WO2014124047A1 - Antimicrobial peptides derived from hepatitis b virus core protein arginine-rich domain - Google Patents

Abstract

A pharmaceutical composition for use in killing and/or inhibiting the growth and/or proliferation of a microorganism in a subject in need thereof, or for treating a subject afflicted with a microbial infection is disclosed. The composition comprises: (a) an effective amount of an isolated peptide, wherein the peptide comprises the arginine-rich carboxy-terminal region of hepatitis B virus core protein (HBc) and exhibits an antimicrobial activity; and (b) a pharmaceutically acceptable carrier. The peptide exhibits an activity against Gram-negative bacteria, Gram-positive bacteria, and/or fungi.

Description

ANTIMICROBIAL PEPTIDES DERIVED FROM HEPATITIS B VIRUS CORE PROTEIN

AKGININE-RICH DOM A IN

FIELD OF TH E INVENTION

The present invention relates to antimicrobial peptides,

BACKGROUND OF THE INVENTION

The increase of drug-resistant pathogens caused by the extensive use of traditional antibiotics is serious concern worldwide. There is an urgent need to develop more effective treatment to overcome the drug-resistance problem. Antimicrobial peptides (AMP) are a new class of antibiotics with a new mode of action and remarkable therapeutic effects. In general, they contain 10-50 amino acids, wit an overall positive charge and an amphipathic structure. It is well known that most AMPs can directly bind to bacteria membrane and kill them by disrupting membrane or targeting intracellular components. Most importantly, they are effective to antibiotics-resistant pathogens. This unique feature has encouraged the development of AMPs as novel antibiotics in the last few decades.

Prior to the present invention, no literature has reported that peptides derived front hepatitis B virus core protein (HBc) possess antimicrobial activities. Hepatitis B virus core protein {21 KDsi) is essential for viral replication. It contains a capsid assembly domain at N-terminus (residue I to 1 9) and an arginine-rich domain (ARD) at C-terminus (residues 150 to 1 S3) (Bimbaum et al . (1990) 1 Virol 64: 3319-3330; Nassal M (1992} j Virol 66: 4107-41 16). ARD contains 16 argm es separated into four arginine-rich clusters (ARD I, II, Hi, IV) and has a function of binding to nucleic acids. When it binds to HBV pre-genomic RKA or polyanions, HBc can assemble into a stable capsid. In addition, ARD contains important signals for nuclear export and import of HBc core protein and particles. It was unexpectedly discovered that the growth of £ eoli expressing HBc! -183 was much slower than that of E. call expressing HBcl-149 (unpublished results).

SUMMARY OF THE INVENTION

in one aspect, the invention relates to a pharmaceutical composition comprisi ng:

(a) an effective amount of an isolated peptide, wherein the peptide comprises the arginine- rich carboxy-terminai region of hepatitis B virus core protein (HBc) and exhibits an ants microbial activity; and

(b) a pharmaceutically acceptable carrier.

In another aspect the invention relates to a pharmaceutical composition comprising.

(a) an effective amount of an isolated peptide comprising a fragment of HBc, the fragment comprising more than one arginine-rich domain (ARD) selected from the group consisting of (i), (ii) and (iii) as follows:

(i) HBc ARD [-IV; (it) HBc ARD Ϊ-ΠΪ; or

(iii) HBc ARD H-IV;

wherein the peptide exhibits an antimicrobial activity, and

(b) a pharmaceutically acceptable carrier,

In another aspect, the in ention relates to a pharmaceutical composition comprising effective amount of an isolated peptide, the peptide comprising an argintne-rich sequence derived from the C- terminal region of hepatitis B vims core protein (HBc), wherein the peptide is characterized by having an antimicrobial activity- Further in another aspect, the invention relates to a pharmaceutical composition as

aforementioned for use in killing and/or inhibiting the growth and/or proliferation of a

microorganism by causing the composition as aforementioned to be in contact with the

microorganism.

Yet in another aspect, the invention relates to a pharmaceutical composition as aforementioned for use in killing and/or inhibiting the growth and/or proliferation of a microorganism in a subject in need thereof or for treating a subject afflicted with a microbial infection. The subject is afflicted wi th Staphylococcias aureus or A' pneumoniae infection.

These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings, although variations and modifications therein may be affected withou departing from the spirit and scope of the novel concepts of the disclosure.

The accompanying drawings illustrate one or more embodiments of the invention and, together with the written description, serve to explain the principles of the invention. Wherever possibie, the same reference numbers are used throughout the drawings to refer to the same or like elements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. I shows amino acid sequences of various HBc ARD peptides tested for bactericidal activity. The lower panel presents various phosphorylated peptides and an R-to-A mutant peptide with a total of four Arg-to-Ala substitutions in AR -Ιϋ and ARD-IV of HBc 147-183.

FIG. 2 shows killing kinetics of HBcl.47-183 against. P. aeruginosa, K. pneumoniae, K colt and S. aureus. Bacteria were treated with HBc 147- 183 (IxMBC). The viability of bacteria was measured at indicated time points. Samples were measured in triplicates.

FIG. 3 shows localization of FITC-HBc 147-183 peptide on bacteria. Approximate 10⁷ CFU of P. aeruginosa ATCC9027, ATCC27853 (A and B), K, pneumoniae ATCC13884 (C), £ coli

ATCC25922 (D), and S.aumm ATCC 19636, ATCC25923 and ATCC 29213 (E, F and G) were incubated with HBc^'147-183 (O.Sx BC) for 1 hour. The bacteria were washed, fixed and stained with DAPl (blue). Images were taken using eonfocal microscopy.

FIG. 4 shows possible bactericidal mechanisms of HBcl 47-1.83. (A) SYTOX Green uptake of P aeruginosa, K. pneumoniae, E. coH, and S. aureus by HBc^'147-183. Measurements of the fluorescence were recorded every minute. (B) Dose-dependent curves of membrane penneabiiizaiion of P. aeruginosa by HBc l 47-183 and HBc 153-176 at 0.5, 1 and 2 μΜ. Two uM melitiin was used as a positive control. Samples were measured in triplicates. (C) DNA-binding activity of HBcl 47- 183. HBc^'147-183 was mixed with pSUPER. plasraid D^'NA at indicated N P ratio for 30 minutes. The mobility of NA was determined by a gel retardation assay.

FIG. 5 shows dose response effects of LPS and LPS antibody on the bactericidal activity of HBcl47-i 83. LPS from P. aeruginosa and E coli, and LPS antibody were mixed with P. aeruginosa and HBc.147-183 (lxMBC) for 3 hours. The bacteria were then plated on MH agar for the measurement of viability. Samples were measured in triplicates.

FIG. 6 shows the ARD peptide HBcl47~183 being capable of binding to LPS and Lipid A in several different in vitro binding assays. Samples were measured in triplicates in each assay. (A.) The cartoon illustrates the in vitro assays of peptide-LPS and peptide-Lipid A binding as well as LPS/Iiptd A competition. (B) Constant amount of LPS was incubated with increasing concentrations of biotinylated ARD HBc 147-583 peptide on the streptavidine-conjugated beads (0, 0.004, 0.02, 0.1, 0.5 and 2.5 uM). Unbound LPS in the supernatant was measured with the LAL ELISA assay. The EU values were normalized with a control without peptide treatment. HBcl 47-183 8p (containing 8 phosphorylated amino acids) was also included as a control peptide due to its poor binding, with LPS.

