U.S. patent application number 16/303464 was filed with the patent office on 2019-05-16 for modified antimicrobial peptide derived from an arginine-rich domain.
The applicant listed for this patent is Academia Sinica Office Of Public Affairs (Technology Transfer)(05), Chiaho Shih. Invention is credited to Heng-Li Chen, Chiaho Shih, Pei-Yi Su.
Application Number | 20190144510 16/303464 |
Document ID | / |
Family ID | 60411947 |
Filed Date | 2019-05-16 |
United States Patent
Application |
20190144510 |
Kind Code |
A1 |
Shih; Chiaho ; et
al. |
May 16, 2019 |
MODIFIED ANTIMICROBIAL PEPTIDE DERIVED FROM AN ARGININE-RICH
DOMAIN
Abstract
An antimicrobial peptide, the peptide comprising 2 to 20
variable domains, each variable domain is a sequence of 2 to 20
consecutive basic amino acids, wherein (a) the variable domains are
separated from each other by a variable linker, (b) the variable
linker can have 1 to 20 any amino acids other than two or more
consecutive basic amino acids, and (c) the peptide has no more than
100 amino acids.
Inventors: |
Shih; Chiaho; (Taipei City,
TW) ; Chen; Heng-Li; (Taipei City, TW) ; Su;
Pei-Yi; (Taipei City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Shih; Chiaho
Academia Sinica Office Of Public Affairs (Technology
Transfer)(05) |
Taipei |
|
US
TW |
|
|
Family ID: |
60411947 |
Appl. No.: |
16/303464 |
Filed: |
May 25, 2017 |
PCT Filed: |
May 25, 2017 |
PCT NO: |
PCT/US2017/034489 |
371 Date: |
November 20, 2018 |
Current U.S.
Class: |
514/2.4 |
Current CPC
Class: |
A01N 63/00 20130101;
C12N 2730/10133 20130101; A01N 37/46 20130101; A61P 31/04 20180101;
C07K 7/08 20130101; A61K 38/00 20130101; C12N 2730/10122 20130101;
C07K 14/005 20130101 |
International
Class: |
C07K 14/005 20060101
C07K014/005; A61P 31/04 20060101 A61P031/04 |
Claims
1. An antimicrobial peptide, the peptide comprising 2 to 20
variable domains, each variable domain is a sequence of 2 to 20
consecutive basic amino acids, wherein (a) the variable domains are
separated from each other by a variable linker, (b) the variable
linker can have 1 to 20 any amino acids other than two or more
consecutive basic amino acids, and (c) the peptide has no more than
100 amino acids.
2. The antimicrobial peptide of claim 1, wherein the peptide has at
least 3 variable domains.
3. The antimicrobial peptide of claim 1, wherein the peptide has a
C-terminal cysteine.
4. The antimicrobial peptide of claim 1, wherein at least one of
the basic amino acids in the variable domains is an arginine.
5. The antimicrobial peptide of claim 4, wherein all of the basic
amino acids in each variable domain are arginine residues.
6. The antimicrobial peptide of claim 1, wherein at least one of
the basic amino acids is a chemically-modified amino acid.
7. The antimicrobial peptide of claim 6, wherein the
chemically-modified amino acid is a D-amino acid.
8. The antimicrobial peptide of claim 7, wherein the D-amino acid
is D-arginine.
9. The antimicrobial peptide of any of claims 1-8, wherein the
variable domains and the variable linkers are derived from the
arginine-rich domain of a hepadnavirus core protein (HBcARD).
10. The antimicrobial peptide of claim 9, wherein the HBcARD
contains a sequence from residue 147 to the C-terminal residue of a
hepadnavirus core protein.
11. The antimicrobial peptide of claim 10, wherein each variable
domain has three or four arginine residues and each variable linker
has 2 to 4 amino acids.
12. The peptide of claim 11, wherein the peptide contains a
consensus sequence selected from the group consisting of: (i)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.sub.8P(X.s-
ub.9), wherein each of X.sub.1, X.sub.3, X.sub.6, and X.sub.9,
individually, is a variable domain, and each of X.sub.2, X.sub.4,
X.sub.5, X.sub.7, and X.sub.8, individually, is any amino acid or
absent, (ii) (X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6),
wherein each of X.sub.1, X.sub.3, and X.sub.6, individually, is a
variable domain, and each of X.sub.2, X.sub.4, and X.sub.5,
individually, is any amino acid or absent, and (iii)
(X.sub.1)X.sub.2PX.sub.3P(X.sub.4X.sub.5QX.sub.6P(X.sub.7), wherein
each of X.sub.1, X.sub.4, and X.sub.7, individually, is a variable
domain, and each of X.sub.2, X.sub.3, X.sub.5, and X.sub.6,
individually, is any amino acid or absent,
13. The antimicrobial peptide of claim 12, wherein the peptide has
a C-terminal cysteine.
14. The antimicrobial peptide of claim 12, wherein the peptide has
a consensus sequence selected from the group consisting of: (i)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.sub.8P(X.s-
ub.9)X.sub.10C, and (ii)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.sub.8P(X.s-
ub.9)X.sub.10Q, wherein each of X.sub.1, X.sub.3, X.sub.6, and
X.sub.9, individually, is a variable domain, and each of X.sub.2,
X.sub.4, X.sub.5, X.sub.7, X.sub.8, and X.sub.10, individually, is
any amino acid or absent.
15. The antimicrobial peptide of claim 12, wherein the peptide
contains a sequence selected from the group consisting of:
TABLE-US-00008 (i) TVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC, in which
at least one of the arginine residues is D-arginine, (ii)
RRRGRSPRRRTPSPRRRRSQSPRRRRSC, (iii) RRRGRSPRRRTPSPRRRRSQSPRRRRSQ,
(iv) RRRGRPRRRPPRRRRQPRRRRC, (v) RRRGRSPRRRTPSPRRRRC, (vi)
RRRGRPRRRPPRRRRC, (vii) RRRTPSPRRRRSQSPRRRRC, or (viii)
RRRPPRRRRQPRRRRC.
16. The antimicrobial peptide of claim 15, wherein the peptide
contains the sequence of RRRGRSPRRRTPSPRRRRSQSPRRRRSC, in which
each of the arginine residues in the sequence is L-arginine.
17. The antimicrobial peptide of claim 15, wherein the peptide
contains the sequence of RRRGRSPRRRTPSPRRRRSQSPRRRRSC, in which at
least one of the arginine residues in the sequence is
D-arginine.
18. The antimicrobial peptide of claim 17, wherein each of the
arginine residues in the sequence is D-arginine.
19. The antimicrobial peptide of claim 17, wherein the sequence is
rRrGRSPrRrTPSPrRrRSQSPrRrRSC, in which R is L-arginine and r is
D-arginine.
20. The antimicrobial peptide of claim 17, wherein the sequence is
RrRGRSPRrRTPSPRrRrSQSPRrRrSC, in which R is L-arginine and r is
D-arginine.
21. The antimicrobial peptide of claim 15, wherein the peptide
contains the sequence of RRRGRPRRRPPRRRRQPRRRRC, in which at least
one of the arginine residues in the sequence is D-arginine.
22. The antimicrobial peptide of claim 15, wherein the sequence is
rRrGRPrRrPPrRrRQPrRrRC, in which R is L-arginine and r is
D-arginine.
23. The antimicrobial peptide of any of claims 15-22, wherein the
sequence is at the C-terminus of the peptide.
24. The antimicrobial peptide of any of claims 1-23, further
comprising a non-HBcARD peptide.
25. The antimicrobial peptide of claim 24, wherein the non-HBcARD
peptide is an affinity tag, a signal sequence, a ligand, or another
antimicrobial peptide or fragment thereof.
26. The antimicrobial peptide of claim 25, wherein the non-HBcARD
peptide is a poly-histidine or an analog thereof.
27. The antimicrobial peptide of claim 26, wherein the peptide has
a sequence selected from the group consisting of: TABLE-US-00009
(i) RRRGRSPRRRTPSPRRRRSQSPRRRRSHHHHHH, (ii)
HHHHHHRRRGRSPRRRTPSPRRRRSQSPRRRRS, (iii)
RRRGRPRRRPPRRRRQPRRRRHHHHHH, and (iv)
HHHHHHRRRGRPRRRPPRRRRQPRRRR
28. The antimicrobial peptide of any of claims 1-4, wherein at
least one variable domain has a lysine.
29. The antimicrobial peptide of any of claims 1-4, wherein at
least one variable domain has a histidine.
30. The peptide of any of claims 1-29, wherein the peptide has a
cyclic structure.
31. The antimicrobial peptide of any of claims 1-30, wherein the
peptide exhibits a broad spectrum antimicrobial activity against a
gram-positive bacteria, gram-negative bacteria, fungus, parasite,
or virus.
