Compositions and methods for making amphipathic polypeptides

Abstract

This invention provides amphipathic polypeptide compositions, and methods of making and using thereof, having improved characteristics including but not limited to increased antimicrobial activity and decreased toxicity. The unexpected discovery is that the amphipathic polypeptide compositions have greatly increased anticancer activity.

Description

Docket No. 10504-103WG1COMPOSITIONS AND METHODS FOR MAKING AMPHIPATHIC POLYPEPTIDESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 650,557, filed May 22, 2024, the entirety of which is hereby incorporated by reference herein for all purposes.STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government support under grant AI172861 awarded by the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.FIELD OF THE INVENTION

[0003] This invention provides amphipathic polypeptide compositions and methods of making and using thereof.INCORPORATION BY REFERENCE

[0004] The contents of the XML file named “10504-103W01-ST26” which was created on May 20, 2025, and is 149 KB in size, are hereby incorporated by reference in their entirety.BACKGROUND OF THE INVENTION

[0005] Natural antimicrobial peptides (AMPs; e.g., the defensins, the cathelicidins, the protegrins) are short cationic peptides (usually less than 100 amino acids) found in the epithelial lining, blood, and lymphatic tissues that serve as one of the first defenses against pathogens. Many AMPs permeabilize the bacterial membrane to cause significant damage or small defects, which in turn reduces the transmembrane potential, leading to cell death. AMPs can also inhibit bacterial cell-wall formation, break down DNA or RNA in bacterial plasma, and cause protein defragmentation or degradation.Docket No. 10504-103W01

[0006] However, natural AMP concentrations in vivo tend to be low and AMPs are unstable under physiological conditions due to proteolysis, acid hydrolysis, and salt effects.

[0007] lite development of synthetic amphipathic polypeptides that have increased antimicrobial and / or anti-cancer activity is therefore urgently needed.SUMMARY OF THE INVENTION

[0008] It has surprisingly been discovered herein methods of producing amphipathic polypeptides (AP) having numerous beneficial effects compared to their helical amphipathic counterpart peptides of identical composition and / or natural antimicrobial peptides (AMP).

[0009] The methods described herein are a surprisingly effective means for creation of amphipathic polypeptides having linear amphipathicity and improved characteristics such as increased antimicrobial activity, increased anticancer activity, greater stability, and / or decreased toxicity. In some embodiments, the method is iterative and involves a cyclical process of creating a group of amphipathic polypeptides, each differing by the addition of one cationic or hydrophobic amino acid to a cationic or hydrophobic end, respectively. The group of amphipathic polypeptides are then analyzed for one or more improved characteristics such as increased antimicrobial activity, increased anticancer activity and decreased toxicity. This method preferably allows for a structure-function titration wherein cationic and / or hydrophobic motifs within the amphipathic polypeptides can be analyzed relative to one another. In some embodiments, preferred cationic and / or hydrophobic motifs are identified and combined in an amphipathic antimicrobial polypeptide sequence.

[0010] In one aspect, provided herein is a method of creating an AP sequence comprising: (a) obtaining an AP comprising a sequence having the formula of CiHi wherein C is a cationic amino acid, H is a hydrophobic amino acid, and wherein the polypeptide has a cationic amino acid end and a hydrophobic amino acid end; (b) adding one cationic amino acid at the cationic amino acid end of the AP to create a cationic AP extension product, and cyclically adding one same or different cationic amino acid at the cationic amino acid end of each cationic AP extension product to create a group of cationic extension polypeptides having the formula of Ci+xHi, wherein x is equal to or between I and 16; and / or (c) adding one hydrophobic amino acid at the hydrophobic amino acidDocket No. 10504-103W01 end of the AP to create a hydrophobic AP extension product, and cyclically adding one same or different hydrophobic amino acid at the hydrophobic amino acid end of each hydrophobic AP extension product to create a group of hydrophobic extension polypeptides having the formula of CiHi+x, wherein x is equal to or between 1 and 16; and (d) adding one cationic amino acid at the cationic amino acid end of one or more of the cationic extension polypeptides; and / or (e) adding one hydrophobic amino acid at the hydrophobic amino acid end of one or more of the hydrophobic extension polypeptides; and (f) end-to-end cyclizing each of the group of polypeptides of step b), step c), step d) and / or step e) to create a group of cyclized polypeptides.

[0011] In another aspect, provided herein is a method of selecting for an AP sequence comprising: (a) obtaining an AP comprising a sequence having the formula of CiHi wherein C is a cationic amino acid, H is a hydrophobic amino acid, and wherein the polypeptide has a cationic amino acid end and a hydrophobic amino acid end; (b) adding one cationic amino acid at the cationic amino acid end of the AP to create a cationic AP extension product, and cyclically adding one same or different cationic amino acid at the cationic amino acid end of each cationic AP extension product to create a group of cationic extension polypeptides having the formula of Ci+xHi, wherein x is equal to or between 1 and 16; and / or (c) adding one hydrophobic amino acid at the hydrophobic amino acid end of the AP to create a hydrophobic AP extension product, and cyclically adding one same or different hydrophobic amino acid at the hydrophobic amino acid end of each hydrophobic AP extension product to create a group of hydrophobic extension polypeptides having the formula of CiHi+x, wherein x is equal to or between 1 and 16; and (d) adding one hydrophobic amino acid at the hydrophobic amino acid end of one or more of the cationic AP extension polypeptides to create a second group of cationic extension polypeptides having the formula of Ci+xHi+ywherein x and y are the same or different and equal to or between 1 and 16; and / or (e) adding one cationic amino acid at the cationic amino acid end of one or more of the hydrophobic extension polypeptides to create a second group of hydrophobic extension polypeptides having the formula of Ci+yHi+xwherein x and y are the same or different and equal to or between 1 and 16; and (f) end-to-end cyclizing each of the group of polypeptides of step b), step c), step d) and / or step e) to create a group of cyclized polypeptides.

[0012] In an alternative aspect, provided herein is a method of creating an AP sequence comprising: (a) obtaining a sequence having the formula of Ci+yHi+Xwherein Ci+yrepresents the number of cationic amino acids forming a cationic motif on one end ofDocket No. 10504-103WG1 the AP, wherein Hi+Xrepresents the number of hydrophobic amino acids forming a hydrophobic motif on the other end of the AP; and wherein x and y are the same or different and equal to any number between 0 and 16; (b) varying y or adding one cationic amino acid at either end of the cationic motif of the AP to create a cationic AP extension product while x remains unchanged results in linear APs, which can become cyclic by joining the cationic end terminus and the hydrophobic end terminus or by adding a cysteine residue at each end terminus to join the 2 end termini via a disulfide bridge; and / or (c) varying x or adding one hydrophobic amino acid at either end of the hydrophobic motif of the AP to create a hydrophobic AP extension product while y remains unchanged results in linear APs, which can become cyclic by joining the cationic end terminus and the hydrophobic end terminus or by adding a cysteine residue at each end terminus to join the 2 end termini via a disulfide bridge.

[0013] A summary of embodiments of the invention is described in further detail below.BRIEF DESCRIPTION OF FIGURES

[0014] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate certain examples of the present disclosure and together with the description, serve to explain, without limitation, the principles of the disclosure. Like numbers represent the same elements throughout the figures.