(C) Beads-bound LPS was released into the supernatant by overnight digestion with trypsin agarose. Free LPS in the supernatant was analyzed with the LAL ELISA assay. Released LPS in the supernatant appeared to be in proportion to the amount of ARD peptide HBc l 47-183 on the beads.

(D) Constant amount of Lipid A was incubated with increasing concentrations of HBc 147- 183 and HBc 147- 183 8P, respectively. The supernatant was also detected with LAL ELISA reagent. The result here is consistent with the notion that Lipid A can bind to HBcl 7-183 directly. (E) LPS Lipid A competition assay. Constant amount of LPS (1 ug) was coated on each well on the ELISA plate, and then incubated with a reaction mixture containing constant amount of 10 nM HBc^'147-183 and. increasing concentrations of Lipid A. The gradual increase of Lipid A reduced the amount of plate- bound ARD peptide HBcl 7-1 S3 in a dose dependent manner.

FIG. 7 shows cytotoxicity assays of ARD peptide HBcl 7-183. (A) Hemolytic, activities of HBc^'147-183 and melitiin were measured with 10% human red blood cells (RBC). Compared to meliitifi, HBcl 47-183 showed no hemolytic activity. (B) Huh.7, HepG2, Veto and HEK293 cells were incubated with varying concentrations (0 to 100 μ ) of HBc 147-183 and melittin for 1 hour at 37°C. The effects on cell viability were determined by MTT assay. Melittin was used as a positive control . BBe 147-1 S3 showed no detectable effect on cell viabilitv, while .melittin exhibited strone toxicity. (C) Kidney cells, Vero and HEK293 were stained with CFS.E and seeded at day 0, At day I , cells were incubated with varying concentrations (0 to 100 μΜ) of HBc 1.47-183 for ! hour. Cell proliferation at day 1 and day3 were determined by flow cytometry. Similar to the mock control experiment, no significant effect on Vero and HEK293 ceils was detected. Samples assayed in FIGs. 7A-C were measured in triplicates. (D)

(D) In vivo toxicity of ARD peptide HBc 147- 183 was determined using three-week old. male ICR mice. The mice were injected intraperiionealSy with peptide (10 and 20 mg/kg of body weight). All mice were alive after 7 days.

FIG. 8 shows in vivo studies of the protection acti vity of ARD peptide HBc! 47-183 agai nst S. aureus. (A) Three-week old male ICR mice were challenged with a lethal dose of S. aureus ATCC 19636 and then divided into five separate groups for five different time points. At each indicated time point (n - 5), blood samples were collected, diluted and plated on BH1 agar. The number of bacteria was counted the following day. A. maximal bacterial load in the blood was observed at 2 h post-inoculation. The data were shown in mean 6 SD. (B) ICR mice inoculated with a lethal dose of S. aureus as described above were treated by intraperitoneal injection with ARD peptide (10 mg/kg) at 1, 1.5 or 2 h post-inoculation, respectively. Each group contained 10 mice. Ail mice (100%) treated with the PBS control died at day 1, while treatment of ARD peptide at 1, 1.5 or 2 h post- inoculation protected the mice with survival rates of 100%, 70% and 40% after 7 days, respectively. (C) As described above, ICR mice were i.p. inocuiated with S. aureus, followed by i.p. injection with PBS (n ^:::: 5) or 10 mg/kg ARD peptide ( ^::: 5) at 1 h post-inoculation. At 4 h post-inoculation, blood, liver and spleen were collected. Liver and spleen samples were homogenized, diluted and, together with blood samples, plated on BHI agar. The number of bacteria was counted the following day. in comparison to mice treated with PBS, treatment of ARD peptide effectively reduced the bacterial load in blood, liver and spleen. (D) Quantitative comparison of bacterial loads in blood, liver and spleen samples of mice treated with PBS (open circle, diamond and square) versus ARD peptide HBc 1.47- i 83 (solid circle, diamond and square). The line indicated the mean of bacterial load.

**P,0.01 (Mann-Whitney U test) for PBS and ARD peptide HBc J 47-183.

FIG. 9 shows an IVIS analysis of in vivo antimicrobial activity of ARD peptide against K.

pneumoniae. (A) K. pneu tua&Anfected mice were treated with either PBS (n = 5) or 10 mg/k ARD peptide (n. ~ 5) at 1 hour post-inocuiation. Four hours post-inoculation, mice were anesthetized and imaged. Bacteria! load was displayed in the photographic image with an overlay of bioluminescence. False color imaging represents intense luminescence in red, moderate luminescence in green and lo luminescence in blue and purple, (B Total flux was quantified by JVIS imaging software. **P_}0.01 (Mann- Whitney U test) for PBS and ARD peptide HBc 147- 83.

FIG. 10 is a table showing antimicrobial activity of HBC ARD peptides.

FIG. 1 1 is a table showing antimicrobial acti vity of ARD peptide HBcI47-183 against colistin- resistant and sensitive /*, aeruginosa and A. baimiatmii.

FIG. 12 shows sequence alignments of human hepatitis B virus fHBV) core protein (HBc) argtnine rich domain (ARD) HBc ARD domain is highly conserved among different serotypes isolated from patients of different geographic areas.

FIGs. I3A-B show sequence alignments of HBc ARD domains of hepadnaviruses from primate, rodent and avian origins. HBc ARD sequences are highly conserved among human, wooly monkey, ground squirrel,, woodchuck, and bat. The arginine (positive charge) clustering subdomaias at HBc ARD are designated as ARD-I, ARD- if ARD -III, and ARD-IV. FIG. 13 B further shows that there are also four clustering positi e charge amino acids in the core protein C-terminus of duck, heron, parrot. Ross's goose and snow goose hepatitis B virus. Despite the sequence divergence and evolutionary distance between the primate, rodent, and avian hepadnaviruses, it is contemplated that these positive charge-rich domain at the C-terminus of core protein from rodent (A) and avian (B) hepadna viruses could also contain antimicrobial activities (cf Table 1 ).

DETAILED DESCRIPTION OF THE INVENTION

The present, invention is more particularly described in the following examples that are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art. Various embodiments of the invention are now described in detail. Referring to the drawings, like numbers indicate like components throughout the views. As used in the description herein and throughout, the claims that follow, the meaning of " ", "an", and "the" includes plural reference unless the context clearly dictates otherwise. Also, as used in the

description herein and throughout the claims that follow, the meaning of "in" Includes "in" and "on" unless the context clearly dictates otherwise. Moreover, titles or subtitles .may be used in the specification for the convenience of a reader, which shall have no influence on the scope of the present invention. Additionally, some tenns used in this specification are more specifically defined below.

DEFINITIONS

The terms used in this specification generally have their ordinary meanings in the art, within the context of the invention, and in the specific context where each term is used. Certain terras that are used to describe the invention are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner regarding the description of the invention. For convenience, certain terms may be highlighted, for example using italics and/or quotation marks. The use of highlighting has no influence on the scope and meaning of a term; the scope and meaning of a tenn is the same, in the same context, whether or not it is highlighted. It wiiS be appreciated that same thing can be said in more than one way. Consequently, alternative language and synonyms may be used for any one or more of the terms dtscussed herein, nor is any special significance to he placed upon whether or not a term is elaborated or discussed herein. Synonyms for certain terms are provided. A recital of one or more synonyms does not exclude the use of other synonyms. The use of examples anywhere in this specification including examples of any terms discussed herein is illustrative only, and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to various embodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the case of conflict, the present document, including definitions will control .

As used herein, "around", "about" or "approximately" shall generally mean within 20 percent, preferably within 10 percent, and more preferably within 5 percent of a given value or range.