32. An antimicrobial peptide conjugate, the conjugate comprising
the peptide of any of claims 1-31 and a non-peptide moiety.
33. A pharmaceutical composition comprising the antimicrobial
peptide of any of claims 1-31 and a pharmaceutically acceptable
carrier.
34. A pharmaceutical composition comprising the antimicrobial
peptide conjugate of claim 32 and a pharmaceutically acceptable
carrier.
35. A method of treating a microbial infection, the method
comprising administering the composition of claim 33 or 34 to a
subject in need thereof.
Description
BACKGROUND
[0001] Antibiotics have been used for the treatment of bacterial
infection for more than 60 years. Recently, the increasing number
of antibiotics-resistant bacteria has become a major threat to
public health. The development of new antibiotics for clinical
treatment is an urgent need. Antimicrobial peptides (AMPs) from
various species can serve as a defense weapon of the host against
pathogenic microbes. Because they can kill bacteria and fungi via
different mode of actions, they have been considered as potential
candidates to overcome the problem of antibiotic-resistance.
SUMMARY
[0002] In one aspect, described herein is an antimicrobial peptide.
The peptide contains 2 to 20 variable domains, each variable domain
is a sequence of 2 to 20 consecutive basic amino acids, wherein (a)
the variable domains are separated from each other by a variable
linker, (b) the variable linker can have 1 to 20 any amino acids
other than two or more consecutive basic amino acids, and (c) the
peptide has no more than 100 amino acids. In one embodiment,
peptide has at least 3 or 4 variable domains. The peptide can have
a C-terminal cysteine. In some embodiments, at least one of the
basic amino acids in the variable domains is an arginine. For
example, all of the basic amino acids in each variable domain in
the peptide can be arginine residues. Alternatively or in addition,
at least one variable domain in the antimicrobial peptide has a
lysine. In one embodiment, at least one variable domain has a
histidine. The peptide can have a cyclic structure.
[0003] At least one of the basic amino acids in the peptide can be
a chemically-modified amino acid. In one embodiment, the
chemically-modified amino acid is a D-amino acid, e.g., D-arginine.
The variable domains and the variable linkers can be derived from
the arginine-rich domain of a hepadnavirus core protein (HBcARD).
In one embodiment, the HBcARD contains a sequence from residue 147
to the C-terminal residue of a hepadnavirus core protein. Each
variable domain in the peptide can have three or four arginine
residues and each variable linker in the peptide can have 2 to 4
amino acids. The peptide can exhibit a broad spectrum antimicrobial
activity against a gram-positive bacterium, gram-negative
bacterium, fungus, parasite, or virus.
[0004] In one embodiment, the antimicrobial peptide contains a
consensus sequence selected from the group consisting of:
[0005] (i)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.-
sub.8P(X.sub.9) (SEQ ID NO: 1), wherein each of X.sub.1, X.sub.3,
X.sub.6, and X.sub.9, individually, is a variable domain, and each
of X.sub.2, X.sub.4, X.sub.5, X.sub.7, and X.sub.8, individually,
is any amino acid or absent,
[0006] (ii) (X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)
(SEQ ID NO:2), wherein each of X.sub.1, X.sub.3, and X.sub.6,
individually, is a variable domain, and each of X.sub.2, X.sub.4,
and X.sub.5, individually, is any amino acid or absent, and
[0007] (iii)
(X.sub.1)X.sub.2PX.sub.3P(X.sub.4)X.sub.5QX.sub.6P(X.sub.7) (SEQ ID
NO: 3), wherein each of X.sub.1, X.sub.4, and X.sub.7,
individually, is a variable domain, and each of X.sub.2, X.sub.3,
X.sub.5, and X.sub.6, individually, is any amino acid or absent.
Each variable domain is a sequence of 2 to 20 consecutive basic
amino acids.
[0008] The peptide can have a consensus sequence selected from the
group consisting of:
[0009] (i)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.-
sub.8P(X.sub.9)X.sub.10C (SEQ ID NO: 4), and
[0010] (ii)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.sub.8P(X.s-
ub.9)X.sub.10Q (SEQ ID NO: 5),
wherein each of X.sub.1, X.sub.3, X.sub.6, and X.sub.9,
individually, is a variable domain, and each of X.sub.2, X.sub.4,
X.sub.5, X.sub.7, X.sub.8, and X.sub.10, individually, is any amino
acid or absent.
[0011] In one embodiment, the antimicrobial peptide contains a
sequence selected from the group consisting of:
TABLE-US-00001 (i) (SEQ ID NO: 6)
TVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC, in which at least one of the
arginine residues is D-arginine, (ii) (SEQ ID NO: 7)
RRRGRSPRRRTPSPRRRRSQSPRRRRSC, (iii) (SEQ ID NO: 8)
RRRGRSPRRRTPSPRRRRSQSPRRRRSQ, (iv) (SEQ ID NO: 9)
RRRGRPRRRPPRRRRQPRRRRC, (v) (SEQ ID NO: 10) RRRGRSPRRRTPSPRRRRC,
(vi) (SEQ ID NO: 11) RRRGRPRRRPPRRRRC, (vii) (SEQ ID NO: 12)
RRRTPSPRRRRSQSPRRRRC, and (viii) (SEQ ID NO: 13)
RRRPPRRRRQPRRRRC.
[0012] For example, the antimicrobial peptide can contain the
sequence of RRRGRSPRRRTPSPRRRRSQSPRRRRSC (SEQ ID NO: 7), in which
each of the arginine residues in the sequence is L-arginine.
Alternatively, the peptide can have the sequence of
RRRGRSPRRRTPSPRRRRSQSPRRRRSC (SEQ ID NO: 7), in which at least one
of the arginine residues in the sequence is D-arginine.
[0013] In one embodiment, the antimicrobial peptide contains the
sequence of rRrGRSPrRrTPSPrRrRSQSPrRrRSC (SEQ ID NO: 7), in which R
is L-arginine and r is D-arginine. Alternatively, the peptide can
contain the sequence of RrRGRSPRrRTPSPRrRrSQSPRrRrSC (SEQ ID NO:
7), in which R is L-arginine and r is D-arginine.
[0014] The antimicrobial peptide can include the sequence of
RRRGRPRRRPPRRRRQPRRRRC (SEQ ID NO: 9), in which at least one of the
arginine residues (e.g., 20%, 30%, 40%, or 50% of the arginine
residues) in the sequence is D-arginine. For example, the sequence
can be rRrGRPrRrPPrRrRQPrRrRC (SEQ ID NO: 9), in which R is
L-arginine and r is D-arginine.
[0015] In one embodiment, the antimicrobial peptide further
contains a non-HBcARD peptide (e.g., an affinity tag, a signal
sequence, a ligand, or another antimicrobial peptide or fragment
thereof). The non-HBcARD peptide can be a poly-histidine or an
analog thereof. In one embodiment, the peptide has a sequence
selected from the group consisting of:
TABLE-US-00002 (i) (SEQ ID NO: 14)
RRRGRSPRRRTPSPRRRRSQSPRRRRSHHHHHH, (ii) (SEQ ID NO: 15)
HHHHHHRRRGRSPRRRTPSPRRRRSQSPRRRRS, (iii) (SEQ ID NO: 16)
RRRGRPRRRPPRRRRQPRRRRHHHHHH, and (iv) (SEQ ID NO: 17)
HHHHHHRRRGRPRRRPPRRRRQPRRRR.
[0016] In another aspect, described herein is an antimicrobial
peptide conjugate, which contains the antimicrobial peptide
disclosed herein and a non-peptide moiety.
[0017] In yet another aspect, a pharmaceutical composition is
described herein. The composition includes the antimicrobial
peptide or the antimicrobial conjugate, and a pharmaceutically
acceptable carrier.
[0018] Also contemplated herein is a method of treating an
infection in a subject in need thereof. The method includes
administering to the subject an antimicrobial peptide, an
antimicrobial peptide conjugate, or a pharmaceutical composition
described herein.
[0019] The details of one or more embodiments are set forth in the
accompanying drawing and the description below. Other features,
objects, and advantages of the embodiments will be apparent from
the description and drawing, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS
[0020] FIG. 1 includes two sets of sequence alignments of HBcARD
domains. (A) HBcARD sequences are highly conserved among human (SEQ
ID NO: 6), wooly monkey (SEQ ID NO: 18), ground squirrel (SEQ ID
NO: 19), woodchuck (SEQ ID NO: 20), and bat (SEQ ID NO: 21). (B)
There are also four positive charge clusters separated in the HBc
C-terminus of duck (SEQ ID NO: 22), heron (SEQ ID NO: 23), parrot
(SEQ ID NO: 24), Ross's goose (SEQ ID NO: 25) and snow goose
hepatitis B virus (SEQ ID NO: 26).
