxis DL- Sphingolipid;
Probable antibacterial and antifungal 0.485 Dihydrosphingosine
activity via inhibition of cell wall N-palmitoyl-L-serine Induces
platelet aggregation 0.486 phosphoric acid Compounds that had no
effect on filament formation Butamben Local anesthetic; contains
p-aminobenzoate which 0.105 decreases the activity of coenzyme Q
dependent electron transport Pentolinium bitartrate Inhibitor of
catecholamine secretion 0.227 Hydrocortisone Glucocorticoid 0.300
hemisuccinate Ambroxol Na.sup.+ channel blocker/Pain
reliever/Mucolytic agent; 0.303 hydrochloride promotes microbicidal
activity of monocytes; anti- inflammatory activity Clopamide
Thiazide diuretic; enhances oxidative stress 0.333 Brinzolamide
Antiglaucomatic/Carbonic anhydrase inhibitor; 0.346 antibacterial
Streptomycin sulfate Antibiotic 0.364 Benfluorex Insulin
sensitizer; activates transcription factors in 0.375 hydrochloride
Saccharomyces cerevisiae Enoxacin Fluoroquinolone; in vitro synergy
with antifungals 0.379 Practolol Beta-blocker; enhances free
radical production by 0.381 neutrophils Betamethasone
Glucocorticoid; inhibits glucose-beta-phosphate 0.417
dehydrogenase, an enzyme present in yeasts Amoxicillin
Antibacterial; enhances colonization of 0.429 gastrointestinal
tract with Candida Pregnenolone sulfate, Neurosteroid; induces
apoptotic cell death on 0.438 sodium salt retinal cells via
activation of caspase-2 and-3 and cytochrome c release Clomiphene
citrate Antiestrogen; decreases fatty acid production in 0.440
(Z,E) Saccharomyces cerevisiae Dobutamine Inotropic agent;
stimulates biofilm formation by 0.455 hydrochloride Staphylococcus
epidermidis Probenecid Induces apoptosis 0.457 Adenosine 5'-
Induces yeast-mycelium transition in C. albicans 0.471
monophosphate monohydrate Azathymine, 6 Antimetabolite; a
derivative of this compound has 0.480 antibacterial and antifungal
activity Benzbromarone Increases uric acid excretion from kidneys;
0.483 Probably inhibits apoptosis of proximal tubular renal
cells
Example 5
High-Throughput Chemical Screens Using Arabidopsis thaliana
Seedlings
[0259] We have also developed protocols to perform automated,
high-throughput (HT) chemical screens using Arabidopsis thaliana
(Arabidopsis) seedlings as a host. Again, the described methods
enable quantitative analyses of a wide range of biological
processes such as the response to different types of biotic
(pathogens) or abiotic (heavy metals, ultraviolet radiation, heat)
stresses that affect viability, as well as other traits. We
optimized methods to perform screens in liquid media in 96-well
plates.
[0260] The exemplary set-up described below highlights key steps in
setting up pathogenicity assays with seedlings--all steps of which
are amenable to automation, typically using or adapting existing
robotics platforms. These examples highlight the ability of this
platform to screen using a wide variety of pathogens (in this case
the human opportunistic pathogen P. aeruginosa and the
phytopathogen P. syringae--the latter being an example of a
bacterial pathogen causing diseases in agricultural setting, are
highlighted).
[0261] For this purpose seeds of an ecotype (natural accession) of
Arabidopsis that is susceptible to the pathogen being screened is
sterilized using standard protocols (soaking in ethanol for 5
minutes followed by 20% bleach treatment for 5 minutes and
extensively rinsed with water). These seeds are vernalized at
4.degree. C. for two days and dispensed into 96-well plates with
each well containing 100 .mu.l MS (Murashige and Skoog) medium and
covered with transparent lids and edges sealed with Parafilm. The
seeds are germinated and grown with 50 .mu.E fluorescent light at
22.degree. C. with replacement of medium at day 8 and subsequently
inoculated with the bacterial pathogen on day 10. In order to
highlight a strategy for automated quantitative read-out of the
extent of disease in one case (FIG. 23), seeds from a transgenic
Arabidopsis plant expressing a luminescent reporter (luciferase)
was used. For other screens using certain pathogens, the use of
mutants in known defense pathways (such as but not limited to npr1,
pad4, sid2 (ics1), ein2, jar1) might make the disease symptoms and
the read-out used more robust.