[0015] FIG. 1A-1E is a schematic showing an example of a linear amphipathic antimicrobial peptide, cyclization by end-to-end amidation, cyclization by cysteine mediated bridging, and peptoidization. Cyclization can be mediated by cysteine residues (S-S) to form cyclic, bicyclic, or multicyclic compounds. Both linear and cyclic sequences can be modified into protease-resistant D-compounds, peptoids, or even peptide mimics. All linear APs will have corresponding cyclized sequences by end-to-end amidation or via cysteine (S-S) bridging. Bicyclic cyclic-AP can also be generated for comparative structure-function correlations using 2 S-S bridges. Prior to advancement, once lead candidates are identified based on having SEO >8, the lead candidates will also be compared for SEO with their corresponding peptoid- (FIG. 1C), unconventional- (FIG. ID), and D-amino acid-sequences (FIG. IE).

[0016] FIG. 2A-2B is a schematic showing one example demonstrating the iterative nature of the method disclosed herein. Cationic peptides will undergo elongation byDocket No. 10504-103W01 addition of either 1 H (hydrophobic, horizontal arrow) or 1 C (cationic, vertical arrow) (r) at a time to generate ternary or binary as indicated (FIG. 2A). In some embodiments, end-to end cyclization results in the conversion of a linear cationic peptide antibiotic to a circular cationic peptide antibiotic. In some embodiments, the generation of circular cationic peptide antibiotic can be also mediated by di-sulfide bridges. An example is shown as CRL series (FIG. 2B) with increasing arginine (R, Arg) (charge 4-7) content while the H domain remains constant; H = 2C + 6L. The following sequences are illustrated: CRRRRLLLLLLC (SEQ ID NO: 1 ); CRRRRRLLLLLLC (SEQ ID NO: 2); CRRRRRRLLLLLLC (SEQ ID NO: 3); and CRRRRRRRLLLLLLC (SEQ ID NO:4).

[0017] FIG. 3A-3D shows the antimicrobial activity of various AMPs created using the methods disclosed herein. WLBU2 (+13) (SEQ ID NO: 107) and indolicidin (+3) (SEQ ID NO: 108) were chosen as controls for activity in MHB2 containing 15% FBS. The CRL pilot sequences indicate that Trp (W) may not be necessary to achieve potency in conditions that are challenging to natural APs. By varying the charge alone, optimal activity was achieved against MDR strains of E. coli (FIG. 3A), A. baumannii (FIG. 3C), and S. aureus (FIG. 3D), with lower potency against P. aeruginosa (FIG. 3B). This shows the importance of the systematic use of the 6 hydrophobic amino acids for structure-function studies with different approaches to design rather than just focusing on tryptophan (Trp, W) or phenylalanine (F, Phe). Not shown is the suppressed activity of H-RL and L-RL series in this test medium. The following sequences are illustrated: CRRRRLLLLC (“CRL1” or “CCR4L4”; SEQ ID NO: 5); CRRRRRLLLLC (“CRL2” or “CC-R5L4”; SEQ ID NO: 6); CRRRRRRLLLLC (“CRL3” or “CC-R6L4”; SEQ ID NO: 7); CRRRRRRRLLLLC (“CRL4” or “CC-R7L4”; SEQ ID NO: 8); CRRRRLLLLLC (“CRL5” or “CC-R4L5”; SEQ ID NO: 9); CRRRRRLLLLLC (“CRL6” or “CC-R5L5”; SEQ ID NO: 10); CRRRRRRLLLLLC (“CRL7” or “CC- R6L5”; SEQ ID NO: 11); CRRRRRRRLLLLLC (“CRL8” or “CC-R7L5”; SEQ ID NO: 12); CRRRRLLLLLLC (“CRL9” or “CC-R4L6”; SEQ ID NO: 1); CRRRRRLLLLLLC (“CRL10” or “CC-R5L6”; SEQ ID NO: 2);CRRRRRRLLLLLLC (“CRL11” or “CC-R6L6”; SEQ ID NO: 3);CRRRRRRRLLLLLLC (“CRL12” or “CC-R7L6”; SEQ ID NO: 4);CRRRRLLLLLLLC (“CRL13” or “CC-R4L7”; SEQ ID NO: 13);CRRRRRLLLLLLLC (“CRL14” or “CC-R5L7”; SEQ ID NO: 14);CRRRRRRLLLLLLLC (“CRL15” or “CC-R6L7”; SEQ ID NO: 15);CRRRRRRRLLLLLLLC (“CRL 16” or “CC-R7L7”; SEQ ID NO: 16).Docket No. 10504-103W01

[0018] FIG. 4 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRLLLLC (“CRL1” or “CCR4L4”; SEQ ID NO: 5); CRRRRRLLLLC (“CRL2” or “CC-R5L4”; SEQ ID NO: 6); CRRRRRRLLLLC (“CRL3” or “CC-R6L4”; SEQ ID NO: 7); CRRRRRRRLLLLC (“CRL4” or “CC-R7L4”; SEQ ID NO: 8); CRRRRLLLLLC (“CRL5” or “CC-R4L5”; SEQ ID NO: 9); CRRRRRLLLLLC (“CRL6” or “CC-R5L5”; SEQ ID NO: 10); CRRRRRRLLLLLC (“CRL7” or “CC-R6L5”; SEQ ID NO: 11); CRRRRRRRLLLLLC (“CRL8” or “CC-R7L5”; SEQ ID NO: 12); CRRRRLLLLLLC (“CRL9” or “CC-R4L6”; SEQ ID NO: 1); CRRRRRLLLLLLC (“CRL10” or “CC- R5L6”; SEQ ID NO: 2); CRRRRRRLLLLLLC (“CRL11” or “CC-R6L6”; SEQ ID NO: 3); CRRRRRRRLLLLLLC (“CRL12” or “CC-R7L6”; SEQ ID NO: 4); CRRRRLLLLLLLC (“CRL13” or “CC-R4L7”; SEQ ID NO: 13);CRRRRRLLLLLLLC (“CRL14” or “CC-R5L7”; SEQ ID NO: 14);CRRRRRRLLLLLLLC (“CRL15” or “CC-R6L7”; SEQ ID NO: 15); CRRRRRRRLLLLLLLC (“CRL16” or “CC-R7L7”; SEQ ID NO: 16).|0019| FIG. 5 shows the charge-dependent lytic effects of various AMPs created using the methods disclosed herein. CRL12 (SEQ ID NO: 4) displayed >20% RBC lysis at 32pM even though it is slightly more potent than CRL11 (+6) (SEQ ID NO: 3), which displays a preliminary selectivity index (SI20>32 / mean MIC) (*P=0.04, obtained by multiple t-tests using GraphPad).

[0020] FIG. 6A-6B show the antimicrobial activity of various AMPs created using the methods disclosed herein. The impact of L-AP cyclization is shown by the comparison of MICs of the linear L-RWV12 (SEQ ID NO: 102) and the circularized C-RWV12 (SEQ ID NO: 103) and CRL11 (SEQ ID NO: 3), (FIG. 6A) against 30 linezolid- resistant clinical isolates from the CDC. The cyclic AMPs show much lower mean MIC with C-RWV16 (SEQ ID NO: 105) (FIG. 6B) displaying the sharpest contrast to the linear AP counterpart L-RWV16 (SEQ ID NO: 104);**P = 0.00043 and ***P<0.0001.