Numerical quantities given herein are approximate, meaning that the term "around", "about" or "approximately" can be i nferred if not expressly stated.

Antimicrobial activity refers to the activity to kill or inhibit the growth of microorganisms such as bacteria, fungi and/or protozoans.

As used herein, the term "the arginine-nch carboxy-terminal region of hepatitis B virus core protei n (HBc)" refers to a highly conserved arginine-nch C-terminal region of HBc (FIGs. 12 and 13 A-B) and is characterized by having an antimicrobial activity. It is contemplated that the argtnine- rich earboxy-tenninai regions from avian and/or rodent hepadnaviruses share the same antimicrobial activities. The C-terminal end of HBc contains four arginine-rieh clusters designated as ARD 1-lV. Each arginine-rich cluster or domain (ARD) contains 2 or more arginine residues in continuity or in close proximity (e.g., being separated by one or two different amino acid residues). In one embodiment, each ARD may contain 2,3 or 4 continuous arginine residues.

As used herein, "an amino acid sequence derived from HBc" refers to "an amino acid sequence originates from hepatitis B virus core protein and possess an antimicrobial activity", it may be a fragment of HBc, with or without a modification, which contains ARD and possess an antimicrobial, activity . A fragment of HBc wi th a modification includes, but not limited to PEGylation at either N- or C-terminus. The term "two clusters" and "two repeats" are interchangeable. The terra "two clusters of

SPRRRR" means "2 repeats of SPRRRR", or "2 SPRRRR".

The term "amphipathic structure "refers to a molecule having hydrophobic and hydrophi!ic regions,

The HBV is divided into four major serotypes (adr, adw, ayr, ay w) based on antigenic epitopes present on its envelope proteins. The term "serotype" or "serovar" refers to distinct variations within a species of bacteria or viruses or among immune cells of different individuals.

The tenn "protecting group^" refers to a functional group that is attached to a therapeutic protein or peptide to prolong its circulator}.' time. A protecting group includes, but not limited, to a

polyethylene glycol (PEG), PBGylation can also provide water solubility to hydrophobic proteins or peptides.

The tenn "treating" or "treatment" refers to administration of an effective amount of a

therapeutic agent to a subject in need thereof with the purpose of cure, alleviate, relieve, remedy, ameliorate, or prevent the disease,, the symptoms of it, or the predisposition towards it. Such a subject can be identified by a health csxe professional based on results from any suitable diagnostic method.

"An effective amount" refers to the amount of an active agent that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on routes of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.

The "Guidance for Industry and Reviewers Estimating the Safe Starting Dose in Clinical Trials for Therapeutics in Adult Healthy Volunteers" published by the U.S. Department of Health and Human Services Food and Drug Administration discloses "a human equivalent dose" may be obtained by calculations from the following formula:

HED - animal close in mg kg x (animal weight in kg/human weight in kg) ^{0 ¾},

For example for i.p. administration, if a mouse (20 gram BW) dose is 10 mg/kg, then an. human dose ma be calculated as 10 mg/kg x (0.02/patient's body weight)^0"". A human equivalent effective dose, however, may vary, depending on other factors such as the route of adm nistration.

It was an unexpected discovery and unsolved mystery that the growth of E, coti expressing HBcl-183 was very poor and much slower than that of E, coU expressing HBc I -149. it appeared that it was HBc 150-183 that somehow retarded the growth of E coli, and reduced HBc 1-183 protein yield dramatically Here, we disclose the in vitro antimicrobial activities of HBcl47-.! 83 against a wide variety of bacteria, including multidrug resistant (MDR) and coiistin (polymyxin E)-resistant A bimman i. The antimicrobial peptides from HBV core protein (HBc) arginine-rich domain (A D) are mainly composed o SPRRR repeats and are effective against both Gram-positive and Gram- negative bacteria, as well as fungi . Using a peritoneal sepsis mouse model, it was demonstrated further thai ARD peptides can effectively protect ail the mice challenged with a lethal dose of Staphylococcus aureus. Treatment of ARD peptides also caused significant reduction of bacterial load of S. ureus and K, pneumoniae in infected mice. Potential mechanisms for the bactericidal activit were investigated. The ARD peptides appeared to be capable of direct binding to the Lipid A moiety of iipopolysac haride (LPS) in several different binding assays. In summary, with high antimicrobial activity and very low toxicity against human ceils and animal models, these HBc ARD peptides may have a therapeutic potential in the future (Chen et a! . " identification of a Novel Antimicrobial Peptides from Human Hepatitis B Vims Core Protein Argimne-Rich Domain (ARD) " PLoS Paihog 9(6): e S.Q(B425, which is incorporated herein by reference in its entirety),

in one aspect, the invention relates to a pharmaceutical composition comprising:

( a) an effective amount of an isolated peptide, wherein the pepti de comprises the arginme- rich carboxy-terminai regi on of hepatitis B viais core protein (HBc) and exhibits an antimicrobial activity; and

(b) a pharmaceutically acceptable carrier.

In another aspect, the invention relates to a pharmaceutical composition comprising:

(a) an effective amount of an isolated peptide comprising a .fragment of HBc, the fragment comprising more than one arginine-rich domain (ARD) selected from the group consisting of (i), (ii) and (Hi) as follows:

(i) HBc ARD 1-I V .

(ii) HBc ARD MIL; or

(Mi) HBc ARD !HV;

wherein the peptide exhibits an antimicrobial activity; and

(b) a pharmaceutically acceptable carrier.

The HBc may be selected from the group consisting of a mammalian HBc and an avian HBc. A mammalian HBc includes, but not limited to, human hepatitis B core protein (HBc), woolly monkey HBc, ground squirrel HBc, woodchucfc HBc, and bat HBc. An avian HBc includes, but not limited to. duck, heron, parrot, Ross's goose, and snow goose.

In another aspect, the invention rel ates to a pharmaceutical composition compri sing an effective amount of an isolated peptide, the peptide comprising an arginme-rich sequence derived from the C- terminai region of hepatitis B virus core protein (HBc), wherein the peptide is characterized by having an antimicrobial activity.

Further in another aspect, the invention relates to a pharmaceutical composition as

aforementioned for use in ki iling and/or inhibiting the growth and/or proliferation of a microorganism by causing the composition to be in contact with the microorganism. The microorganism may be present in a subject.

Yet in another aspect, the invention relates to a pharmaceutical composition as aforementioned for use in killing and/or inhibiting the growth and/or proliferation of a microorganism in a subject in need thereof or for treating a subject afflicted with a microbial infection. The subject may be afflicted with Stetphylocoecus aureus or K. pneumoniae infection.

The antimicrobial peptide according to the invention contains few or no hydrophobic amino acids, and thus has no amprripathic structure.

In one embodiment of the invention, the peptide comprises the amino acid sequence of Ser Pro Arg Arg Arg Arg (SPRRRR; SEQ ID NO: 13) or Arg Arg Arg Ser (RRRS; SEQ 3D NO: 1 )

Alternatively, the peptide may comprise two clusters of SPRRRR (SEQ ID NO: 13), or three clusters of SPRRR (SEQ ID NO: 15). The peptide may comprise RRRS (SEQ ID NO: 14).

In another embodiment of the invention, the peptide comprises at feast 2 clusters of Pro Arg (PR) located upstream to the sequence RRRS (SEQ ID NO: 14). The at least 2 clusters of PR may be immediately adjacent to, or nearby the RRRS sequence with a few residues apart, such as 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, .16, 17 or I S amino acid residues apart.