[0021] FIG. 2 is a set of graphs showing comparison of serum
resistance between L- and D-HBcARD peptides. Peptides (L-HBcARD and
D-HBcARD) were incubated with MBC buffer (10 mM sodium phosphate
and 50 mM sodium chloride, pH 7.2) containing 5% fetal bovine serum
(A), 5% mouse serum (B) or human serum (male and female) (C) at
37.degree. C. for 3 hours. The amounts of peptides were determined
using SDS PAGE electrophoresis and green angel staining. (D)
Peptide D-HBcARD exhibited 10,000-fold higher potency than that of
L-HBcARD in MBC assay. Peptides (L-HBcARD and D-HBcARD) were
incubated with S. aureus ATCC19636 with or without 5% mouse serum
at 37.degree. C. for 3 hours. The antimicrobial activity was
determined by colony formation assay. ***P<0.0001.
[0022] FIG. 3 is a graph showing comparison of hemolytic effect
between L- and D-HBcARD peptides. Human red blood cells were
incubated with different concentrations of the peptides. Hemolysis
is presented as the percentage of Triton X-100-induced hemolysis.
***P<0.0001.
[0023] FIG. 4 is a set of graphs showing comparison of in vivo
protection activities between L- and D-form HBcARD peptides in a
mouse sepsis model infected with S. aureus. (A) Three week-old ICR
mice were inoculated with S. aureus (4.times.10.sup.6 CFU/mouse)
and i.p. injected with PBS, L-HBcARD, or D-HBcARD at 2 hours
post-inoculation. Each group contained ten mice. (B) and (C) Three
week-old ICR mice were immunized with L- and D-HBcARD peptides (5
mg/kg), respectively, at day 0, 3 and 6. On day 14, the mice were
inoculated with S. aureus (4.times.10.sup.6 CFU/mouse) and i.p.
injected with PBS, L-HBcARD or D-HBcARD (10 mg/kg) at 1 hour
post-inoculation, respectively. Another group of mice were treated
with PBS in parallel as a control. Each group of animals contained
five mice. *P<0.05; ***P<0.0001; ns, no significance.
[0024] FIG. 5 is a graph showing in vivo protection efficacies of
various modified HBcARD peptides at different doses in an ICR mouse
sepsis model.
[0025] FIG. 6 is a graph showing in vivo protection efficacies of
150-177C and 150-177Q peptides in a BALB/c mouse lung infection
model. Colistin-resistant A. baumannii was inoculated via
intra-tracheal route.
[0026] FIG. 7 is a set of graphs showing lower in vivo toxicity of
HBcARD peptide D-150-177C in comparison with polymyxin B. At day 0,
male ICR mice (5 mice/group) were ip. injected with different doses
of D-150-177C peptide (20-80 mg/kg) and polymyxin B (50 mg/kg),
respectively. (A) The survival rates of all groups were monitored
for 7 days. (B) Serum samples collected from mice treated with
D-150-177C and polymyxin B were determined for alanine
aminotransferase activity (ALT) at day 1. The dash line represents
the mean of ALT value (45 U/L) of ICR mice (Charles River
Laboratories).
[0027] FIG. 8 is a graph showing in vivo protection efficacies of
all-D-arginine and partial-D-arginine substituted 150-177C peptides
in a mouse sepsis model. Two hours after the inoculation, the mice
(n=10/group) were treated with 5 mg/kg of various peptides D-, DL-,
LD-150-177C, and PBS, respectively. Peptides D- and DL-150-177C
protected all S. aureus-infected mice from death, while peptide
LD-150-177C protected only 80% of mice.
[0028] FIG. 9 is a graph showing that deletion of serine and
threonine residues from peptide DL-150-177C improved the in vivo
protection efficacies at different doses in a mouse sepsis
model.
DETAILED DESCRIPTION
[0029] It was unexpectedly discovered that certain modified
peptides derived from an arginine-rich domain of HBV core protein
exhibited broad spectrum antimicrobial activities.
[0030] Described herein is an antimicrobial peptide. It contains at
least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20) variable domains. The variable domains are
in tandem and each separated from another by a variable linker. The
antimicrobial peptide can have a maximum length of 100 amino acids
(e.g., less than 10, 10, 14, 15, 20, 21, 22, 25, 28, 30, 35, 37,
40, 45, 47, 50, 55, 57, 60, 65, 70, 75, 80, 85, 90, 95, or 100
amino acids). In one embodiment, the peptide has a C-terminal
cysteine.
[0031] Each of the variable domains, individually, is a sequence of
at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20) consecutive basic amino acids, e.g.,
arginine, histidine, and lysine. Each variable domain can contain a
sequence of identical basic amino acids or different basic amino
acids. For example, the antimicrobial peptides can contain 2 to 20
identical or different variable domains, each being
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7X.sub.8X.sub.9X.sub.10X.-
sub.11X.sub.12X.sub.13X.sub.14X.sub.15X.sub.16X.sub.17X.sub.18X.sub.19X.su-
b.20 (SEQ ID NO: 27), wherein each of X.sub.1-X.sub.20,
individually, is an arginine, histidine, or lysine (natural or
chemically modified) and any of X.sub.3-X.sub.20 can be present or
absent. For example, each variable domain can have 2 to 10 basic
amino acids.
[0032] In one embodiment, at least one variable domain in the
peptide consists solely of arginine residues. In another
embodiment, all of the variable domains in the peptide contain only
arginine residues. Alternatively, the peptide can contain at least
one variable domain that has one or more histidine or lysine
residues.
[0033] Each variable linker has at least one amino acid (e.g., 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20) and can contain any amino acid. It cannot have two or more
consecutive basic amino acids.
[0034] The term "amino acid" refers to any of the 20 standard amino
acids (i.e., alanine, arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, and valine). The term can also
refer to a non-standard, non-proteinogenic, or chemically-modified
amino acid, or an amino acid analog. An amino acid can be in the L-
or D-form stereoisomer.
[0035] The term "basic amino acid" refers to arginine, lysine, or
histidine, the L- or D-form thereof, or an analog thereof.
[0036] Non-standard amino acids include selenocysteine,
pyrrolysine, N-formylmethionine, non-proteinogenic amino acids,
amino acid analogs, and chemically-modified amino acids. A
chemically-modified amino acid or amino acid analog typically has a
different side chain from its naturally-occurring counterpart Amino
acid analogs and methods of incorporating them into a polypeptide
are known in the art. See, e.g., Nguyen et al., Biochemica et
Biophysica Acta 1808 (2011), 2297-2303; Knappe, Antimicrobial
Agents and Chemotherapy 54(9) 2010, 4003-4005; U.S. Pat. Nos.
7,879,979; 5,972,940; 8,835,162; and US20080199964.
[0037] Amino acid analogs are also commercially available. Amino
acid analogs can be incorporated in the antimicrobial peptide to
improve its stability, bioavailability, pharmacokinetics, tissue
distribution, safety, tolerability, and/or efficacy.
[0038] Any of the antimicrobial peptides described herein can
contain one or more residues that are not one of the twenty
standard amino acids. In particular, one or more (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2%, 3%,
5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, or 100%) of the basic amino acids in the
variable domains or in the entire antimicrobial peptide can be a
D-form of natural arginine, lysine, or histidine, or an analog of
natural arginine, lysine, or histidine.
[0039] The antimicrobial peptide can be derived from the
arginine-rich domain of a hepadnavirus core protein (HBcARD).
HBcARD refers to a highly conserved arginine-rich C-terminal region
of a core protein (HBc). See, FIG. 1. The HBc can be a mammalian
HBc or an avian HBc. A mammalian HBc can be human HBc, woolly
monkey HBc, ground squirrel HBc, woodchuck HBc, and bat HBc. An
avian HBc can be a duck, heron, parrot, Ross's goose, or snow goose
HBc. The HBc can be from a hepadnavirus of any genotype.
[0040] For example, the antimicrobial peptide can include a
fragment of the HBcARD or a variant thereof (e.g., containing one
or more amino acid substitutions, deletions, or insertions). The
variable domains and the linkers can be derived from an HBcARD. For
example, the sequence between any two arginine repeats in an HBcARD
or a variant thereof can be used as a linker.