[0021] FIG. 7A-7B shows the antimicrobial activity of various AMPs created using the methods disclosed herein. Oic and Tic are unnatural amino acids that mimic Tryptophan (W). They replace W in the L-RWV14 sequence; R = Arginine. Linear APs with atypical amino acids (FIG. 7A) displayed broad activity (FIG. 7B, mean MIC) against MDR CDC AR isolates. Derived from L-RWV14 (SEQ ID NO: 111), L-Oicl4 or LTicl4 (FIG. 7A) is composed of arginine (Arg, R), and the atypical amino acids similar to tryptophan (Oic or Tic) and valine (Nva). The AR isolates are from the CDC.Docket No. 10504-103WG1These linear AP derivatives display no RBC lysis at any of the test concentrations. Depending on the bacterial pathogen, activity was enhanced; no toxicity to white blood cells. The following sequences are illustrated: RRRRRRR-Oic-Oic-Oic-Oic-Nva-Nva- Nva (“L-Oicl4”; SEQ ID NO: 17); RRRRRRR-Tic-Tic-Tic-Tic-Nva-Nva-Nva (“L- Ticl4”; SEQ ID NO: 18).

[0022] FIG. 8 shows that linear amphipathic peptides are less toxic to mammalian cells than their helical amphipathic counterparts. Numbers at the end of the peptide names represent the number of residues in length. H- represents helical amphipathic, whereas L- at the beginning of the names stands for linear amphipathic. Trend of RBC lysis between sequences of helical and linear amphipathicity. SAAP-148 (SEQ ID NO: 112), a 24r WLBU2-like AP derived from LL37 (SEQ ID NO: 106), displayed highest RBC lysis, followed by WLBU2 (SEQ ID NO: 107) and h-RWV16 (E35). Just like indolicidin (Indo) (SEQ ID NO: 108), the linear / circular APs displayed background RBC lysis at their MOL. The following sequences are illustrated: RR-Nva-Oic-R-Oic- Nva-RR-Nva-Oic-R-Oic-Nva-RR (“E2-35R” or “H-Oicl6”; SEQ ID NO: 19); RR- Nva-Tic-R-Tic-Nva-RR-Nva-Tic-R-Tic-Nva-RR (“E2-53R” or “H-Ticl6”; SEQ ID NO: 20).

[0023] FIG. 9 shows the antimicrobial activity of various AMPs created using the methods disclosed herein against a linezolid-resistant MRSA strain after incubation in plasma. Peptides were incubated in human plasma for Ih or 4h (37°C) and then tested for activity against 5. aureus (CDC strain SA 710). L- stands for linear; c- stands for cyclic; CRL11 (SEQ ID NO: 3) is cyclized via 2 cysteine residues. Activity appeared to be enhanced for L-WRV12 and to a lesser extent C-WRV12 and C-WRV16. Activities of the helical lead peptide E35 and CRL11 (SEQ ID NO: 3) remain unchanged; this is preliminary result from the first experimental trial. Inset: A flat curve (top) indicates no growth at the MIC. Not shown is no effect of control.

[0024] FIG. 10 shows treatment of mice infected with linezolid-resistant MRSA with either L-WRV12 or CC-R6L6 (i.e., CRL11; SEQ ID NO: 3). All peptide- treated mice (N =5) survived, whereas 2 of 5 PBS-treated mice survived. PBS-treated mice did not recover their initial weight; peptide-treated mice regained their initial weight prior to infection. CD-I mice, injected with cyclophosphamide (150 mg / kg i.p.) at 48 and 8 hours prior to i.p. injection of 1.5 x 107CFU of SH730 (linezolid-resistant 5. haemolyticus'), were treated i.v. with 100 ng (~4.5mg / kg) of each peptide and monitored for survival. Bacterial dose was slightly suboptimal as only 2 (x) of the 5 mock- treatedDocket No. 10504-103W01 mice died. While the AP-treated groups lost weight on the first day, they progressively gained weight following the second dose, 24h after bacterial exposure. By contrast, weight gain was observed beginning only on day 5 in the 3 mice that survived in the mock-treated group. Statistical significance was established by two-way ANOVA.

[0025] FIG. 11 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRLLLLC (“CRL1” or “CCR4L4”; SEQ ID NO: 5); CRRRRRLLLLC (“CRL2” or “CC-R5L4”; SEQ ID NO: 6); CRRRRRRLLLLC (“CRL3” or “CC-R6L4”; SEQ ID NO: 7); CRRRRRRRLLLLC (“CRL4” or “CC-R7L4”; SEQ ID NO: 8); CRRRRLLLLLC (“CRL5” or “CC-R4L5”; SEQ ID NO: 9); CRRRRRLLLLLC (“CRL6” or “CC-R5L5”; SEQ ID NO: 10); CRRRRRRLLLLLC (“CRL7” or “CC-R6L5”; SEQ ID NO: 11); CRRRRRRRLLLLLC (“CRL8” or “CC-R7L5”; SEQ ID NO: 12); CRRRRLLLLLLC (“CRL9” or “CC-R4L6”; SEQ ID NO: 1); CRRRRRLLLLLLC (“CRL10” or “CC- R5L6”; SEQ ID NO: 2); CRRRRRRLLLLLLC (“CRL11” or “CC-R6L6”; SEQ ID NO: 3); CRRRRRRRLLLLLLC (“CRL12” or “CC-R7L6”; SEQ ID NO: 4); CRRRRLLLLLLLC (“CRL13” or “CC-R4L7”; SEQ ID NO: 13); CRRRRRLLLLLLLC (“CRL14” or “CC-R5L7”; SEQ ID NO: 14); CRRRRRRLLLLLLLC (“CRL15” or “CC-R6L7”; SEQ ID NO: 15); CRRRRRRRLLLLLLLC (“CRL16” or “CC-R7L7”; SEQ ID NO: 16).

[0026] FIG. 12 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWC (“CRW1” or “CC-R4W4”; SEQ ID NO: 21); CRRRRRWWWWC (“CRW2” or “CC- R5W4”; SEQ ID NO: 22); CRRRRRRWWWWC (“CRW3” or “CC-R6W4”; SEQ ID NO: 23); CRRRRRRRWWWWC (“CRW4” or “CC-R7W4”; SEQ ID NO: 24); CRRRRWWWWFC (“CRWF5” or “CC-R4W4F1”; SEQ ID NO: 25);CRRRRRWWWWFC (“CRWF6” or “CC-R5W4F1”; SEQ ID NO: 26);CRRRRRRWWWWFC (“CRWF7” or “CC-R6W4F1”; SEQ ID NO: 27); CRRRRRRRWWWWFC (“CRWF8” or “CC-R7W4F1”; SEQ ID NO: 28); CRRRRWWWWFFC (“CRWF9” or “CC-R4W4F2”; SEQ ID NO: 29);CRRRRRWWWWFFC (“CRWF10” or “CC-R5W4F2”; SEQ ID NO: 30); CRRRRRRWWWWFFC (“CRWF11” or “CC-R6W4F2”; SEQ ID NO: 31); CRRRRRRRWWWWFFC (“CRWF12” or “CC-R7W4F2”; SEQ ID NO: 32); CRRRRWWWWFFFC (“CRWF13” or “CC-R4W4F3”; SEQ ID NO: 33); CRRRRRWWWWFFFC (“CRWF14” or “CC-R5W4F3”; SEQ ID NO: 34);Docket No. 10504-103W01CRRRRRRWWWWFFFC (“CRWF15” or “CC-R6W4F3”; SEQ ID NO: 35); CRRRRRRRWWWWFFFC (“CRWF16” or “CC-R7W4F3”; SEQ ID NO: 36).