The peptide has no RRGGRRRR sequence (SEQ ID NO: 17) at the C -terminus thereof. The peptide may comprise a protecting group.

in another embodiment of the invention, the peptide has a cysteine (C) at the C-teraiinus thereof in another embodiment of the invention, the peptide has at least 1 amino acids but no more than7 amino acids in length.

In another embodiment of the invention, the pepti de is characterized by having an activity against Gram-negative bacteria. Gram-positive bacteria, and/or fungi .

In another embodiment, of the invention, the peptide comprises more than one HBc argi nine-rich domain selected from the group consisting of (i), (u) and (iii ) as follows:

<i) HBc ARD l IX III, and IV;

(ii) HBc ARD f II and III: or

(iii) HBc ARD It, III, and IV.

The peptide comprises 3 or 4 ARD. in another embodiment of the invention, the peptide exhibits an activity against colistin-resistant baumanmi.

in another embodiment of the invention, the peptide is free of the sequence of SQSRESQC (SEQ ID NO: 1 6) at the C-terminus thereof and is characterized by having an activity against Gram- negative bacteria. Alternatively, the peptide may comprise HBc ARD 11-1 but without HBc ARD Ϊ and exhibits an activity against P. aeruginosa, or the peptide may comprise HBc ARD i-Iil but without HBc ARD IV and exhibits an activit against. K. ptmm ia.

In another embodiment of the invention, the peptide exhibits the following characteristics:

i) a reduced antimicrobial activity provided that the Ser in one of the two SPR RR {SEQ ID NO:

13) clusters is phosphorylated; and/or

ii) a loss of antimicrobial activity provided that the Ser in each SPRRR (SEQ ID NO: 15) cluster is phosphorylated.

The peptide exhibits bactericidal activity and has no cytotoxicity to red blood cells, kidney cells, and/or !iver cells

in another embodiment of the invention, the aforementioned peptide compri ses: i) Ser or Pro amino acid residues downstream to the ARD IV at the C-terminal portion of the peptide. The peptide may further comprise: ii ) Ser and/or Pro amino acid residues between each ARD (i.e., between ARD 1 and II, between ARD ΙΪ and III, and between ARD 10 and IV.

in another embodiment of the invention, the Ser residue in the amino acid sequence of the peptide is not phosphorylated.

The composition may be formulated for topical, aerosol, oral, systemic intravenous, ocular, or rectal administration, or for inhalation administration.

In another embodiment of the invention, the peptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, and any serotype thereof.

The amount of the peptide in the composition is effective in killing and/or inhibiting the growth and/or proliferation of Gram-negative bacteria, Gram-positive bacteria, and/or fungi,

EXAMPLES

Without intent to limit the scope of the invention, exemplary instruments, apparatus, methods and their related results according to the embodiments of the present invention are given below. Note that titles or subtitles may be used in the exampl es for convenience of a reader, which in no way should limit the scope of the invention. Moreover, certain theories are proposed and disclosed herein; however, in no way they, whether they are right or wrong, should limit the scope of the invention so long as the invention is practiced according to the invention without regard for any particular theory or scheme of action.

MATERIALS AND METHODS

All animal experiments were conducted under protocols approved by Academia Sinica

Institutional Animal Care & Utilization Committee (ASIACUC permit number 12-02-322). Research was conducted in compliance with the principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, Ϊ 96.

Bacterial Isolates

The antimicrobial acti vities of HBc ARD peptides were tested on numerous bacterial strains front ATCC, including Psettdomotas aeruginosa Migula strain (ATCC 27853, ampicillin-resistant), Pseudomonas aeruginosa Migula strain (ATCC 9027, ampici 11 in-resi stent), Klebsiella pneumoniae strain (ATCC 17593), Escherichia coli strain (ATCC 25922), Staphylococcus aureus subsp. strain (ATCC 25923, methici l!in-resistant), Staphylococcus aureus subsp. strain (ATCC 29213,

memici!lin-resistant). Staphylococcus aureus subsp. strain (ATCC 19636, meraicHlinresistant), and Candida albicans strain (ATCC 1023 1).

Clinical, isolates ot Pseudomonas aeruginosa (NHRl-01 , NHRI-02 AND NHRl-04) were obtained through the program of Taiwan Surveillance of Antimicrobial Resistance, National Health Research Institutes, Taiwan. Acimlob cier baumamtii (ATCC 17989, ATCC 17978 CR,

ATCC 1 606, ATCC 1 606 CR, TCGH 45530 AND TCGH 6709) were obtained from Tzu-Chi Buddhist General Hospital in Taiwan, and clinical isolates (TCGH 45530 AND TCGH 46709) were identified using the Vitek. system (Biomerieux Vitek, Inc., MO, USA). A. baamanmi is defined as multidrug-resi stant when the organism is resistant to piperacillin, piperaciUm-tazobactam, ampiciilin/sulbactam, imipenem, ceftazidime, gexitamiein, amikacin, tetracycline, chloramphenicol, ciprofloxacin, and cotr noxazole. Susceptibility to colistin was determined using the broth-dilution method, in accordance with the guidelines of the Clinical and Laboratory Standards Institute.

Antimicrobial activity

Al l peptides were purchased from Yao-Hong Biotechnology Inc. (Taipei, Taiwan). Vendors provided data of peptide characterizations, including HPLC and Mass. Antimicrobial activity was determined as described with some modifications as detailed below. Bacteria were grown overnight in Mueller -Hinton broth (D.ifco) at 37°C, and during the mid-logarithmic phase, bacteria were diluted to 10^(> CFU (colony formation unitVinl in phosphate bufter (10 mM sodium phosphate and 50 mM sodium chloride, pH 7.2). Peptides were serially diluted in the same buffer. Fifty microliter (ul) of bacteria was mixed with fifty ul of peptides at varying concentrations followed by incubation at 37*C for 3 hours without shaking. At the end of .incubation, bacteria were placed on Muelier-Binton broth agar plates, and allowed growth at 37'^!C overnight for measurement of minimal bactericidal concentration (MBC). The lowest peptide concentration on the agar plate, which displayed no bacterial growth (zero colony), was defined as MBC. All peptides were tested n triplicate.

For measurement of killing kinetics, bacteria and peptides were prepared as described above. Fifty u! of bacteria were mixed with fifty μΙ of peptides at the concentrations corresponding to MBC and were incubated at 37°€, At the indicated time, bacteria were serially diluted and placed on Mueiler-Hi.nto.ti broth agar plates for viability measurement

Co!ifocal fluorescence microscopy

The localization of peptide was monitored by confocal fluorescence microscopy. Bacteria were grown to mid-logarithmic phase and collected by centrifugals on. Approximate I0⁷ CFU were resuspended in a phosphate buffer containing FiTC-!abeled HBcl47~183 at a concentration corresponding to O.SxMBC. Followi g incubation for 1 hour at 37^c'€, cells were washed, fixed, and immobilized on poiy-L-iysine coated glass slides. ProLong Gold aniifade reagent with DAPI

(Invitrogen) was added to the slides prior to mounting. Localization of iabeled-pepttde was observed using an Olympus Ultraview confocat microscopy equipped with a lOOx oil immersion lens.

SYTOX Green uptake

Briefly, bacteria (10⁷ CPU) were prepared and mixed with 1 μΜ SYTOX Green (Invitrogen) for 1 5 minutes in the dark. After the addition of peptides to the final concentrations corresponding to their respective MBC, fluorescence intensity was measured at 37°C using wavelengths 485 nm and 520 nm filters for excitation and emission. Melittin (Sigma), a major toxin of bee venom, was used as a positive control to provide maxima! permeabilizatkm.