[0041] In one embodiment, the antimicrobial peptide contains a
consensus sequence selected from the group consisting of:
[0042] (i)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.-
sub.8P(X.sub.9) (SEQ ID NO: 1), wherein each of X.sub.1, X.sub.3,
X.sub.6, and X.sub.9, individually, is a variable domain, and each
of X.sub.2, X.sub.4, X.sub.5, X.sub.7, and X.sub.8, individually,
is any amino acid or absent,
[0043] (ii) (X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)
(SEQ ID NO:2), wherein each of X.sub.1, X.sub.3, and X.sub.6,
individually, is a variable domain, and each of X.sub.2, X.sub.4,
and X.sub.5, individually, is any amino acid or absent, and
[0044] (iii)
(X.sub.1)X.sub.2PX.sub.3P(X.sub.4)X.sub.5QX.sub.6P(X.sub.7) (SEQ ID
NO: 3), wherein each of X.sub.1, X.sub.4, and X.sub.7,
individually, is a variable domain, and each of X.sub.2, X.sub.3,
X.sub.5, and X.sub.6, individually, is any amino acid or absent.
Each variable domain is a sequence of 2 to 20 consecutive basic
amino acids.
[0045] The peptide can have a consensus sequence selected from the
group consisting of:
[0046] (i)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.-
sub.8P(X.sub.9)X.sub.10C (SEQ ID NO: 4), and
[0047] (ii)
(X.sub.1)GRX.sub.2P(X.sub.3)X.sub.4PX.sub.5P(X.sub.6)X.sub.7QX.sub.8P(X.s-
ub.9)X.sub.10Q (SEQ ID NO: 5),
wherein each of X.sub.1, X.sub.3, X.sub.6, and X.sub.9,
individually, is a variable domain, and each of X.sub.2, X.sub.4,
X.sub.5, X.sub.7, X.sub.8, and X.sub.10, individually, is any amino
acid or absent.
[0048] The antimicrobial peptide can be a fusion or chimeric
peptide that further contains a non-HBcARD peptide. A non-HBcARD
peptide is not derived from any HBcARD and does not contain at
least two variable domains connected by a linker as described
above. A non-HBcARD peptide can be a peptide derived from another
source (e.g., from a protein other than an HBcARD), an engineered
peptide (e.g., another antimicrobial peptide), an affinity tag
(e.g., a FLAG, poly-His, Myc, HA, CBP, HBH, or V5 tag), a signal
sequence (e.g., a leader sequence or a localization signal), or a
ligand (e.g., a receptor ligand).
[0049] For example, the antimicrobial peptide can have up to 100
amino acids and contain a sequence selected from the group
consisting of:
TABLE-US-00003 (i) (SEQ ID NO: 6)
TVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC, in which at least one of the
arginine residues is D-arginine, (ii) (SEQ ID NO: 7)
RRRGRSPRRRTPSPRRRRSQSPRRRRSC, (iii) (SEQ ID NO: 8)
RRRGRSPRRRTPSPRRRRSQSPRRRRSQ, (iv) (SEQ ID NO: 9)
RRRGRPRRRPPRRRRQPRRRRC, (v) (SEQ ID NO: 10) RRRGRSPRRRTPSPRRRRC,
(vi) (SEQ ID NO: 11) RRRGRPRRRPPRRRRC, (vii) (SEQ ID NO: 12)
RRRTPSPRRRRSQSPRRRRC, (viii) (SEQ ID NO: 13) RRRPPRRRRQPRRRRC. (ix)
(SEQ ID NO: 14) RRRGRSPRRRTPSPRRRRSQSPRRRRSHHHHHH, (x) (SEQ ID NO:
15) HHHHHHRRRGRSPRRRTPSPRRRRSQSPRRRRS, (xi) (SEQ ID NO: 16)
RRRGRPRRRPPRRRRQPRRRRHHHHHH, and (xii) (SEQ ID NO: 17)
HHHHHHRRRGRPRRRPPRRRRQPRRRR.
[0050] As described herein, the antimicrobial peptide can have one
or more modified amino acids. For example, in any of the
above-described sequences or consensus sequences, one or more of
the basic amino acids in the variable domains or in the entire
antimicrobial peptide can be a D-form of natural arginine, lysine,
or histidine, or an analog of natural arginine, lysine, or
histidine.
[0051] Any of the above-described sequences or consensus sequences
can be at the N- or C-terminus of the antimicrobial peptide.
[0052] The antimicrobial peptide described herein can also be
conjugated to a non-peptide moiety at the N- or C-terminus to form
a peptide conjugate. A non-peptide moiety can be a polymer (e.g., a
polyethylene glycol polymer), oligosaccharide, lipid, glycolipid,
solid support (e.g., a bead or nanoparticle), small molecule drug,
biotin, nucleic acid molecule, antibody, vitamin, carrier protein
(e.g., KLH, BSA, or OVA), or detectable label (e.g., fluorescent,
radioactive, or enzymatic label). The peptide conjugate also
exhibits an antimicrobial activity. Methods of generating peptide
conjugates are known in the art.
[0053] The antimicrobial peptide or peptide conjugate described
herein can be mixed with a pharmaceutically acceptable carrier to
form a pharmaceutical composition.
[0054] The composition can be formulated with a pharmaceutically
acceptable carrier such as a phosphate buffered saline, a
bicarbonate solution, and/or an adjuvant. Suitable pharmaceutical
carriers and diluents, as well as pharmaceutical necessities for
their use, are known in the art. This composition may be prepared
as an injectable, liquid solution, emulsion, or another suitable
formulation.
[0055] An effective amount of the composition described above may
be administered by intranasal inhalation, topical application, or
parenteral routes, e.g., intravenous injection, subcutaneous
injection or intramuscular injection. Alternatively, other modes of
administration including suppositories and oral formulations may be
desirable. For suppositories, binders and carriers may include, for
example, polyalkalene glycols or triglycerides. Oral formulations
may include normally employed incipients such as pharmaceutical
grades of saccharine, cellulose, magnesium carbonate and the like.
These compositions take the form of solutions, suspensions,
tablets, pills, capsules, sustained release formulations or
powders.
[0056] The above-described composition can be administered to a
subject (e.g., a human, another mammal, or a laboratory animal) to
treat a microbial infection or to inhibit growth of a microbe in
the subject. The composition can be used to treat an infection
caused by a Gram-positive or Gram-negative bacteria, fungus,
parasite, or virus, e.g., Pseudomonas aeruginosa, Klebsiella
pneumoniae, Shigella dysenteriae, Escherichia coli, Staphylococcus
aureus, Acinetobacter baumannii, Clostridium Difficile, Candida,
Aspergillus, Blastomyces, Cryptococcus neoformans, Cryptococcus
gattii, Coccidioides, Histoplasma, Pneumocystis jirovecii,
ringworm, Sporothrix, Exserohilum, or Cladosporium.
[0057] The specific disclosure below is to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present disclosure to its fullest extent. All
publications cited herein are hereby incorporated by reference in
their entirety.
Modified Antimicrobial Peptides Derived from Human HBcARD
[0058] A novel antimicrobial peptide from human hepatitis B virus
(HBV) core protein (HBc) arginine-rich domain (ARD) was previously
identified. See Chen et al. (2013), PLoS Pathog 9:e1003425
doi:10.1371/journal.ppat.1003425, and Chen et al., (2016), Appl
Microbiol Biotechnol. 100(21): 9125-9132. This HBcARD peptide
showed a broad spectrum antimicrobial activity against
Gram-negative and Gram-positive bacteria. In our mouse sepsis
model, injection of HBcARD peptide at 2 hour postinoculation can
protect 40% of S. aureus-infected mice from death. See Chen et al.
(2013).
[0059] The HBcARD peptides are highly conserved in the
hepadnaviruses of different species. To improve the antimicrobial
efficacy of HBcARD, we compared the sequences and antimicrobial
activities of various HBcARD peptides of mammalian, rodent, and
avian hepadnaviruses. HBcARD peptide of human hepadnaviruses
displayed the strongest antimicrobial activity.
[0060] To further improve the potency of our lead compound HBcARD,
we tested the antimicrobial activity of each of several HBcARD
derivatives, including peptides modified by truncation and
D-arginine substitution for L-arginine. See Table 1.