[0027] FIG. 13 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRFFFFC (“CRF1” or “CCR4F4”; SEQ ID NO: 37); CRRRRRFFFFC (“CRF2” or “CCR5F4”; SEQ ID NO: 38); CRRRRRRFFFFC (“CRF3” or “CCR6F4”; SEQ ID NO: 39); CRRRRRRRFFFFC (“CRF4” or “CCR7F4”; SEQ ID NO: 40); CRRRRFFFFFC (“CRF5” or “CCR4F5”; SEQ ID NO: 41); CRRRRRFFFFFC (“CRF6” or “CCR5F5”; SEQ ID NO: 42); CRRRRRRFFFFFC (“CRF7” or “CCR6F5”; SEQ ID NO: 43); CRRRRRRRFFFFFC (“CRF8” or “CCR7F5”; SEQ ID NO: 44); CRRRRFFFFFFC (“CRF9” or “CCR4F6”; SEQ ID NO: 45); CRRRRRFFFFFFC (“CRF10” or “CCR5F6”; SEQ ID NO: 46); CRRRRRRFFFFFFC (“CRF11” or “CCR6F6”; SEQ ID NO: 47); CRRRRRRRFFFFFFC (“CRF12” or “CCR7F6”; SEQ ID NO: 48); CRRRRFFFFFFFC (“CRF13” or “CCR4F7”; SEQ ID NO: 49); CRRRRRFFFFFFFC (“CRF14” or “CCR5F7”; SEQ ID NO: 50); CRRRRRRFFFFFFFC (“CRF15” or “CCR6F7”; SEQ ID NO: 51); CRRRRRRRFFFFFFFC (“CRF16” or “CCR7F7”; SEQ ID NO: 52).

[0028] FIG. 14 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWPC (“CRWP1” or “CC-R4W4P1”; SEQ ID NO: 53); CRRRRRWWWWPC (“CRWP2” or “CC-R5W4P1”; SEQ ID NO: 54); CRRRRRRWWWWPC (“CRWP3” or “CC-R6W4P1”; SEQ ID NO: 55); CRRRRRRRWWWWPC (“CRWP4” or “CC-R7W4P1”; SEQ ID NO: 56); CRRRRWWWWPPC (“CRWP5” or “CC-R4W4P2”; SEQ ID NO: 57); CRRRRRWWWWPPC (“CRWP6” or “CC-R5W4P2”; SEQ ID NO: 58); CRRRRRRWWWWPPC (“CRWP7” or “CC-R6W4P2”; SEQ ID NO: 59); CRRRRRRRWWWWPPC (“CRWP8” or “CC-R7W4P2”; SEQ ID NO: 60); CRRRRWWWWLC (“CRWL1” or “CC-R4W4L1”; SEQ ID NO: 61); CRRRRRWWWWLC (“CRWL2” or “CC-R5W4L1”; SEQ ID NO: 62); CRRRRRRWWWWLC (“CRWL3” or “CC-R6W4L1”; SEQ ID NO: 63); CRRRRRRRWWWWLC (“CRWL4” or “CC-R7W4L1”; SEQ ID NO: 64); CRRRRWWWWLLC (“CRWL5” or “CC-R4W4L2”; SEQ ID NO: 65); CRRRRRWWWWLLC (“CRWL6” or “CC-R5W4L2”; SEQ ID NO: 66);Docket No. 10504-103W01CRRRRRRWWWWLLC (“CRWL7” or “CC-R6W4L2”; SEQ ID NO: 67); CRRRRRRRWWWWLLC (“CRWL8” or “CC-R7W4L2”; SEQ ID NO: 68).

[0029] FIG. 15 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWVC (“CRWV1” or “CC-R4W4V1”; SEQ ID NO: 69); CRRRRRWWWWVC (“CRWV2” or “CC-R5W4V1”; SEQ ID NO: 70); CRRRRRRWWWWVC (“CRWV3” or “CC-R6W4V1”; SEQ ID NO: 71); CRRRRRRRWWWWVC (“CRWV4” or “CC-R7W4V1”; SEQ ID NO: 72); CRRRRWWWWVVC (“CRWV5” or “CC-R4W4V2”; SEQ ID NO: 73); CRRRRRWWWWVVC (“CRWV6” or “CC-R5W4V2”; SEQ ID NO: 74); CRRRRRRWWWWVVC (“CRWV7” or “CC-R6W4V2”; SEQ ID NO: 75); CRRRRRRRWWWWVVC (“CRWV8” or “CC-R7W4V2”; SEQ ID NO: 76); CRRRRWWWWVVVC (“CRWV9” or “CC-R4W4V3”; SEQ ID NO: 77); CRRRRRWWWWVVVC (“CRWV10” or “CC-R5W4V3”; SEQ ID NO: 78); CRRRRRRWWWWVVVC (“CRWV11” or “CC-R6W4V3”; SEQ ID NO: 79); CRRRRRRRWWWWVVVC (“CRWV12” or “CC-R7W4V3”; SEQ ID NO: 80); CRRRRWWWWVVVVC (“CRWV13” or “CC-R4W4V4”; SEQ ID NO: 81); CRRRRRWWWWVVVVC (“CRWV14” or “CC-R5W4V4”; SEQ ID NO: 82); CRRRRRRWWWWVVVVC (“CRWV15” or “CC-R6W4V4”; SEQ ID NO: 83); CRRRRRRRWWWWVVVVC (“CRWV16” or “CC-R7W4V4”; SEQ ID NO: 84).

[0030] FIG. 16 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWVC (“CRWV1” or “CC-R4W4V1”; SEQ ID NO: 69); CRRRRRWWWWVC (“CRWV2” or “CC-R5W4V1”; SEQ ID NO: 70); CRRRRRRWWWWVC (“CRWV3” or “CC-R6W4V1”; SEQ ID NO: 71); CRRRRRRRWWWWVC (“CRWV4” or “CC-R7W4V1”; SEQ ID NO: 72); CRRRRWWWWVVC (“CRWV5” or “CC-R4W4V2”; SEQ ID NO: 73); CRRRRRWWWWVVC (“CRWV6” or “CC-R5W4V2”; SEQ ID NO: 74); CRRRRRRWWWWVVC (“CRWV7” or “CC-R6W4V2”; SEQ ID NO: 75); CRRRRRRRWWWWVVC (“CRWV8” or “CC-R7W4V2”; SEQ ID NO: 76); CRRRRWWWWVVVC (“CRWV9” or “CC-R4W4V3”; SEQ ID NO: 77); CRRRRRWWWWVVVC (“CRWV10” or “CC-R5W4V3”; SEQ ID NO: 78); CRRRRRRWWWWVVVC (“CRWV11” or “CC-R6W4V3”; SEQ ID NO: 79); CRRRRRRRWWWWVVVC (“CRWV12” or “CC-R7W4V3”; SEQ ID NO: 80);Docket No. 10504-103W01CRRRRWWWWVVVVC (“CRWV13” or “CC-R4W4V4”; SEQ ID NO: 81); CRRRRRWWWWVVVVC (“CRWV14” or “CC-R5W4V4”; SEQ ID NO: 82); CRRRRRRWWWWVVVVC (“CRWV15” or “CC-R6W4V4”; SEQ ID NO: 83); CRRRRRRRWWWWVVVVC (“CRWV16” or “CC-R7W4V4”; SEQ ID NO: 84).