Gel retardation assay

The proportion between amino nitrogen (Ν1¾ ) of HBcl47-l S3 and phosphate (PO ) of DNA was defined as N/P ratio. Briefly. HBcl47-l83 was incubated with pSUPE plasorid DNA at different N P ratio (0, 0.2, 0.4, 0.6, 0.8, 1, 2, 3 and 4} for 30 minutes at 37°C. The mobility of pSUPER piasmid DNA was analyzed by electrophoresis on 1% agarose gel.

ift vitro binding assay between ARD peptides and LPS LipidA

Several kinds of peptide-LPS or peptide-Lipid A. binding assays were performed as follows:

1 ) Streptavidine-conjugated beads (Dynabeads My One Streptavidin Tl, invitrogen) were blocked by P. aeruginosa LPS (Sigma) at 37°C for 1.5 hour. After washing with PBST (PBS, pH 7.4 containing 0.1% (w/v) Tween-20), altquots containing 250 pmol streptavidine-conjugated beads were incubated with a reaction mixture overnight at 4°C. The reaction mixture was prepared by mixing increasing amounts of biotinylated peptide HBcl47-183 (0, 0.004, 0.02, 0.1 , 0.5 and 2.5 μΜ) and 5 p.g/ml P. aeruginosa LPS (Sigma) or 200 pg/^'nii E. eoii lipid. A (Sigma), at 37^C'C for 3 hour. After incubation overnight at 4°C, the reduction of LPS (or Lipid A) in the supematants were measured by the Limulus Amebocyte Lysate (LAL) test (Charles River Endosafe) with an ELISA reader (Molecular Devices). The amount (EU/inl) of LPS was calculated according to the standard curve prepared with Endosafe Control Standard Endotoxin. 2) To directly measure the increasing amounts of LPS bound to the increasing amounts of peptide HBcl 47-183 on the pep tide-coated beads, the beads were then washed with PBST three times and incubated with 100 ui of PBS containing 0..I S units of trypsin agarose (Sigma) for overnight digestion at 37°C. After trypsinization, the trypsin-rel eased LPS in the supernatants were collected and measured by the Limulus Aniebocvte Lysate (LAI.) test. Similar results were obtained by another LPS testing method: Endosafe- PST Cartridges (Charles Ri ver Laboratories).

3) To perform the LPS Lipid A competition assay, one ug of LPS was coated on High Binding EL ISA plates (Coming) overflight at 4°C. The LPS-coated plates were washed by PBST and then blocked with PBST containing 5% BSA for 1 hour at 37°C. After washing, HBci 47- 183 ( 10 « ), mixed with varying concentrations of coli Lipid A (0 to 10 ug mt), were added i to each well and incubated for 1 hour at 37°C. Plate-bound HB l 47-183 was measured by streptavidin conjugated with HRP (1: 1.0000 dilittion) for 1 hour at 37°C. T B substrates were added into each well for color development. The absorption was measured at 450 nm with a reference wavelength at 655 nm. Hemolytic activit

The hemolytic activities of peptides were determined by hemolysis against human red blood cells (hRBCs). Human blood was obtained in EDT A -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°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- l cO, respectively.

Cytotoxicity

Cytotoxicity was measured in HepG2, Huh7, HEK293, and Vera cells by TT assay. Cells were seeded at It)⁴ cells/well in a 96-weO plate and serial dilutions of peptides were added into each well . PBS was used as a negative control and meltttin was used as a positive control. After 1 hour of incubation, the medium were replaced by fresh medium containing 10% MTT soluiion

(PROMEGA^TM), and the plate was incubated for 4 hours in 5% CO* at 37^"C . The absorbance at the wavelength of 595 nm was measured by an El. ISA reader (BIO-RAD™ model 680).

CFSB cell Proliferation assay

To set up CFSE cell proliferation assay, 293 cells (human kidney origin) and Vero cells (monkey kidney origin) were resuspended in PBS to a final concentration of 10 cells/ml before incubation with 10 μ CFSE dye (CELLTRACE™ CFSE cell proliferation kit, INVITROGEN™) at 37*C for 10 min. To quench the staining; ice-old culture media were then added and incubated on ice for 5 min. Labeled cells were then pelleted and washed three times with a fresh medium containing 10% FBS before seeding into six well plates at a density of 3.3 x 1 ⁽^cells/well. After 20 h, the medium was removed and incubated with a fresh medium containing 5, 25 and .100 μΜ. HBc 1.47-183 for one hour (FlTC-iabeled ARD peptide had been largely internalized in 10 minutes after the addition of ARD peptides to the medium of HepG2 cells). Forty-eight hours later, cells were harvested and analyzed by flow cytometry (FACSCanto, BD Bioscience).

In vivo animal studies

Three-week old male ICR mice (19 to 21 g) were purchased from BioLASCO (Taiwan).

Overnight culture of bacteria in BHI broth (Difco) was subcultured in fresh BHI broth to log phase. Inoculums were diluted in BHI broth to indicated densities. To test, the acute toxicity of ARD peptide in vivo, ICR male mice were inoculated intraperitoneally (Ϊ .p.) with 10 and 20 mg kg HBc 147-183 in PBS, respectively. Each group contained 5 mice. After peptide injection, the number of dead mice was recorded daily for 7 days post-injection. To test t e antimicrobial activity of the ARD peptide in vivo, all mice were inoculated i .p. with Staphylococcus aureus ATCC 1.9636 (4 x l0⁶CFU/tnouse) in BHI broth. Peptide HBc! 47-183 (10 mg/kg) was administered i.p. at 1, 1.5 and 2 hours post- inoculation. PBS (10 ml/kg) control was administered at 1 hour post-inoculation. Each group contained 10 mice. Mortality was monitored daily for 7 days post-inoculation. In a separate experiment to measure the bacterial load, mice were inoculated i.p. with Staphylococcus aureus ATCC 19636 (10° CFU/mouse) in BHI broth. AS! mice were administered at 1 hour post-inocul tion with pepti de HBc 147-183 (10 mg kg) or PBS ( 10 ml/kg) control, and sacrificed at 4 hours post- inoculation. Blood samples (200 ul) were mixed with 100 roM EDTA (10 ul) and were diluted 20- fold in PBS (Ca^2* and Mg^2'" free). Liver and spleen samples (0, 1 g) were homogenized in sterile PBS (500 μ|). Samples were diluted approximatel 100-fold and plated on BHI agar for scoring the colony numbers.

To test the in vim antimicrobial acti vity of the ARD peptide against Gram-negative bacteria, mice were inoculated with Klebsiella pneumoniae Xen39 ( 1 ⁷ cfti mouse) (Cali per LifeSciences), an engineered strain containing a modified Phaiorhahd lnminescerts ImABCDE operon. One hour post-inoculation, mice received either 10 ml/kg PBS (n^~5) or 10 mg/kg ARD peptide (n^~5).

respectively. In viva imaging was carried out at 4 hours post-inoculation. The mice were anesthetized first, before transferring to the IVIS imaging system (IVtS spectrum), and luminescence was measured with an exposure time of i minutes or less. The image system measured the number of photons and translated the data to false color i mages that depicted the region of strong luminescence with red, moderate luminescence with yellow and green, and mild luminescence with blue.

Decreasing bioluminescence indicated reduction of bacteria. The images were overlay of photographic images and bioluminescence using a computer-generated color scale. Total flux (RL^'U) of region of interest (ROI) was quantified by the lVl^'S imaging software.