TABLE-US-00004 TABLE 1 Sequences of various HBcARD peptides tested
for their bactericidal activity HBcARD peptide amino acid sequence
L-147-183 TVVRRRGRSPRRRTPSPRRRRSQSPRRRRSQSRESQC (SEQ ID NO: 6)
D-147-183 TVVrrrGrSPrrrTPSPrrrrSQSPrrrrSQSrESQC (SEQ ID NO: 6)
150-177C RRRGRSPRRRTPSPRRRRSQSPRRRRSC (SEQ ID NO: 7) 150-177Q
RRRGRSPRRRTPSPRRRRSQSPRRRRSQ (SEQ ID NO: 8) 150-171C
RRRGRSPRRRTPSPRRRRSC (SEQ ID NO: 10) 157-177C RRRTPSPRRRRSQSPRRRRSC
(SEQ ID NO: 12) 164-177C RRRRSQSPRRRRSC (SEQ ID NO: 28) D-150-177C
rrrGrSPrrrTPSPrrrrSQSPrrrrSC (SEQ ID NO: 7) DL-150-177C
rRrGRSPrRrTPSPrRrRSQSPrRrRSC (SEQ ID NO: 7) LD-150-177C
RrRGRSPRrRTPSPRrRrSQSPRrRrSC (SEQ ID NO: 7) DL-dST-150-177C
rRrGRPrRrPPrRrRQPrRrRC (SEQ ID NO: 9) R: L-arginine; r:
D-arginine
Comparison of Sequences and Antimicrobial Activities of Human and
Non-Human HBcARD Peptides
[0061] By multiple sequence alignment, we compared the sequences of
HBcARD peptides of various hepadnaviruses, including human
(AAP31571.1), wooly monkey (AA074859.1), ground squirrel
(AAB08031.1), woodchuck (AAA46761.1), bat (AGT17576.1), duck
(AA049490.1), heron (AAA45737.1), parrot (AFY97786.1), Ross's goose
(AAR89928.1) and snow goose (AAD22001.1). See FIG. 1. Similar to
the HBcARD peptide of human hepatitis B virus, HBcARD peptides of
other mammalian hepadnaviruses (wooly monkey, ground squirrel,
woodchuck and bat) contained four clustering arginine-rich domains.
While the sequence homology of HBcARD peptides between avian and
mammalian hepadnaviruses was low, they both contained four highly
positive-charged domains. To compare their respective antimicrobial
activities, we determined the minimal bactericidal concentrations
of four HBcARD peptides derived from bat, woodchuck, duck and heron
hepadnaviruses. The results indicated that peptides derived from
bat and woodchuck hepadnaviruses exhibited potent antimicrobial
activity comparable to that from human hepatitis B virus. See Table
2. In contrast, peptides derived from avian hepadnaviruses
displayed lower antimicrobial activity. Because HBcARD peptides
from human HBV is shorter in length (147-183; 37 amino acids), we
focused on this peptide in our subsequent modification and
optimization experiments.
TABLE-US-00005 TABLE 2 Antimicrobial activity of human, bat,
woodchuck, duck and heron HBcARD peptides against Gram-positive and
Gram-negative bacteria Minimal bactericidal concentration (mg/L)
Human Bat Woodchuck Duck Heron Bacterial strain.sup.a HBcARD HBcARD
HBcARD HBcARD HBcARD P. aeruginosa 9.2 10 9.8 >70.6 >69.3
ATCC 9027 K. pneumoniae 9.2 5 9.8 >70.6 >69.3 ATCC 13884 E.
coli 18.4 20 19.6 ND ND ATCC 25922 A. baumannii 2.3 2.5 4.9 70.6
69.3 ATCC 17978 S. aureus 18.4 20 19.6 ND ND ATCC 19636 .sup.aATCC:
American Type Culture Collection; ND: not detectable
Comparison of Antimicrobial Activity of L- and D-HBcARD
Peptides
[0062] We used PeptideCutter to predict the potential protease
cleavage sites on the HBcARD 147-183 peptide (37-mer). The results
showed that more than 70% of the protease cleavage sites were
mapped to the arginine residues of HBcARD peptide (data not shown).
To improve the peptide stability in the serum, we synthesized a
modified peptide, D-HBcARD 147-183 peptide, by D-amino acid
replacement, in which all arginine residues were replaced with its
D-isomer.
[0063] To investigate whether D-arginine substitution will improve
the antimicrobial activity, L- and D-HBcARD peptides (37-mer) were
tested side-by-side against a wide variety of bacteria, including
P. aeruginosa, K pneumoniae, A. baumannii, E. coli, S. dysenteriae
and S. aureus. Relative to the L-HBcARD, the D-HBcARD peptide
displayed similar antimicrobial activity against A. baumannii
(MBC=2.3-4.6 mg/L), and better antimicrobial activity against P.
aeruginosa (MBC=4.6 mg/L), K. pneumoniae (MBC=4.6 mg/L), and S.
aureus (MBC=4.6-9.2 mg/L). However, for reasons that remain
unclear, the antimicrobial activity of D-HBcARD peptide against E.
coli and S. dysenteriae was decreased from 18.4 to 73.6 mg/L. See
Table 3.
TABLE-US-00006 TABLE 3 Comparison of minimal bactericidal
concentration (MBC) between wild type HBcARD 147-183 peptide
L-HBcARD and D-HBcARD Minimal bactericidal concentration (mg/L)
L-HBcARD D-HBcARD Bacterial strain.sup.a (37-mer) (37-mer)
Gram-negative P. aeruginosa ATCC 9027 9.2 4.6 P. aeruginosa ATCC
27853 9.2-18.4 4.6 K. pneumoniae ATCC 13884 9.2 4.6 E. coli ATCC
25922 18.4 73.6 A. baumannii ATCC 17978 2.3 2.3 A. baumannii ATCC
17978CR.sup.b 2.3 2.3 A. baumannii ATCC 19606 2.3 2.3 A. baumannii
ATCC 19606CR.sup.b 2.3 2.3 A. baumannii TCGH 45530.sup.b 2.3 2.3 A.
baumannii TCGH 46709.sup.b 4.6 4.6 S. dysenteriae Xen27.sup.c 18.4
73.6 Gram-positive S. aureus ATCC 19636 18.4 9.2 S. aureus ATCC
25923 18.4 4.6 S. aureus ATCC 29213 18.4 ~4.6 .sup.aATCC: American
Type Culture Collection .sup.bColistin-resistant A. baumannii
(Laboratory induced and Clinical isolates from Tzu Chi General
Hospital (Chen et al. PLoS Pathog. 2013; 9(6): e1003425.)
.sup.cCaliper Life Sciences, Inc.
Comparison of Serum Resistance Between L-HBcARD and D-HBcARD
[0064] To compare the stability of the 37-mer L- and D-HBcARD
peptides against serum protease degradation, we performed a
protease resistance assay. After incubation with 5% bovine serum at
37.degree. C. for 3 hours, more than 80% of L-HBcARD peptide was no
longer detectable by staining with Green Angel on SDS-PAGE. In
contrast, the signal intensity of the D-HBcARD peptide was not
reduced under the same condition. See FIG. 2, A. Similar results
were obtained, when L-HBcARD was incubated with 5% mouse or human
(male and female) sera. See FIGS. 2, B and C. These results
suggested that D-HBcARD was more resistant than L-HBcARD to
protease digestion of fetal bovine, mouse and human serums.
[0065] Furthermore, we determined the in vitro antimicrobial
activity of L- and D-HBcARD peptides in the presence of serum. The
results showed that both L-HBcARD and D-HBcARD peptides at 18.4
mg/L concentration were able to kill S. aureus in the MBC assay.
See FIG. 2, D. Upon the addition of 5% mouse serum, there was a
significant difference in the antimicrobial activities between L-
and D-HBcARD peptides (P<0.0001). While the treatment with
L-HBcARD peptide in the presence of 5% mouse serum produced
10.sup.7 bacterial colonies, treatment with D-HBcARD peptide
produced around 10.sup.3 colonies. Therefore, our D-HBcARD peptide
exhibited higher antimicrobial activity than that of the L-HBcARD
peptide in the presence of 5% mouse serum. This observation
suggested that D-HBcARD was more stable and resistant to
proteolytic degradation.
Hemolytic Activity of D-HBcARD Peptide
[0066] In the hemolysis assay, human RBCs were incubated with
serially-diluted doses (0 to 460 mg/L) of D-HBcARD, L-HBcARD, or
melittin, at 37.degree. C. for one hour. As shown in FIG. 3,
melittin caused 100% hemolysis at the concentration of 8.9 mg/L. In
contrast, no hemolytic activity of both D- and L-HBcARD peptides
was detected up to 460 mg/L (P<0.0001).
Comparison of In Vivo Protection Efficacies Between L- and D-Form
HBcARD Peptides in a Mouse Sepsis Model Infected with S. aureus
[0067] To investigate the in vivo protection efficacy of the 37-mer
L- and D-HBcARD peptides, we performed a mouse sepsis model
infected with S. aureus. ICR mice were first i.p. inoculated with
S. aureus (4.times.10.sup.6 CFU/mouse), followed by one single i.p.
injection of L-HBcARD, D-HBcARD and PBS at two hours
post-inoculation, respectively. All mice (n=10) administered with
PBS died at day 1. See FIG. 4, A. At 7 days post-inoculation, 40%
of mice treated with L-HBcARD (10 mg/kg) survived. See FIG. 4, A.
In contrast, D-HBcARD peptide was able to achieve a much higher
survival rate (60% for 5 mg/kg dose, and 100% for 10 mg/kg dose).