[0031] FIG. 17 shows the antimicrobial activity of various AMPs created using the methods disclosed herein, and specifically that CCR6L6 (i.e., CRL11, SEQ ID NO: 3) is a good candidate against 3 ESKAPE bacteria. The following sequences are illustrated: CRRRRLLLLLLC (“CRL9” or “CC-R4L6”; SEQ ID NO: 1); CRRRRRLLLLLLC (“CRL10” or “CC-R5L6”; SEQ ID NO: 2);CRRRRRRLLLLLLC (“CRL11” or “CC-R6L6”; SEQ ID NO: 3);CRRRRRRRLLLLLLC (“CRL12” or “CC-R7L6”; SEQ ID NO: 4).

[0032] FIG. 18 shows the antimicrobial activity of various AMPs created using the methods disclosed herein, and specifically that CCR7W4 (SEQ ID NO: 24) is a good candidate against E. coli and E. faecalis. The following sequences are illustrated: CRRRRWWWWC (“CRW1” or “CC-R4W4”; SEQ ID NO: 21); CRRRRRWWWWC (“CRW2” or “CC-R5W4”; SEQ ID NO: 22); CRRRRRRWWWWC (“CRW3” or “CC- R6W4”; SEQ ID NO: 23); CRRRRRRRWWWWC (“CRW4” or “CC-R7W4”; SEQ ID NO: 24).

[0033] FIG. 19 shows the antimicrobial activity of various AMPs created using the methods disclosed herein, and specifically that the CRF series produced 3 good candidates: CCR4F5 (SEQ ID NO: 41) (E. coli), CCR5F5 (SEQ ID NO: 42) (E. coli), and CCR7F5 (SEQ ID NO: 44) (E. coli and S. aureus). R = arginine, F = phenylalanine. The following sequences are illustrated: CRRRRFFFFFC (“CRF5” or “CCR4F5”; SEQ ID NO: 41); CRRRRRFFFFFC (“CRF6” or “CCR5F5”; SEQ ID NO: 42); CRRRRRRFFFFFC (“CRF7” or “CCR6F5”; SEQ ID NO: 43); CRRRRRRRFFFFFC (“CRF8” or “CCR7F5”; SEQ ID NO: 44).

[0034] FIG. 20 shows the antimicrobial activity of various AMPs created using the methods disclosed herein, and specifically that the CRF6 series produced 3 good candidates: CCR5F7 (SEQ ID NO: 50) (E. coli, E. faecalis, and MRSA), CCR6F6 (SEQ ID NO: 47) (E. coli), and CCR5F6 (SEQ ID NO: 46) (E. faecalis, A. baumannii, & MRSA). R = arginine, F = phenylalanine. The following sequences are illustrated: CRRRRFFFFFFC (“CRF9” or “CCR4F6”; SEQ ID NO: 45); CRRRRRFFFFFFC (“CRF10” or “CCR5F6”; SEQ ID NO: 46); CRRRRRRFFFFFFC (“CRF11” orDocket No. 10504-103W01“CCR6F6”; SEQ ID NO: 47); CRRRRRRRFFFFFFC (“CRF12” or “CCR7F6”; SEQ ID NO: 48).

[0035] FIG. 21 shows the antimicrobial activity of various AMPs created using the methods disclosed herein and the possibility of combining two or more AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRFFFFFFFC (“CRF13” or “CCR4F7”; SEQ ID NO: 49); CRRRRRFFFFFFFC (“CRF14” or “CCR5F7”; SEQ ID NO: 50); CRRRRRRFFFFFFFC (“CRF15” or “CCR6F7”; SEQ ID NO: 51); CRRRRRRRFFFFFFFC (“CRF16” or “CCR7F7”; SEQ ID NO: 52).

[0036] FIG. 22 shows the antimicrobial activity of various AMPs created using the methods disclosed herein and the possibility of combining two or more AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWFC (“CRWF5” or “CC-R4W4F1”; SEQ ID NO: 25); CRRRRRWWWWFC (“CRWF6” or “CC-R5W4F1”; SEQ ID NO: 26); CRRRRRRWWWWFC (“CRWF7” or “CC-R6W4F1”; SEQ ID NO: 27); CRRRRRRRWWWWFC (“CRWF8” or “CC-R7W4F1”; SEQ ID NO: 28).

[0037] FIG. 23 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. P = proline; CC-R4W4P1 (SEQ ID NO: 53) for E. coli; CCR7W4P1 (SEQ ID NO: 56) for E. coli, E. faecalis, & MRSA (methicillin-resistant S. aureus). The following sequences are illustrated: CRRRRWWWWPC (“CRWP1” or “CC-R4W4P1”; SEQ ID NO: 53); CRRRRRWWWWPC (“CRWP2” or “CC- R5W4P1”; SEQ ID NO: 54); CRRRRRRWWWWPC (“CRWP3” or “CC-R6W4P1”; SEQ ID NO: 55); CRRRRRRRWWWWPC (“CRWP4” or “CC-R7W4P1”; SEQ ID NO: 56).

[0038] FIG. 24 shows the antimicrobial activity of various AMPs created using the methods disclosed herein and the possibility of combining two or more AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWLC (“CRWL1” or “CC-R4W4L1”; SEQ ID NO: 61); CRRRRRWWWWLC (“CRWL2” or “CC-R5W4L1”; SEQ ID NO: 62); CRRRRRRWWWWFC (“CRWL3” or “CC-R6W4L1”; SEQ ID NO: 63); CRRRRRRRWWWWLC (“CRWL4” or “CC-R7W4L1”; SEQ ID NO: 64).

[0039] FIG. 25 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWVVVVC (“CRWV13” or “CC-R4W4V4”; SEQ ID NO: 81);Docket No. 10504-103WG1CRRRRRWWWWVVVVC (“CRWV14” or “CC-R5W4V4”; SEQ ID NO: 82); CRRRRRRWWWWVVVVC (“CRWV15” or “CC-R6W4V4”; SEQ ID NO: 83); CRRRRRRRWWWWVVVVC (“CRWV16” or “CC-R7W4V4”; SEQ ID NO: 84).

[0040] FIG. 26 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWVVVC (“CRWV9” or “CC-R4W4V3”; SEQ ID NO: 77); CRRRRRWWWWVVVC (“CRWV10” or “CC-R5W4V3”; SEQ ID NO: 78); CRRRRRRWWWWVVVC (“CRWV1 1” or “CC-R6W4V3”; SEQ ID NO: 79); CRRRRRRRWWWWVVVC (“CRWV12” or “CC-R7W4V3”; SEQ ID NO: 80).