RESULTS

hi vitro antimicrobial activity of HBc peptides

As shown in FIGs. 1 and 10, HBcl47-.l 83 displayed a broad-spectrum activity against Gram- negative bacteria {P. aeruginosa, K. pneumoniae and E, coli), Gram-positive bacteria aurem), and fungi (C, albicans). Among these tested strains, P. aeruginosa and K. pneumonia were the most sensitive to this peptide. The MBCs of HBc^'147-183 were lower than 4 uM for P. aeruginosa and A'. pneumonia, and around 4 μ for £, coli, and S, aureus. C. albicans was the least sensitive to this peptide (MBC ~8 uM).

To further map the active sequences of the antimicrobial activity, various peptides (FIG, 1) in different length were synthesized and tested as before. Peptide HBcl 47-175, with the deletion of the last eight amino acids at the C -terminus, maintained strong activity against Gram-negative bacteria, albeit it lost the activity against S. aureus and C. albicans. We detected no activity against all of the tested bacteria and fungi from peptides ARD (HBc^'147-159) or ARD II1-IV (HBc 164- 176 and HBcl62-.l 75). In contrast, all peptides containing ARD ϊί-lV (HBc 1 53-176, HBcl57-176, HBcl 53- 1 75,, HBcl 55-175, and HBcl 57-175) and ARD Mil (HBcl47-167) exhibited strong activity against P. aeruginosa and K. pneumonia, respectively, albeit they were weak, against £ coli (FIG. 10). Therefore, peptide ARD II-IV and ARD I-HI appeared to be necessary and sufficient for the bactericidal activity against P. aeruginosa and K. pneumonia.

Positive charge of ARD peptides is critical to the bactericidal activity.

Phophory on studies on serine residues SI 55, SI 62, SI 0, SI 6 and S181 revealed that serine phosphorylation in general weakened the potency of antimicrobial activit It was found that all HBc peptides, once phosphorylated, lost their activities against ⁽..^*. albicans (FIG. 1 I ). For bacteria, the phosphorylation on SIS Ϊ showed no effects, whereas phosphorylations on SI 55, I 62, S I 0₅ and S 176 reduced the antimicrobial activity. ^'The MBCs dropped to 8 μΜ for HBcl 55p and HBcl 76p„ and 32 μΜ for HBcI62p and HBcl70p, respectively. When Si 55, S162 and Si 70 were

simultaneously phosphorylated (HBc 155pl 62p 170p), the antimicrobial activity was completely lost (>32 uM). The results suggested that, except for S I S I, serine phosphorylation is generally detrimental to the antimicrobial activity of HBc ARD peptide. To confirm the importance of arginine residues for bactericidal activity, we synthesized and tested peptide HBc 147-1 S3-III-IV AA, which has two R-to-A substitution mutations in each of ARD ill and ARD I V, Similar to phosphorylated HBc ARD peptides, the MBC of HBcl47-183-IlT-iV AA was significantly increased compared to HBc 147-1 S3. The result indicated that arginine residues are required for the antimicrobial activity. Drug Resi stance

The antimicrobial activity of HBcl47-l 83 against colistin-resi slant P. aeruginosa and A.

h ^' mmanttii viss tested. As shown in FIG. 1. 1, while HBc^'147- 1 83 killed eolistin-sensi ve P.

aeruginosa at 4 μΜ, colistin-resistant P. aeruginosa were cross-resistant to HBc 147- 183 (MBC> 16 μΜ). In contrast to . aeruginosa, the MBCs of HBc 147-183 against coJistin-sensitive and colistin- resi slant A. hatm mmi are in a similar range of 0.5- 1 uJvl This result indicates that, for colistin- resistant .4. haumat ii. there is no cross-resistance to our ARD peptide HBc^'147- 183,

Killing kinetics

Time course of bacteria! viability was determined after the tested bacteria (P. aeruginosa, K, pneumonia, E. coli and S. aureus) were treated with HBcl47- ! 83 at the concentrations

corresponding to the MBC (FIG. 2). The results showed that P. aeruginosa was immediately killed within 20 minutes upon the addition ofHBd 47-183 (2 μ ). Although A^', pneumonia and K coli were members of Gram-negative bacteria, they were killed by 4 μΜ HBc 147-183 in 180 minutes. For S. anrem, complete killing by 4 μΜ HBc! 47- 183 was observed in 120 minutes.

Localization and mechanism of HBc 147-183

. aeruginosa, E. coli and S. aureus were treated with FITC-labe!ed HBcl47-! 83 corresponding to O.SxMBC, and the localization of HBcl47~183 was visualized using confocal fluorescence microscopy (FIG. 3). The results showed thai, upon peptide treatment, P. aeruginosa. K. pneumonia and E. coli appeared as hollow rods with fluorescence clearly defined bacteria surface, suggesting that HBc^'147-ί 83 was accumulated on the membrane (FIGs. 3 A-D). To understand better the effect of HBc peptides on the membranes, SYTOX Green uptake assay was performed. A significant degree of membrane perraeabilization was i nduced on P. aeruginosa upon the addition of 2 μΜ HBc147-183 (FKJ 4A), Although it was also accumulated on the membrane of A^", pneumonia and E. coli, 4 Μ HBc 147- i 83 was not able to induce membrane permeabilization as observed on S.

aureus. Consistent with the bactericidal activity of HBc^'147-ί 83 against P, aeruginosa, HBc 153-! 76 caused same membrane permeabilization within 10 minutes in a dose-dependent manner (FIG. 4B). This indicated thai the bactericidal effect of HBc peptides on . aeruginosa is directly through the membrane permeabilization with a fast kinetics similar to that of killing kinetics (FIG. 2). On the other hand, HBc147-l 83 was found to penetrate through the membrane of S. aureus and localized in the cytoplasm (FIGs. 3E-G). To investigate the potential interaction between HBc 147-1 S3 and DNA, HBc 147-183 was mixed with pSUPER plasmid DNA at different /P ratio (Materials and Methods) and analyzed by gel electrophoresis (FIG. 4C). The results showed that the mobility of DNA was decreased when the ratio of pepticie/DN A increased and the plasmid. DNA was completely retarded at the ratio of 1 , suggesting thai HBc 147- 183 has a strong binding activity to piasmid DNA. Overall, it suggests that the bactericidal mechanisms of HBc l47-183 on Gram-positive and Gram- negative bacteria may be completely different.

Direct binding of HBc147-l83 to LPS

To determine whether LPS of Gram-negative bacteria could serve as a potential target of HBc147-183. LPS (0.05 to 50 ug/mi) from either f aerttgi m or K co!i (Sigma) were incubated with both P. aeruginosa and 2 uM HBel47-183 for three hours, respectively. The results showed that the bactericidal activity of HBc^'147-183 was significantly reduced by addition of either LPS at the concentration of 50 ug/ml (FIG. 5). ϊη addition, HBcl47- 183 preferentially bound to the LPS from P. aeruginosa, rather than that from E. coil However, the addition of anti-LPS antibody (Genetex Co.) cannot sufficiently neutralize the bactericidal activity of H.Bcl47-183 (FIG. 5). it suggests that HBcl 47- 183 could bind to not only LPS but also other target molecules on the membrane. Alternatively, HBc147«183 and the anti-LPS polyclonal antibody used here could bind predominantly to two different epitopes on the LPS.