[0262] Bacteria are grown in appropriate medium--King's Broth in
the case of Pseudomonas syringae pv. tomato DC3000 or a mutant
derivative with an insertion in the hrcC gene--DC3000(hrcC) at
30.degree. C. or LB in the case of E. coli strain DH5a, P.
aeruginosa (strain PA14) or a mutant derivative of PA14 with a
deletion in a regulatory gene lasR--PA14(lasR). These mutants serve
to highlight examples of genes that are important for pathogenesis
in relevant hosts and when impaired do indeed show attenuated
disease symptoms.
[0263] Because the dispensing of seeds is a labor-intensive step,
we collaborated with Union Biometrica, Inc. to test their robotic
equipment COPAS XL to do this step automatically. These tests
revealed that single seeds could be dispensed into microplate wells
with over 99% accuracy. The equipment used for this purpose has a
higher size exclusion diameter than the COPAS BioSort robot
described above used to dispense C. elegans, thus allowing the
sorting and dispensing of appropriately sized larvae as well as
Arabidopsis seeds for the organisms described in Example 6. The
addition of media and compounds, and replacement or media can be
done using standard liquid handling devices available in the
market.
[0264] Bacteria were added to ten-day old seedlings and the plates
covered and sealed at the edges as before and were placed at
25.degree. C. at 50 .mu.E light as before. FIGS. 21 and 22 display
visual phenotypes (i.e., extent of disease and damage to
seedlings). As can be seen in these figures both these pathogens
(PA14 and DC3000) cause extensive damage to the seedlings, while a
less pathogenic bacterium such as E. coli DH5.alpha. causes lesser
seedling damage and disease symptoms progress much more slowly (not
shown). A key regulatory mutant of PA14 (lasR) that impairs the
synthesis and secretion of several virulence factors (not shown)
and the hrcC mutant of DC3000-DC3000 (hrcC) which knocks out the
key type III secretion mechanism important for the delivery of many
virulence effectors into the host cell cause significantly less
damage (FIG. 22). These results highlight the relevance and use of
this system to identify anti-infectives using a chemical screen in
this platform.
[0265] Another key aspect for high throughput screening is the need
for readouts that are indicators of damage to the host (seedlings)
that can be either automated using a quantitative assay
(preferably) or a reproducible qualitative assay. One way would be
to adapt a live/dead stain such as Sytox.RTM. described herein in
the context of the C. elegans screens. Alternatively, one can use
fluorescent or luminescent markers expressed ectopically using
constitutive promoters that could be read in multi-well plate
readers.
[0266] As a proof-of-principle seedlings expressing luciferase
constitutively from a CaMV 35S promoter were used in the assay and
the seedling luminescence was read on Day 0 and Day 3--well before
symptoms are visually obvious, using a multi-well luminometer
(TopCount, Perkin Elmer). As can be seen in the graph shown in FIG.
23, the luminescence was almost abrogated by day 3 (about a 3 log
reduction in relative luminescence units), indicative of poor
seedling health in the case of wild-type PA14, whereas the control
untreated plants and the seedlings exposed to the PA14 (lasR)
mutant either did not decrease or increased in luminescence
compared to day 0, respectively.
Example 6
Other Organisms for Identifying Antimicrobial or Antiviral
Compounds as Well as Compounds that Increase Longevity
[0267] Drosophila melanogaster (Fruit Fly)
[0268] The fruit fly, Drosophila melanogaster, may also be used in
the methods described herein (for instance in Example 1) for
identifying antimicrobial or antiviral compounds, as well as
compounds that increase lifespan.
[0269] Fly stocks of, e.g., OregonR or the marked strain yellow
white (yw) are cultured under standard conditions on corn meal
medium. Cultures of P. aeruginosa strain, PA14, and the control, E.
coli DH5a, are grown overnight in King's B medium (King et al.
(1954) J. Lab Clin. Med. 44:301-307). Following overnight
culturing, the cells are diluted 1/10 and grown for an additional
four to five hours. The cells are subsequently washed twice,
resuspended in distilled water, and then added to the corn meal
medium. As such, the pathogen, e.g., P. aeruginosa, is introduced
into the fly larvae by ingestion. Alternatively, direct injection
of the pathogen into an appropriately staged larva (e.g., first,
second, or third instar) may also be used.