[0041] FIG. 27 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWVVC (“CRWV5” or “CC-R4W4V2”; SEQ ID NO: 73); CRRRRRWWWWVVC (“CRWV6” or “CC-R5W4V2”; SEQ ID NO: 74); CRRRRRRWWWWVVC (“CRWV7” or “CC-R6W4V2”; SEQ ID NO: 75); CRRRRRRRWWWWVVC (“CRWV8” or “CC-R7W4V2”; SEQ ID NO: 76).100421 FIG. 28 shows the antimicrobial activity of various AMPs created using the methods disclosed herein. The following sequences are illustrated: CRRRRWWWWVC (“CRWV1” or “CC-R4W4V1”; SEQ ID NO: 69); CRRRRRWWWWVC (“CRWV2” or “CC-R5W4V1”; SEQ ID NO: 70); CRRRRRRWWWWVC (“CRWV3” or “CC-R6W4V1”; SEQ ID NO: 71); CRRRRRRRWWWWVC (“CRWV4” or “CC-R7W4V1”; SEQ ID NO: 72).DETAILED DESCRIPTION

[0043] Provided herein are compositions and methods for selecting for and making amphipathic polypeptides having increased antimicrobial and / or anticancer activity. In some embodiments, the amphipathic polypeptides have increased antimicrobial activity and / or anti-cancer activity and low toxicity.

[0044] Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicants desire that the following terms be given the particular definition as defined below.Docket No. 10504-103W01

[0045] Terminology

[0046] As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0047] The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.

[0048] The term “amphipathic” as used herein refers to a polypeptide having both hydrophobic and hydrophilic parts. In some embodiments, the amphipathic polypeptides described herein have linear amphipathicity. Put another way, the linear amphipathic polypeptides have a contiguous span of hydrophilic or cationic amino acids and a contiguous span of hydrophobic amino acids.

[0049] The term “antimicrobial peptide” or “AMP” refers to naturally occurring peptide agents that act as part of the host’s innate immune system to protect against pathogens, including bacteria, fungi, and viruses.

[0050] Naturally occurring AMPs are ubiquitous agents with a rare ability to both kill multidrug-resistant bacteria and even inhibit endotoxin-induced inflammation. AMPs that are ribosomally synthesized in animal species, referred to as AMPs with classical amphipathic structures, or simply classical AMPs, represent a crucial component of innate immunity; they are able to selectively target negatively charged lipids on bacterial surfaces through electrostatic interactions while they require relatively higher concentrations to similarly interact with eukaryotic cell membranes. Thus, AMPs can either disrupt bacterial cell membranes or, less commonly, target vital intracellular structures like DNA and RNA, leading to cell death.

[0051] A "composition" is intended to include a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.

[0052] A "control" is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive" or "negative."

[0053] A "decrease" or “reduction” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. For example, a decrease can be a change in the symptoms of a disorder such that the symptoms are lessDocket No. 10504-103W01 than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, decreasing or reducing can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100%, or more decrease or reduction so long as the decrease or reduction is statistically significant.

[0054] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCB1 web site or the like). Such sequences are then said to be “substantially identical.” This definition also refers to, or may be applied to, the compliment of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as those that have substitutions. As described below, the preferred algorithms can account for gaps and the like. Preferably, identity exists over a region that is at least about 10 amino acids or 20 nucleotides in length, or more preferably over a region that is 10-50 amino acids or 20-50 nucleotides in length. As used herein, percent (%) amino acid sequence identity is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can be determined by known methods.

[0055] For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates areDocket No. 10504-103W01 designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0056] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. ( 1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1990) J. Mol. Biol. 215:403-410). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0057] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci.Docket No. 10504-103W01USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01.

[0058] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more increase so long as the increase is statistically significant.

[0059] The term "polypeptide" is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, peptoids (such as a-peptide, a- peptoid, [3-peptoid), or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g. ester, ether, etc. As used herein the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs.

[0060] The term “8120” as used herein refers to the maximum test concentration of a polypeptide, for example an amphipathic polypeptide as disclosed herein, which causes less than 20% toxicity (i.e., TC20) divided by the minimum inhibitory concentration (MIC). The term “MIC” as used herein refers to the refers to the lowest concentration of a polypeptide, for example an amphipathic polypeptide as disclosed herein, that prevents the visible growth of a specific microorganism. MIC is a measure of the efficacy of each AP at killing bacteria, where a lower MIC is more efficient.

[0061] The term “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.

[0062] The term “therapeutic index” or “TI” as used herein refers to the maximum tolerable dose (MTD) of a polypeptide, for example an amphipathic polypeptide as disclosed herein, divided by the systemic minimum effective therapeutic dose (MED). The TI is a dose or range of doses at which the polypeptide is effective without unacceptable adverse effects or toxicity.Docket No. 10504-103W01

[0063] Compositions and Methods

[0064] Provided herein are compositions and methods for selecting for and making amphipathic polypeptides having increased antimicrobial and / or anticancer activity. As used herein, “amphipathic” refers to a polypeptide having both hydrophobic and hydrophilic parts. The amphipathic polypeptides described herein have linear amphipathicity, or in other words, the polypeptides have a contiguous span of hydrophilic or cationic amino acids and a contiguous span of hydrophobic amino acids. FIG. 1A-1E demonstrates this concept and shows a schematic of a linear amphipathic polypeptide having a span of hydrophobic (H) amino acids adjacent to a span of cationic (C) amino acids.

[0065] In some embodiments, the amphipathic polypeptide is equal to or between about 8 and 28 amino acids. The amphipathic polypeptide can also be equal to or between about 10 and 26 amino acids, equal to or between about 12 and 24 amino acids, equal to or between about 14 and 22 amino acids, equal to or between about 16 and 20 amino acids, or about 18 amino acids. In some embodiments, the cationic span and the hydrophobic span are of about equal lengths or number of amino acids. In other aspects, the cationic span is between about 25% and 75% of the amphipathic polypeptide. In some embodiments, the cationic span is about 25%, 30%, 40%, 50%, 60%, 70% or 75% of the amphipathic polypeptides. In other or further aspects, the hydrophobic span is between about 25% and 75% of the amphipathic polypeptide. In some embodiments, the hydrophobic span is about 25%, 30%, 40%, 50%, 60%, 70% or 75% of the amphipathic polypeptide.

[0066] As noted above, each amino acid within the amphipathic polypeptide can be either natural or synthetic. Included within the term “amino acid” are D or L optical isomers of amino acids and amino acid analogs. The amino acids may be linked by peptide bonds. In another embodiment, the amino acids may be linked by other bonds, e.g. ester, ether, etc. Amino acid codes known to those skilled in the art and provided in Table 1 will be used throughout this disclosure.

[0067] Table 1 : Amino Acid CodesDocket No. 10504-103W01

[0068] In certain aspects, each cationic amino acid is selected from arginine, lysine, or histidine. In those or other aspects, each hydrophobic amino acid is selected from alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, or glycine. In some embodiments, each hydrophobic amino acid is selected from alanine, isoleucine, leucine, tryptophan, Oic, Tic, phenylalanine, valine, proline, or glycine. The unnatural hydrophobic amino acids Oic and Tic mimic the double ring of tryptophan. In some embodiments, each hydrophobic amino acid is selected from isoleucine, leucine, phenylalanine, or valine. In still other embodiments, each hydrophobic amino acid is tryptophan or phenylalanine. In some embodiments, each cationic amino acid is the same cationic amino acid within the amphipathic polypeptide. In some embodiments, the cationic amino acids of the amphipathic polypeptide are selected from two or moreDocket No. 10504-103W01 different cationic amino acids. In some embodiments, each hydrophobic amino acid is the same hydrophobic amino acid within the amphipathic polypeptide. In some embodiments, the hydrophobic amino acids of the amphipathic polypeptide are selected from two or more different hydrophobic amino acids.