See FIG. 4, A. These results indicated that the in vivo efficacy of
HBcARD can be improved by the D-arginine replacement strategy
(P<0.0001).
Comparison of the Immunogenicity Between L- and D-Form HBcARD
Peptides in a Mouse Sepsis Model Infected with S. aureus
[0068] To investigate whether repeated treatments with the 37-mer
HBcARD peptides may compromise the efficacy by inducing
neutralization antibody in vivo, we immunized 3-week old mice three
times with L- and D-HBcARD peptides, respectively, prior to
bacterial infection. Two weeks after the first immunization,
immunized mice were then inoculated with S. aureus, followed by
i.p. injection with either L- or D-HBcARD peptides 1 hour
post-inoculation. Consistent with our previous report (Chen et al.,
2013), L-HBcARD still protected all mice from death, irrespective
of the prior immunizations with or without L-HBcARD peptide. See
FIG. 4, B. Control mice immunized with PBS and treated with PBS
showed a mortality near 80% within 24 hours after bacterial
challenge (P<0.05). Seven days post-inoculation, all mice
treated with L- and D-HBcARD (10 mg/kg) survived bacterial
challenge, irrespective of the prior immunization with or without
D-HBcARD. See FIG. 4, C. Therefore, prior immunizations with either
D-form or L-form peptides induced no neutralization activity
against the in vivo antimicrobial activity of subsequent treatments
with HBcARD peptides.
Significance of Terminal Cysteine
[0069] We tested the antimicrobial activity of additional HBcARD
derivatives. See Table 1. The antimicrobial activities of these
peptides were determined by minimal bactericidal concentration
(MBC). Unlike the parental peptide HBcARD 147-183 (37-mer) (see
Chen et al., 2013), the derivative peptide HBcARD 150-177Q (28-mer)
showed no detectable bactericidal activity against S. aureus. See
Table 4. To mimic the parental peptide HBcARD 147-183 with a
cysteine at the carboxyl terminus, we designed another 28-mer
derivative HBcARD 150-177C by replacing the terminal Q (glutamine)
residue of HBcARD 150-177Q with a C (cysteine) residue. See Table
1. Interestingly, this Q-to-C substitution effectively rescued the
bactericidal activity against S. aureus. See Table 4.
TABLE-US-00007 TABLE 4 Minimal bactericidal concentrations (MBC) of
modified HBcARD peptides against various Gram-negative and
Gram-positive bacteria MBC (uM) 150- 150- 150- 157- 164- Bacteria
strains 177C 177Q 171C 177C 177C Gram-negative P. aeruginosa
ATCC9027 2 -- -- -- -- P. aeruginosa 2 -- -- -- -- ATCC27853 E.
coli ATCC25922 2 -- -- -- -- A. baumannii ATCC17978 0.5 -- -- -- --
A. baumannii ATCC19606 0.25 -- -- -- -- A. baumannii ATCC45530 0.5
-- -- -- -- A. baumannii ATCC46709 1 -- -- -- -- Gram-positive S.
aureus ATCC19636 2 ND ND ND ND --, not determined; ND, no
detectable antimicrobial activity
Significance of Length or Arginine Content
[0070] Given the fact that the terminal cysteine appears to be
important for the bactericidal activity, we asked if the total
length of the HBcARD 150-177C peptide (28-mer) can be further
reduced. We compared the potencies against S. aureus among peptides
150-171C (20-mer), 157-177C (21-mer), and 164-177C (14-mer). As
shown in Table 4, none of these peptides showed detectable
activity. The results here indicated that shortened peptides with a
reduced number of arginines exhibited no antimicrobial activity
against S. aureus. See Table 4. On the other hand, we have shown
that shorter peptides containing 3 variable domains are sufficient
to kill other bacteria (data not shown).
The 28-Mer Peptide D-150-177C Improved Protection Efficacy in a
Sepsis Mouse Model
[0071] We compared the in vivo protection efficacy of HBcARD
150-177C peptides (28-mer) containing either D-form or L-form
arginines. See Table 1 and FIG. 5. ICR mice (n=10/group) were ip.
inoculated with S. aureus ATCC19636 (4.times.10.sup.6/mouse),
followed by treatment with modified peptides at two hours
post-inoculation. All PBS control mice died on day 1, and only 20%
of the mice treated with control peptide (150-177Q) survived.
Administration of L-150-177C peptide at the dose of 5 and 10 mg/kg
protected 30% and 70% mice from death, respectively. See FIG. 5.
When treated with 5 mg/kg of D-150-177C, all mice (100%)
survived.
The 28-Mer Peptide D-150-177C Protected Mice from Lung Infection
with Colistin-Resistant A. baumannii
[0072] BALB/c mice (n=8/group) were intra-tracheally inoculated
with colistin-resistant A. baumannii TCGH 46709 (3.4.times.10.sup.8
cfu/mouse). These lung-infected mice were ip. treated with colistin
(5 mg/kg/day) or D-150-177C (5 and 10 mg/kg/day), respectively. All
mice treated with colistin died at 60 hours post-inoculation with
drug-resistant A. baumannii. See FIG. 6. In contrast, there was a
dose-dependent protection effect from D-150-177C. A significant
difference was observed when the mice were treated with 10
mg/kg/day of D-150-177C peptide (p<0.05). See FIG. 6.
Comparison of In Vivo Toxicity Between Peptide D-150-177C and
Polymyxin B
[0073] Using the sepsis mouse model, we examined the survival rates
of ICR mice intra-peritoneally (i.p.) injected with the 28-mer
D-150-177C peptide (20-80 mg/kg body weight) and polymyxin B (a
colistin-related compound) (50 mg/kg body weight). See FIG. 7, A.
All mice treated with polymyxin B (50 mg/kg) died at day one, as
expected from the notorious nephrotoxicity of colistin and
polymyxin B to kidney. See FIG. 7, A. In contrast, no acute
toxicity was observed in the mice treated with 20 and 40 mg/kg
D-150-177C. See FIG. 7, A. When the doses were increased to 60 and
80 mg/kg, survival rates of mice were decreased to 80% and 40%,
respectively. See, FIG. 7, A. Liver injury can be detected by the
serum ALT level. Mice treated with D-150-177C in the dose range of
20 to 40 mg/kg showed a higher ALT level than polymyxin B, but the
p value is insignificant. See FIG. 7, B. The ALT levels of mice
treated with 60 mg/kg were significantly higher than those treated
at 20 mg/kg polymyxin B (p<0.01). See FIG. 7, B.
Comparison of In Vivo Protection Efficacies Among Various Modified
150-177C Peptides
[0074] The 28-mer peptide D-150-177C contains a total of 14
L-arginines substituted with 14 D-arginines. See Table 1 and FIG.
6. D-arginine is far more expensive than L-arginine. To reduce the
cost of peptide synthesis, we compared the in vivo protection
efficacies between peptides containing complete or partial
D-arginine substitution. See Table 1 and FIG. 8. Peptide
DL-150-177C is only partially D-arginine substituted, and exhibited
very similar protection efficacy to all-D-arginine substituted
peptide D-150-177C (100% substitution). See FIG. 8. In contrast,
the protection efficacy of peptide LD-150-177C (also partially
substituted) appeared to be less than DL-150-177C. See FIG. 8.
Serine and Threonine are Dispensable
[0075] We engineered a new 22-mer peptide, DL-dST-150-177C (see
Table 1), with two major modifications: (1) partial-D-arginine
substitution by using the backbone of peptide DL-150-177C; and (2)
deletion of 5 serine and one threonine residues from the parental
28-mer peptide DL-150-177C. This 22-mer peptide DL-dST-150-177C
showed remarkably improved protection efficacy compared to the
28-mers DL-150-177C and L-150-177C. See FIG. 9.
Bacterial Isolates
[0076] The antimicrobial activities of HBcARD peptides were tested
using a number of bacterial strains, including Pseudomonas
aeruginosa Migula strains (ATCC 27853, ampicillin-resistant and
ATCC 9027, ampicillin-resistant), Klebsiella pneumoniae strain
(ATCC 13884), Shigella dysenteriae Xen27 (Caliper Co.), Escherichia
coli strain (ATCC 25922), Staphylococcus aureus subsp. strains
(ATCC 25923, methicillin-resistantATCC 29213,
methicillin-resistantandATCC 19636, methicillin-resistant) and
Acinetobacter baumannii strains (ATCC 17978, ATCC 17978 CR,
ATCC19606, ATCC 19606 CR, TCGH 45530 and TCGH 46709). Clinical
isolates TCGH 45530 and TCGH 46709 were obtained from Tzu-Chi
Buddhist General Hospital (TCGH) in Taiwan, and were identified
using the Vitek system (Biomerieux Vitek, Inc., Hazelwood, Mo.,
USA). See Chang et al. (2012), J Microbiol Immunol Infect 45:37-42
doi:10.1016/j.jmii.2011.09.019.