As shown in FIG. 6 A, the potential interaction between BBcl47-183 and LPS (or Lipid A moiety) in vitro was investigated using several different binding assays. In FIG. 6B, when increasing amount of HBcl47-183 was bound to the strepavidine-conjugated Dynabeads and allowed incubation with a constant amount of LPS, gradually increasing amount of LPS appeared to be depleted from the supernatant. IlBc 1.47- 183 8p„ which has eight ser thr phosphorylations, was used in parallel as a control peptide. Similar results were obtained by another LPS testing method:

Endosafe-PTS Cartridges (Charles River Laboratories). In FIG. 6C, the beads-captured LPS were dissociated from the beads by trypsin agarose digestion of the ARD peptide HBc 147-183. The amount of rel eased LPS was measured by the LAL test (Materials and Methods), LPS contai ns mainly the polysaccharide and Lipid A moieties. To determine whether the ARD peptide can bind to Lipid A directly, we tested in FIG. 6D the binding between Lipid A and the ARD peptide in a manner similar to FIG. 6B. As expected, increasing amounts of BBc 1.47-1 S3 on the beads led to decreasing amounts of Lipid A remaining i the supernatant. The inverse correlation between the ARD peptide on the beads and the Lipid A in the supernatant (FIG. 6D) is strikingly similar to what was observed previously between the ARD peptide HBcl47- 183 on the beads and LPS in the supernatant (FIG. 6B). To directly demonstrate that the ARD peptide can bind to the lipid A moiety of LPS, we performed a competition experiment between Lipid A and LPS (FIG. 6E). The LPS- coated ELISA plates were incubated with constant amount of HBcl47-l83 (10 tsM), which was premised with varying concentrations of E. coti Lipid A (0 to 10 ug/ml). After extensive washing, plate-bound (i.e., LPS-bound) biotinylated peptide HBc 147- 183 was measured by streptavidin conjugated with HRP, followed by adding T B substrates and color development. The binding of HBc 147- 183 to LPS was significantly decreased by the increasing concentrations of Lipid A (FIG, 6E). The result here lends support for the notion thai Lipid A moiety of LPS can serve as a direct target for ARD peptide HBc 147-1 S3.

Cytocoxieity

To determine the cytotoxicity of HBc peptides, we measured the hemolytic acti vity of HBcl 47-

183. Compared to the melittin control, no detectable hemolysis by HBcl 47-183 was observed after one hour of incubation (FIG. 7A). in addition, MTT assay was performed to determine the cytotoxicity of HBc 147-183 to hum n hepatoma (Hu 7 and HepG2 cells) and kidney eel is (Yero and HEK2 3 cells). The viability of cells treated with melittin at low dose (3. 125 μΜ) was

significantly decreased. In contrast, HBcl47-I83 caused only a low level of cytotoxicit at the concentrati n of 100 μΜ (FIG. 7B). The CFSE cell proliferation assay was also performed to determine the effect of HBc 147- J.83 on the proliferation of Vero and HBK293 kidney ceils. In comparison to day 1, CFSE intensity of cells treated with HBcl 47- 183 (5, 25 and 00 μΜ) decreased to the same level as the mock control on day 3 (F G. 7C), suggesting that ARD peptide HBcl 47-183 has no significant effect on ceil proliferation.

Animal model

To conduct the experimental infection with bacteria, we i.p. inoculated mice with Stcqtkylococcus aureus ATCC 1 636 (4x10^b cfu/raouse). Bacterial load in blood at i, 2, 4 and 6 hours post- inoculation was determined. As shown in FIG. 8 A, bacteria rapidly transferred to the blood

compartment from peritoneal cavity. Within 2 hours, the number of bacteria in the blood, achieved the ma imum (10" cfu/nil). Thereafter, the number of bacteria in the blood gradually decreased spontaneously. To distinguish the ARD peptide-mediated from the spontaneous clearance, we therefore tested the in vivo protection activity of the ARD peptide within 2 hours post-inoculation. Briefly, mice were i.p. inoculated with Staphylococcus aureus ATCC 19636 and received a single dose of 10 nil/kg PBS or a single dose of 10 mg/kg ARD peptide at 1 , 1.5, 2 hours post-inoculation, respectively. Mice (η= ΪΟ) treated with PBS died within 24 hours post-inoculation (FIG. SB). In contrast, administration of ARD peptide ( 10 mg/kg) at 1 hour post-inoculation can effectively protect all mice (n^~10) from death at day 7. When we administered ARD peptide at 1.5 (n~10) and 2 (n^~ 10) hours post- inoculation, survival rates were decreased to 70% and 40%, respectively. Instead of using death as a surrogate indicator of the antimicrobial activity of ARD peptide, we also determined directly the in vivo effect of ARD peptide on bacterial load of infected mice (FIG. 8C). Mice were inoculated with Staphylococcus aureus as before and treated with 10 ml/kg PBS (n=5) or 10 mg kg ARD peptide (n~5) at 1 hour post-inoculation. Four hours post-inoculation, bacterial load in blood, liver and spleen samples of control mice were in the range of 1 " cfu/rnl (FlGs. 8C and 8D). Administration of ARD peptide significantly reduced the bacteria! load (- 10^' cfu/ml) by 100-fold in blood, liver and spleen than the PBS control mice ( ><0.0l). in addition to Staphylococcus aureus, we also examined the in vim antimicrobial activity of ARD peptide on K. pneumoniae using an IVES imaging system. Similar to the change in bacterial load of S. aureus, bioluminescence of mice inoculated with A^', pneumoniae X.en39 peaked at 2 hour post-inoculation (data not shown) We then treated K, pneumoniae Xen394nfected mice with PBS or ARD peptide at 1 hour post-inoculation, respectively. ^'The results showed that the bioluminescence of ARD pepti de-treated mice was very weak, whereas PBS control showed a more extensive bioluminescence (FIG. 9A). There was a significant difference in the overall RLU values of mice treated with PBS versus ARD peptide

(7^J<0.01) (FIG. 9B). Taken together, the results indicated that HBcl47-183 exhibited significant antimicrobi l activity in vivo. Table 1 shows comparison of minimal bactericidal concentrations of human HBc ARD and woodchuck HBc ARD against Gram-negative and Gram-positive bacteria.

Table i

The invention relates to a novel antimicrobial peptide (HBcI47-1 S3) isolated from the C-terminal domain of HBc. The computer program based on the antimicrobial peptide database (Wang et al. (2004) Nucleic Acids Res 32: D590-592) predicted unfavorably that HBc 147- 183 could serve as an antibacterial peptide, due to its very low content of hydrophobic amino acids. Contrary to the computer prediction, surprisingly, HBcl47-183 exhibited a broad-spectrum antimicrobial activity. While co!istitt-resistant P. aeruginosa exhibited cross-resistance to ARD peptide HBc 147- 183, we found a strong activity of hbcl47-I 83 ( B(M>.5-1 urn) against all tested colistin-resistant 5.

f ^'mimannii. Our ARD peptide could bind to Lipid A of £ coii and LPS of P. aeruginosa (FIG. 6). While only the full-length HBc 147-183 (ARD I-IV) was effective against the tested gram-positive bacteria, S. aureus, ARD IJ-IV (HBe^'153-176) and ARD i-HI (HBcl47-167), in a less than full-length context, exhibited strong activity against gram -negative P. aeruginosa and X. pneumoniae, respect vely (but not coii).