[0069] In some embodiments, the amphipathic polypeptides having linear amphipathicity are end-to-end cyclized. The amphipathic polypeptides can be end-to- end cyclized by any suitable method including, for example but not limited to disulfide bonding (cysteine bridge) and end-to-end amidation. In certain aspects, the amphipathic polypeptides are end-to-end cyclized using disulfide bonding. In certain aspects, the amphipathic polypeptides are end-to-end cyclized by end-to-end amidation. In some embodiments, the end-to-end cyclized amphipathic polypeptides have additional, non- end-to-end disulfide or other bonds (e.g., bicyclic).

[0070] In some embodiments, the amphipathic polypeptides selected from SEQ ID NO: 1-100 having linear amphipathicity are end-to-end cyclized. In some embodiments, the amphipathic polypeptides selected from SEQ ID NO: 1-100 can be end-to-end cyclized by any suitable method including disulfide bonding (cysteine bridge) and end-to-end amidation. In certain aspects, the amphipathic polypeptides selected from SEQ ID NO: 1-100 are end-to-end cyclized using disulfide bonding. In certain aspects, the amphipathic polypeptides selected from SEQ ID NO: 1-100 are end-to-end cyclized by end-to-end amidation. In some embodiments, the end-to-end cyclized amphipathic polypeptides selected from SEQ ID NO: 1-100 have additional, non- end-to-end disulfide or other bonds (e.g., bicyclic). In some embodiments, the amphipathic polypeptides selected from SEQ ID NO: 1-100 are bicyclic.

[0071] In some embodiments, two or more APs generated using the methods disclosed herein are combined to create a novel AP polypeptide molecule. In some embodiments, the two or more APs can be conjugated by end-to-end amidation or via cysteine (S-S) bridging. In some embodiments, the two or more APs are conjugated via a peptide bond.

[0072] The amphipathic polypeptides described herein have increased antimicrobial activity and / or increased anticancer activity as compared to a control. As used herein, the terms “antimicrobial,” “anti-bacterial,” or “anti-viral” refer to the ability of the amphipathic polypeptides described herein to prevent, inhibit, or kill one or more microbes including but not limited to a bacterium, virus, fungus, and parasite. The term “anticancer” refers to the ability of the amphipathic polypeptides described herein to reduce the size, number, and / or metastasis of a cancer. In some embodiments,Docket No. 10504-103WG1“antimicrobial” and / or “anticancer” refers to an increase in survival of a subject to which a composition comprising an amphipathic polypeptide described herein has been administered. The increase in survival is determined as compared to a control, such as an untreated subject or a general study population.

[0073] In some embodiments, the bacterium is selected from M. tuberculosis, M. bovis, M. bovis strain BCG, BCG substrains, M. avium, M. intracellular, M. africanum, M. kansasii, M. marinum, M. ulcerans, M. avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Salmonella typhi, other Salmonella species, Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, other Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Bacillus anthracis, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, or other Yersinia species. In some embodiments, the bacterium is S’. aureus, E. faecalis, E. coli or A. baumannii. In certain aspects, the bacterium has increased antibiotic resistance as compared to other strains of the bacterium species. In some embodiments the S. aureus is methicillin resistant (MRSA).

[0074] In some embodiments, virus is selected from Herpes Simplex virus- 1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus, Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1,Docket No. 10504-103W01Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, or Human Immunodeficiency virus type-2.

[0075] In some embodiments, the fungus is selected from Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, or Altemaria altemata.

[0076] In some embodiments, the parasite is selected from Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Schistosoma mansoni, other Schistosoma species, or Entamoeba histolytica.

[0077] In some embodiments, the cancer includes, but is not limited to acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarcinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuseDocket No. 10504-103W01 large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa-associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom’s macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia / lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP- NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA),Docket No. 10504-103W01 melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer, or vulvar cancer (e.g., Paget’s disease of the vulva).

[0078] The amphipathic polypeptides described herein can also have decreased toxicity as compared to a control. In some embodiments, decreased toxicity is decreased lysis of red blood cells as compared to a control.

[0079] The methods described herein are a surprisingly effective means for creation of amphipathic polypeptides having linear amphipathicity and improved characteristics such as increased antimicrobial activity, increased anticancer activity, and / or decreased toxicity. In some embodiments, the method is iterative and involves a cyclical process of creating a group of amphipathic polypeptides, each differing by the addition of one cationic or hydrophobic amino acid to a cationic or hydrophobic end, respectively. The group of amphipathic polypeptides are then analyzed for one or more improved characteristics such as increased antimicrobial activity, increased anticancer activity and decreased toxicity. This method preferably allows for a structure-function titration wherein cationic and / or hydrophobic motifs within the amphipathic polypeptides can be analyzed relative to one another. In some embodiments, preferred cationic and / or hydrophobic motifs are identified and combined in an amphipathic antimicrobial polypeptide sequence.

[0080] Accordingly, provided herein is a method of creating an amphipathic polypeptide sequence comprising: a) obtaining an amphipathic polypeptide (AP) comprising a sequence having the formula of CiHi wherein C is a cationic amino acid, H is a hydrophobic amino acid, and wherein the polypeptide has a cationic amino acid end and a hydrophobic amino acid end; b) adding one cationic amino acid at the cationic amino acid end of the AP to create a cationic AP extension product, and cyclically adding one same or different cationic amino acid at the cationic amino acid end of each cationic AP extensionDocket No. 10504-103W01 product to create a group of cationic extension polypeptides having the formula of Ci+xHi, wherein x is equal to or between 1 and 16; and / or c) adding one hydrophobic amino acid at the hydrophobic amino acid end of the AP to create a hydrophobic AP extension product, and cyclically adding one same or different hydrophobic amino acid at the hydrophobic amino acid end of each hydrophobic AP extension product to create a group of hydrophobic extension polypeptides having the formula of CiHi+x, wherein x is equal to or between 1 and 16; and d) adding one cationic amino acid at the cationic amino acid end of one or more of the cationic extension polypeptides to create a second group of cationic extension polypeptides having the formula of Ci+xHi+ywherein x and y are the same or different and equal to or between 1 and 16; and / or e) adding one hydrophobic amino acid at the hydrophobic amino acid end of one or more of the hydrophobic extension polypeptides to create a second group of hydrophobic extension polypeptides having the formula of Ci+yHi+xwherein x and y are the same or different and equal to or between 1 and 16; and f) end-to-end cyclizing each of the group of polypeptides of step b), step c), step d) and / or step e) to create a group of cyclized polypeptides.

[0081] In some embodiments, the method further comprises the following steps: g) determining antimicrobial activity, anticancer activity and / or toxicity of each of the group of polypeptides of step b), step c), step d) and / or step e) and of each of the group of cyclized polypeptides of step f); and h) creating an amphipathic polypeptide comprising the one or more polypeptide sequences associated with an expected antimicrobial activity and / or expected anticancer activity.

[0082] In some embodiments, the method further comprises the following steps: g) determining antimicrobial activity, anticancer activity and / or toxicity of each of the group of cyclized polypeptides of step f); and h) creating an amphipathic polypeptide comprising the one or more polypeptide sequences associated with an expected antimicrobial activity and / or expected anticancer activity.Docket No. 10504-103W01

[0083] It should be understood that each or any of steps a) through e) can be performed using any computer implemented method, including but not limited to, artificial intelligence methods.