[0270] Following bacterial infection, the fly larvae are placed in,
e.g., 24-well, 48-well, 96-well, or 384-well microtiter plates,
each well containing liquid media and the compound or compounds to
be tested, incubated at 18.degree. C. to 28.degree. C., and
monitored for death as assayed by a lack of movement.
[0271] Galleria mellonella (Greater Wax Moth)
[0272] The larvae of Galleria mellonella (greater wax moth) may
also be used in the screening methods described herein.
[0273] A pathogen, e.g., Pseudomonas aeruginosa, is injected into
G. mellonella larvae as follows. Cultures of P. aeruginosa are
grown overnight in King's B medium (King et al. (1954) J. Lab.
Clin. Med. 44:301-307). This culture is then diluted 1:100 in the
same medium and cultured. After two hours of growth, the cultures
are harvested by centrifugation, and the cells are resuspended in
an equal volume of 10 mM MgSO.sub.4. Each culture is subsequently
diluted to an OD.sub.600 of 0.1 (approximately 10.sup.8 cells/ml).
Using a Hamilton syringe, five microliter volumes of serial 10-fold
dilutions (10.degree. to 10.sup.-6) are injected into one of the
abdominal parapodia of G. mellonella (Lysenko (1963) Journal of
Insect Pathology, 5:78-82). Bacterial counts are determined by
plating according to standard methods. G. mellonella larvae are
purchased from Van der Horst Wholesale, St. Mary's, OH.
[0274] After injection of bacteria, G. mellonella larvae are placed
in, e.g., 24-well microtiter plates, each well containing liquid
media and the compound or compounds to be tested, and incubated at
25.degree. C. Lethality is visually assessed after forty-eight
hours by monitoring the color change (from white to black) of each
larva, and by determining larval motility. Each single non-motile
black larva is scored as dead.
[0275] Plutella xylostella (Diamondback Moth)
[0276] The larvae of Plutella xylostella (diamondback moth) may
also be used in the screening methods described herein.
[0277] Larvae of Plutella xylostella are fed mustard leaves
infiltrated with Pseudomonas aeruginosa as follows. P. aeruginosa
was cultured in King's B medium (King et al. (1954) J. Lab. Clin.
Med. 44:301-307). Overnight cultures are pelleted in a
microcentrifuge and were then washed twice in 10 mM MgSO.sub.4.
Cells are then resuspended in 10 mM MgSO.sub.4 and are diluted to
an OD.sub.600 of 0.1. Pl. xylostella larvae are maintained on a
semisynthetic wheat germ based diet according to standard methods
(Shelton et al. (1991) J. Ent. Sci., 26:17-26).
[0278] Mustard greens (available from grocery stores) are cut into
pieces of about 10 cm.sup.2 and are submersed in 10 mM MgSO.sub.4
containing P. aeruginosa. The submersed leaves are placed under
vacuum, and the vacuum is released suddenly to infiltrate the
bacterial solution into the leaves. As a control, leaves are also
infiltrated with only 10 mM MgSO.sub.4. Infiltrated leaf material
is incubated at 23.degree. C. in a petri dish with twenty Pl.
xylostella larvae, which are allowed to feed at will. After
feeding, Pl. xylostella larvae are placed in, e.g., 24-well or
48-well microtiter plates, each well containing liquid media and
the compound or compounds to be tested, and incubated at 25.degree.
C. Deceased larvae are scored after forty-eight hours. Larvae which
do not move after being touched with a pipette tip are scored as
dead.
OTHER EMBODIMENTS
[0279] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each independent publication or patent
application was specifically and individually indicated to be
incorporated by reference.
[0280] While the invention has been described in connection with
specific embodiments thereof, it will be understood that it is
capable of further modifications and this application is intended
to cover any variations, uses, or adaptations of the invention
following, in general, the principles of the invention and
including such departures from the present disclosure that come
within known or customary practice within the art to which the
invention pertains and may be applied to the essential features
hereinbefore set forth, and follows in the scope of the claims.
[0281] Other embodiments are within the claims.
* * * * *