Antimicrobial Assay
[0077] L- and D-HBcARD peptides were purchased from Yao-Hong
Biotechnology Inc. (Taipei, Taiwan). Antimicrobial activity was
determined as described. See, Chen et al., 2013. Briefly, bacteria
were grown in MH broth (Difco) to mid-logarithmic phase at
37.degree. C., and were diluted to 10.sup.6 CFU (colony formation
unit)/ml in phosphate buffer (10 mM sodium phosphate and 50 mM
sodium chloride, pH 7.2). Peptides were serially diluted in the
same buffer. Fifty microliters (.mu.l) of bacteria were mixed with
fifty .mu.l of peptides at varying concentrations, followed by
incubation at 37.degree. C. for 3 hours without shaking. At the end
of incubation, bacteria were placed on Mueller-Hinton broth agar
plates, and allowed to grow at 37.degree. C. overnight for
measurement of minimal bactericidal concentration (MBC). The lowest
peptide concentration that displayed no bacterial growth (zero
colony) was defined as MBC. All peptides were tested in
triplicate.
Stability to Proteases
[0078] L- and D-HBcARD peptides (0.5 nmol) were mixed with MBC
buffer in the presence or absence of 5% serum collected from
bovine, mouse and human origins. After incubation at 37.degree. C.
for 3 hours, the amounts of peptides surviving the protease
digestion were determined by SDS-PAGE electrophoresis and staining
with Green Angel. The images were quantified using image J software
and the intensities were normalized with the no serum control. To
investigate the effect of serum on the antimicrobial activity of
HBcARD peptides, L- and D-HBcARD peptides at their respective MBC
concentrations were incubated with S. aureus ATCC19636 strain
(10.sup.6 CFU/ml) at 37.degree. C. for three hours. Bacteria were
plated on MH agar and the antimicrobial activity was determined by
colony formation.
Hemolytic Activity
[0079] The hemolytic activities of peptides were determined by
hemolysis against human red blood cells (hRBCs). Human blood was
obtained in EDTA-containing tube and was centrifuged at 450 g for
10 min. The pellet was washed three times with PBS buffer, and a
solution of 10% hRBCs was prepared. hRBCs solution was mixed with
serial dilutions of peptides in PBS buffer, and the reaction
mixtures were incubated for 1 h at 37.degree. C. After
centrifugation at 450 g for 10 min, the percentage of hemolysis was
determined by measuring the absorbance at the wavelength of 405 nm
of the supernatant. Blank and 100% hemolysis were determined in PBS
buffer and in the presence of 1% Triton X-100, respectively.
In Vivo Animal Studies
[0080] Three-week old male ICR mice (19 to 21 g) were purchased
from BioLASCO (Taiwan). To test the in vivo protection efficacy of
the HBcARD peptides, all mice were inoculated intraperitoneally
with S. aureus ATCC 19636 (4.times.10.sup.6 CFU/mouse). HBc147-183
(L- or D-HBcARD) or the PBS control was administered
intraperitoneally at 2 hours after bacterial inoculation,
respectively. Each group contained 10 mice. Mortality was monitored
daily for 7 days following the bacterial inoculation.
[0081] To investigate the potential immunogenicity of HBcARD
peptides, we determined the in vivo antimicrobial activity of
HBcARD peptide in peptide-immunized mice. Briefly, three week-old
male mice were immunized three times with 0.2 ml of L- and D-HBcARD
peptides (5 mg/kg) at day 0, 3, 6, respectively. Immunized mice
were inoculated with S. aureus ATCC 19636 (4.times.10.sup.6
CFU/mouse) at day 14, and were administered with 0.2 ml of PBS, or
L- and D-HBcARD peptide (10 mg/kg) one-hour postinoculation.
Another group of mice received the identical protocol with PBS as a
control. Each group contained 5 mice. Mortality was monitored daily
for 7 days following the bacterial inoculation.
Statistical Analysis
[0082] Statistical analysis was performed using Graphpad software.
The results were shown in mean.+-.SD, and the difference between
individual groups was analyzed by student t test. Survival curves
were plotted by Kaplan-Meier method and analyzed by log-rank
test.
OTHER EMBODIMENTS
[0083] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless explicitly
stated otherwise, each feature disclosed is only an example of a
generic series of equivalent or similar features.
[0084] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the described
embodiments, and without departing from the spirit and scope
thereof, can make various changes, derivatives, and modifications
of the embodiments to adapt it to various usages and conditions.
Thus, other embodiments are also within the claims.
Sequence CWU 1
1
28116PRTArtificial SequenceSynthetic
polypeptideMISC_FEATURE(1)..(1)Xaa can be 2 to 20 amino acids
selected from arginine, lysine, and
histidineMISC_FEATURE(4)..(4)Xaa can be any amino acid or
absentMISC_FEATURE(6)..(6)Xaa can be 2 to 20 amino acids selected
from arginine, lysine, and histidineMISC_FEATURE(7)..(7)Xaa can be
any amino acid or absentMISC_FEATURE(9)..(9)Xaa can be any amino
acid or absentMISC_FEATURE(11)..(11)Xaa can be 2-20 amino acids
each selected from arginine, lysine, and
histidineMISC_FEATURE(12)..(12)Xaa can be any amino acid or
absentMISC_FEATURE(14)..(14)Xaa can be any amino acid or
absentMISC_FEATURE(16)..(16)Xaa can be 2-20 amino acids each
selected from arginine, lysine, and histidine 1Xaa Gly Arg Xaa Pro
Xaa Xaa Pro Xaa Pro Xaa Xaa Gln Xaa Pro Xaa1 5 10
15211PRTArtificial SequenceSynthetic
polypeptideMISC_FEATURE(1)..(1)Xaa can be 2 to 20 amino acids
selected from arginine, lysine, and
histidineMISC_FEATURE(4)..(4)Xaa can be any amino acid or
absentMISC_FEATURE(6)..(6)Xaa can be 2 to 20 amino acids selected
from arginine, lysine, and histidineMISC_FEATURE(7)..(7)Xaa can be
any amino acid or absentMISC_FEATURE(9)..(9)Xaa can be any amino
acid or absentMISC_FEATURE(11)..(11)Xaa can be 2 to 20 amino acids
selected from arginine, lysine, and histidine 2Xaa Gly Arg Xaa Pro
Xaa Xaa Pro Xaa Pro Xaa1 5 10311PRTArtificial SequenceSynthetic
polypeptideMISC_FEATURE(1)..(1)Xaa can be 2 to 20 amino acids
selected from arginine, lysine, and
histidineMISC_FEATURE(2)..(2)Xaa can be any amino acid or
absentMISC_FEATURE(4)..(4)Xaa can be any amino acid or
absentMISC_FEATURE(6)..(6)Xaa can be 2 to 20 amino acids selected
from arginine, lysine, and histidineMISC_FEATURE(7)..(7)Xaa can be
any amino acid or absentMISC_FEATURE(9)..(9)Xaa can be any amino
acid or absentMISC_FEATURE(11)..(11)Xaa can be 2 to 20 amino acids
selected from arginine, lysine, and histidine 3Xaa Xaa Pro Xaa Pro
Xaa Xaa Gln Xaa Pro Xaa1 5 10418PRTArtificial SequenceSynthetic
polypeptideMISC_FEATURE(1)..(1)Xaa can be 2 to 20 amino acids
selected from arginine, lysine, and
histidineMISC_FEATURE(4)..(4)Xaa can be any amino acid or
absentMISC_FEATURE(6)..(6)Xaa can be 2 to 20 amino acids selected
from arginine, lysine, and histidineMISC_FEATURE(7)..(7)Xaa can be