Comparisons with other A MPs it is surprising that the ARD domain of HBc protein (HBc 147- 183) exhibits novel and broad spectrum antimicrobial activity, ^'ibis peptide shares some degree of similarity with several antimicrobial peptides in literature, such as protamine

{PilRRRSSS PYf R RRPRVSRRllR RGGRRi R; SEQ ID NO: 1 1) and drosocin

(G PRPYSPRPTSf lPRPlRV; SEQ ID NO: 12). A radial diffusion assay showed that a single axgifline-rich domain (RRRR) is sufficient for antimicrobial activity, especially against gram- negative bacteria Lesmes et al. (2009) Peptides 30: 2150-2160). Unlike protamine, the arginine-rich domain of HBc 1 7-183, such as ARD HI and ARD III -IV, were not sufficient for the antimicrobial activity. In addition, sequence alignment by anitimicrobial peptide database revealed that HBcl 53- 176 shares 44% amino acid sequence homology with Drosocin, which is a proline-rich peptide isolated from Drosophila.. However, except for P. aeruginosa, Drosocin is predominately active against most Gram-negat ve bacteria. Drosocin kills bacteria via an apparently non-membranolytic mechanism. The HBc ARD is a novel peptide with a broad spectrum bactericidal activity quite distinct from other known argi nine-rich antimicrobial peptides. Protamine contains RRGGRRRR (SEQ ID NO: 17), while HBcI47- 183 contains SQSRESQC (SEQ ID NO: 16) at the C-ierrninus of HBc.

Bacterid dal M echani sms

The results showed the membrane localization of HBc 147- 183 on Gram-negative bacteria (FIG . 3) and the neutralization activity of LPS from either P. aeruginosa oris, coli (FIG. 5). It suggests that HBc^'147-183 could have a strong binding activity to LPS. The results revealed a direct binding of HBc^'147-1 S3 to LPS and Lipid A (FIGs. 6B, 6C and 6D). Furthermore, Lipid A moiety of LPS was shown to be one major direct target of HBc i 47- 183 (FIG. 6E), However, incubation of LPS antibody with P. aeruginosa and HBc.147-183 failed to neutralize the bactericidal activity of HBc ARD peptide (FIG. 5). One interpretation for this negative result is that HBc^'147-183 could bind not only LPS but also some other molecules on the bacterial membrane. We found a better neutralization effect of the LPS from P. aeruginosa than that from E. coli (FI G. 5). The preference of bi ndi ng by HBc147-183 for the LPS of P. aeruginosa is correlated with its stronger bactericidal activity against P. aeruginosa.

The mode of action of HBc i 47-183 on P. aeruginosa could be related to membrane

permeabi!ization based on the fast killing kinetics (FIG. 2) and its membrane localization (FIGs. 3A and 3B). This speculation is also supported by the results of SYTOX Green uptake experiment (FIG. 4A). Like P. aeruginosa. HBc 147- 183 was also accumulated on the membrane of K, pneumonia and R coli. However, the killing kinetics and S YTOX Green uptake experiments of K. pneumonia and K coli did not support for a mechanism of membrane permeabiiization. It remains to be further investigated how bacteria can be killed by the ARD peptides using a mechanism other than membrane permeabilization .

In the case of Gram -positive bacteria, we found that HBcl47-183 was not accumulated on the membrane (FIG. 3), Instead, it can enter the cytoplasm of S, aureus without any apparent

development of membrane permeabilization (FIG. 4A). In addition to LPS, HBc 1.47- 183 can also bind strongly to plasmid DNA {FIG. 4C). Taken together, the bactericidal mechanism of HBcl47- 183 against Gram-positive bacteria appeared to be more similar to Buforin II, which was reported to kill bacteria by binding to DNA and UNA after penetrating bacterial membrane.

Although HBc^'147-183 can penetrate through the cell membrane of Huh 7 and HepG2 cells (data not shown), we observed no significant cytotoxic effect on human hepatoma cells Huh 7 and HepG2, and kidney cells Vero and HE 293, even at a high peptide concentration (100 μΜ) by MTT assay (FIG. 7BJ and proliferation assay (FIG. 7C). Taken together with the results from the hemolytic assay (FIG. 7 A), HBc 147- Ί 83 appears to be much safer relative to melittm in ceil culture. The animal model study showed no apparent in vivo toxicity of ARD peptide at 20 mg/kg dose in the ICR mice by ί .p. injection (FIG. 7D). At as low as 10 mg/kg level, treatment of ARD peptide can protect mice from death (FIG. SB). 3n contrast, all mice receiving the PBS control were dead soon after bacterial inoculation. In addition to the sepsis survival model, treatment of ARD peptide (10 mg/kg) also resulted in a significant reduction of bacterial load of S. aureus and K. pneumoniae, whereas PBS control mice showed high levels of bacterial load (FIGs. 8C-D and FIG. 9). The results demonstrated the in vivo antimicrobial, potency of HBc ARD peptide.

Claims

CLAIMS What is claimed is:

1 . A pharmaceutical composition comprising;

(a) an effective amount of an isolated peptide, wherein the peptide comprises the arginine- rich carboxy-terminal region of hepatitis B virus core protein (HBc) and exhibits art anti icrobial activity; and

(b) a pharmaceutically acceptable carrier,

2. The composition of claim 1 , wherein the peptide comprises the amino acid sequence of Ser Pro Arg Arg Arg Arg (SPRRRR; SEQ ID NO: 13) or Arg Arg Arg Ser (RRRS; SEQ ID NO:

14).

3. The composition of claim 2, wherein the peptide comprises two repeats of SPRRRR (SEQ ID NO: 13).

4. The composition of claim 1 , wheretn the peptide comprises three clusters of SPRRR (SEQ ID NO: 15).

5. The composition of c!aim ] , wherein the peptide comprises RRRS (SEQ ID NO: 14).

6. The composition of claim 5, wherein the peptide comprises 2 clusters of proline Argimne (PR) sequences located upstream to the sequence RRRS (SEQ ID NO: 14),

7. The composition of claim 5, wherein the peptide has no RRGGRRRR sequence (SEQ ID NO: 17) at the C-iermin s thereof.

8. The composition of claim I, wherein the peptide has a cysteine (C) at the C-teitmnus thereof.

9. The composition of claim 1 , wherein the peptide comprises no amphipathic structure.

10. The composition of chirr 1 , wherein the peptide comprises the amino acid sequence of SEQ ID NO: 1.

11. The composition of claim I , wherein the peptide is characterized by having an activity

against Gram-negative bacteria. Grain-positive bacteria., and/or fungi.

12. The composition of claim 1 , wheretn the peptide comprises a protecting group.

13. The composition of claim 1 , wheretn the peptide comprises more than one arginine-rich domain selected from the group consisting of (i), (ii) and (hi) as follows:

(i) HBc ARD I H, 111, and IV;

(it) HBc ARD 1, 0 and 10; or

(iii) BBc ARD II, HI, and IV. The composition of claim 1 , wherein the peptide exhibits an activity against colistin-resistant

A. haumatmii.

The composition of claim I, wherein the peptide is free of the sequence of SQSRESQC (SEQ ID NO: 16) at the C-terminus thereof and is characterized by having an activity against Grai i-negative bacteria.

The composition of claim 14, wherein the peptide comprises HBc ARD IWV but without

HBc ARD I and exhibits an activity against /⁵, aeruginosa.

The composition of claim 14, wherein the peptide comprises HBc ARD l-l!l but without HBc ARD IV and exhibits an activity against K. pneumonia.

The composition of any one of claims 1 to 17, wherein the composition is formulated for topical, aerosol, oral, systemic intravenous, intraperitoneal, ocular, or rectal administration, or i n.halation ad mi ni strati on .

A pharmaceutical composition as claimed in any one of clai ms 1 to 17, fonts© in killing and/or inhibiting the growth and/or proliferation of a microorganism in a subject i n need thereof, or for treating a subject afflicted with a microbial infection.