any amino acid or absentMISC_FEATURE(9)..(9)Xaa can be any amino
acid or absentMISC_FEATURE(11)..(11)Xaa can be 2 to 20 amino acids
selected from arginine, lysine, and
histidineMISC_FEATURE(12)..(12)Xaa can be any amino acid or
absentMISC_FEATURE(14)..(14)Xaa can be any amino acid or
absentMISC_FEATURE(16)..(16)Xaa can be 2 to 20 amino acids selected
from arginine, lysine, and histidineMISC_FEATURE(17)..(17)Xaa can
be any amino acid or absent 4Xaa Gly Arg Xaa Pro Xaa Xaa Pro Xaa
Pro Xaa Xaa Gln Xaa Pro Xaa1 5 10 15Xaa Cys518PRTArtificial
SequenceSynthetic polypeptideMISC_FEATURE(1)..(1)Xaa can be 2 to 20
amino acids selected from arginine, lysine, and
histidineMISC_FEATURE(4)..(4)Xaa can be any amino acid or
absentMISC_FEATURE(6)..(6)Xaa can be 2 to 20 amino acids selected
from arginine, lysine, and histidineMISC_FEATURE(7)..(7)Xaa can be
any amino acid or absentMISC_FEATURE(9)..(9)Xaa can be any amino
acid or absentMISC_FEATURE(11)..(11)Xaa can be 2 to 20 amino acids
selected from arginine, lysine, and
histidineMISC_FEATURE(12)..(12)Xaa can be any amino acid or
absentMISC_FEATURE(14)..(14)Xaa can be any amino acid or
absentMISC_FEATURE(16)..(16)Xaa can be 2 to 20 amino acids selected
from arginine, lysine, and histidineMISC_FEATURE(17)..(17)Xaa can
be any amino acid or absent 5Xaa Gly Arg Xaa Pro Xaa Xaa Pro Xaa
Pro Xaa Xaa Gln Xaa Pro Xaa1 5 10 15Xaa Gln637PRTArtificial
SequenceSynthetic polypeptide 6Thr Val Val Arg Arg Arg Gly Arg Ser
Pro Arg Arg Arg Thr Pro Ser1 5 10 15Pro Arg Arg Arg Arg Ser Gln Ser
Pro Arg Arg Arg Arg Ser Gln Ser 20 25 30Arg Glu Ser Gln Cys
35728PRTArtificial SequenceSynthetic polypeptide 7Arg Arg Arg Gly
Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg1 5 10 15Arg Arg Ser
Gln Ser Pro Arg Arg Arg Arg Ser Cys 20 25828PRTArtificial
SequenceSynthetic polypeptide 8Arg Arg Arg Gly Arg Ser Pro Arg Arg
Arg Thr Pro Ser Pro Arg Arg1 5 10 15Arg Arg Ser Gln Ser Pro Arg Arg
Arg Arg Ser Gln 20 25922PRTArtificial SequenceSynthetic polypeptide
9Arg Arg Arg Gly Arg Pro Arg Arg Arg Pro Pro Arg Arg Arg Arg Gln1 5
10 15Pro Arg Arg Arg Arg Cys 201019PRTArtificial SequenceSynthetic
polypeptide 10Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser
Pro Arg Arg1 5 10 15Arg Arg Cys1116PRTArtificial SequenceSynthetic
polypeptide 11Arg Arg Arg Gly Arg Pro Arg Arg Arg Pro Pro Arg Arg
Arg Arg Cys1 5 10 151220PRTArtificial SequenceSynthetic polypeptide
12Arg Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg1
5 10 15Arg Arg Arg Cys 201316PRTArtificial SequenceSynthetic
polypeptide 13Arg Arg Arg Pro Pro Arg Arg Arg Arg Gln Pro Arg Arg
Arg Arg Cys1 5 10 151433PRTArtificial SequenceSynthetic polypeptide
14Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr Pro Ser Pro Arg Arg1
5 10 15Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser His His His His
His 20 25 30His1533PRTArtificial SequenceSynthetic polypeptide
15His His His His His His Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg1
5 10 15Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg
Arg 20 25 30Ser1627PRTArtificial SequenceSynthetic polypeptide
16Arg Arg Arg Gly Arg Pro Arg Arg Arg Pro Pro Arg Arg Arg Arg Gln1
5 10 15Pro Arg Arg Arg Arg His His His His His His 20
251727PRTArtificial SequenceSynthetic polypeptide 17His His His His
His His Arg Arg Arg Gly Arg Pro Arg Arg Arg Pro1 5 10 15Pro Arg Arg
Arg Arg Gln Pro Arg Arg Arg Arg 20 251836PRTWooly monkey HepB virus
18Thr Val Val Arg Arg Arg Arg Pro Ser Gly Arg Arg Thr Pro Ser Pro1
5 10 15Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser Gln Ser
Pro 20 25 30Ala Ser Ser Cys 351941PRTGround squirrel HepB virus
19Thr Val Ile Arg Arg Arg Gly Ser Ala Arg Val Val Arg Ser Pro Arg1
5 10 15Arg Arg Thr Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg
Arg 20 25 30Arg Pro Gln Ser Pro Ala Ser Asn Cys 35
402042PRTWoodchuck HepB virus 20Thr Val Ile Arg Arg Arg Gly Gly Ala
Arg Ala Ser Arg Ser Pro Arg1 5 10 15Arg Arg Thr Pro Ser Pro Arg Arg
Arg Arg Ser Gln Ser Pro Arg Arg 20 25 30Arg Arg Ser Gln Ser Pro Ser
Ala Asn Cys 35 402143PRTBat HepB virus 21Thr Ile Val Arg Arg Arg
Gly Gly Ser Arg Ala Thr Arg Ser Pro Arg1 5 10 15Arg Arg Thr Pro Ser
Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg 20 25 30Arg Arg Ser Gln
Ser Pro Ala Ser Ser Asn Cys 35 402236PRTDuck HepB virus 22Arg Lys
Pro Arg Gly Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr1 5 10 15Val
Val Tyr Gly Arg Arg Arg Ser Lys Ser Arg Glu Arg Arg Ala Pro 20 25
30Thr Pro Gln Arg 352336PRTHeron HepB virus 23Arg Lys Pro Arg Gly
Leu Glu Pro Arg Arg Arg Lys Val Lys Thr Thr1 5 10 15Val Val Tyr Gly
Arg Arg Arg Ser Lys Ser Arg Gly Arg Arg Ser Ser 20 25 30Pro Ser Gln
Arg 352436PRTParrot HepB virus 24Arg Lys Pro Arg Gly Leu Glu Pro
Arg Arg Arg Lys Val Lys Thr Thr1 5 10 15Val Val Tyr Gly Arg Arg Arg
Ser Lys Ser Arg Glu Arg Ser Ser Ser 20 25 30Ser Pro Gln Arg
352536PRTRoss' Goose HepB virus 25Arg Lys Pro Arg Gly Leu Glu Pro
Arg Arg Arg Lys Val Lys Thr Thr1 5 10 15Val Val Tyr Gly Arg Arg Arg
Ser Lys Ser Arg Glu Arg Arg Ala Pro 20 25 30Thr Pro Gln Arg
352636PRTSnow goose HepB virus 26Arg Lys Pro Arg Gly Leu Glu Pro
Arg Arg Arg Lys Val Lys Thr Thr1 5 10 15Val Val Tyr Gly Arg Arg Arg
Ser Lys Ser Arg Glu Arg Arg Ala Ser 20 25 30Ser Pro Gln Arg
352720PRTArtificial SequenceSynthetic
polypeptideMISC_FEATURE(1)..(1)Xaa can be arginine, lysine, or
histidineMISC_FEATURE(2)..(2)Xaa can be arginine, lysine, or
histidineMISC_FEATURE(3)..(3)Xaa can be arginine, lysine, or
histidine or absentMISC_FEATURE(4)..(4)Xaa can be arginine, lysine,
or histidine or absentMISC_FEATURE(5)..(5)Xaa can be arginine,
lysine, or histidine or absentMISC_FEATURE(6)..(6)Xaa can be
arginine, lysine, or histidine or absentMISC_FEATURE(7)..(7)Xaa can
be arginine, lysine, or histidine or absentMISC_FEATURE(8)..(8)Xaa
can be arginine, lysine, or histidine or
absentMISC_FEATURE(9)..(9)Xaa can be arginine, lysine, or histidine
or absentMISC_FEATURE(10)..(10)Xaa can be arginine, lysine, or
histidine or absentMISC_FEATURE(11)..(11)Xaa can be arginine,
lysine, or histidine or absentMISC_FEATURE(12)..(12)Xaa can be
arginine, lysine, or histidine or absentMISC_FEATURE(13)..(13)Xaa
can be arginine, lysine, or histidine or
absentMISC_FEATURE(14)..(14)Xaa can be arginine, lysine, or
histidine or absentMISC_FEATURE(15)..(15)Xaa can be arginine,
lysine, or histidine or absentMISC_FEATURE(16)..(16)Xaa can be
arginine, lysine, or histidine or absentMISC_FEATURE(17)..(17)Xaa
can be arginine, lysine, or histidine or
absentMISC_FEATURE(18)..(18)Xaa can be arginine, lysine, or
histidine or absentMISC_FEATURE(19)..(19)Xaa can be arginine,
lysine, or histidine or absentMISC_FEATURE(20)..(20)Xaa can be
arginine, lysine, or histidine or absent 27Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa1 5 10 15Xaa Xaa Xaa Xaa
202814PRTArtificial SequenceSynthetic polypeptide 28Arg Arg Arg Arg
Ser Gln Ser Pro Arg Arg Arg Arg Ser Cys1 5 10
* * * * *