[0084] Also provided herein is a method of selecting for an amphipathic polypeptide sequence comprising: a) obtaining an amphipathic polypeptide (AP) comprising a sequence having the formula of C1H1 wherein C is a cationic amino acid, H is a hydrophobic amino acid, and wherein the polypeptide has a cationic amino acid end and a hydrophobic amino acid end; b) adding one cationic amino acid at the cationic amino acid end of the AP to create a cationic AP extension product, and cyclically adding one same or different cationic amino acid at the cationic amino acid end of each cationic AP extension product to create a group of cationic extension polypeptides having the formula of Ci+xHi, wherein x is equal to or between 1 and 16; and / or c) adding one hydrophobic amino acid at the hydrophobic amino acid end of the AP to create a hydrophobic AP extension product, and cyclically adding one same or different hydrophobic amino acid at the hydrophobic amino acid end of each hydrophobic AP extension product to create a group of hydrophobic extension polypeptides having the formula of CiHi+x, wherein x is equal to or between 1 and 16; and d) adding one hydrophobic amino acid at the hydrophobic amino acid end of one or more of the cationic AP extension polypeptides to create a second group of cationic extension polypeptides having the formula of Ci+xHi+ywherein x and y are the same or different and equal to or between 1 and 16; and / or e) adding one cationic amino acid at the cationic amino acid end of one or more of the hydrophobic extension polypeptides to create a second group of hydrophobic extension polypeptides having the formula of Ci+yHi+xwherein x and y are the same or different and equal to or between 1 and 16; and f) end-to-end cyclizing each of the group of polypeptides of step b), step c), step d) and / or step e) to create a group of cyclized polypeptides.

[0085] In some embodiments, the method further comprises: g) determining antimicrobial activity, anticancer activity and / or toxicity of each of the group of polypeptides of step b), step c), step d) and / or step e) and of each of the group of cyclized polypeptides of step f); andDocket No. 10504-103W01 h) selecting one or more of the group of cyclized polypeptides of step g) having a higher antimicrobial activity, a higher anticancer activity and / or a lower toxicity as compared to other cyclized polypeptides in the group and thereby selecting for an amphipathic polypeptide sequence.

[0086] In some embodiments, the method further comprises: g) determining antimicrobial activity, anticancer activity and / or toxicity of each of the group of cyclized polypeptides of step f); and h) selecting one or more of the group of cyclized polypeptides of step g) having a higher antimicrobial activity, a higher anticancer activity and / or a lower toxicity as compared to other cyclized polypeptides in the group and thereby selecting for an amphipathic polypeptide sequence.

[0087] As used herein, the term “AP extension product” refers to any amphipathic polypeptide that has a one amino acid addition at an end as compared to another amphipathic polypeptide in the group. A “cationic AP extension product” has a cationic amino acid added at the cationic end of amphipathic polypeptide within the group. A “hydrophobic AP extension product” has a hydrophobic amino acid added at the hydrophobic end of amphipathic polypeptide within the group. For example, the amphipathic polypeptide of CCCCCHHHH (C5H4), wherein C refers to a cationic amino acid and H refers to a hydrophobic amino acid, is a cationic AP extension product of CCCCHHHH (C4H4). The amphipathic polypeptide of CCCCCCHHHH (C6H4) is a cationic AP extension product of CCCCCHHHH (C5H4). Similarly, CCCCHHHHH (C4H5) is a hydrophobic AP extension product of CCCCHHHH (C4H4), and CCCCHHHHHH (C4H6) is a hydrophobic AP extension product of CCCCHHHHH (C4H5).

[0088] In some embodiments, the AP polypeptide sequence begins with a template comprising 8 residues, wherein the 8 residues consist of four (4) cationic residues and four (4) hydrophobic residues.

[0089] In some embodiments, the charge of the resulting AP polypeptide sequence is between +4 and +10. In some embodiments, the length of the AP polypeptide sequence is between 8 residues and 18 residues.

[0090] It should be understood that “end” refers to either end of a cationic or hydrophobic region. For example, an amino acid addition at a cationic end of the amphipathic polypeptide of CCCH with four amino acid positions, can be an additionDocket No. 10504-103W01 of a cationic amino acid at the first position (before the first C amino acid) or the fourth position (between the third C amino acid and the H amino acid).

[0091] “Cyclically adding” refers to successive addition of one cationic or hydrophobic amino acid to a cationic or hydrophobic end, respectively, of a prior created AP extension product. Accordingly, the present disclosure includes methods wherein multiple cycles of single amino acid additions are made to extend a prior produced AP extension product by one cationic or hydrophobic amino acid, creating a group of amphipathic polypeptides that differ from one other amphipathic polypeptide in the group by one amino acid. In these embodiments, within each member of the group of amphipathic polypeptides, the original C or H amino acids in the amphipathic polypeptide are kept constant once added. In some embodiments, only one motif is elongated at a time. For example, one such group of cationic AP extension product polypeptides comprises RRRRLLLL (SEQ ID NO: 85), RRRRRLLLL (SEQ ID NO: 86), RRRRRRLLLL (SEQ ID NO: 87), and RRRRRRRLLLL (SEQ ID NO: 88). Another such group comprises RRRRLLLL (SEQ ID NO: 89), KRRRRLLLL (SEQ ID NO: 90), KKRRRRLLLL (SEQ ID NO: 91) and KKKRRRRLLLL (SEQ ID NO: 92). A still further group comprises RRRRLLLL (SEQ ID NO: 93), RRRRLLLLL (SEQ ID NO: 94), RRRRLLLLLL (SEQ ID NO: 95), and RRRRLLLLLLL (SEQ ID NO: 96). An exemplary group of hydrophobic AP extension product polypeptides comprises RRRRLLLLV (SEQ ID NO: 97), RRRRLLLLVV (SEQ ID NO: 98), RRRRLLLLVVV (SEQ ID NO: 99), and RRRRLLLLVVVV (SEQ ID NO: 200). Accordingly, included herein are methods wherein each member of the group of AP extension products of step b) and / or step c) differs from at least one other member of the group by a single end amino acid difference; and the C or H residues can be a single amino acid (e.g., R or / and K for the C motif; L , P, F, V, I, or / and W for the H motif) or different.

[0092] Further, step d) and / or step e) refer to adding single cationic or hydrophobic amino acids to the cationic AP extension polypeptides and / or the hydrophobic AP extension polypeptides. As one example, a hydrophobic amino acid valine is added to a hydrophobic amino acid end of RRRRRRRLLLL (SEQ ID NO: 88) (a cationic AP extension product) to create RRRRRRRLLLLV (SEQ ID NO: 99). In another example, a cationic amino acid arginine is added to a cationic amino acid end of RRRRLLLLVVVV (SEQ ID NO: 98) (a hydrophobic AP extension product) to create RRRRRLLLL VVVV (SEQ ID NO: 100). Through cyclical additions of single aminoDocket No. 10504-103W01 acids to multiple AP extension products the method can allow for structure-function titration, delineating the roles of the C or H motifs relative to each other. Therefore, while the present method can begin with Ci+xHi or CiHi+x, it can evolve as Ci+xHyand CxHi+y. An example could be C4H4, C4H5, C4H6 followed by C5H5, CsHg, C5H7 followed by CeHi, C6H2, CGH^ followed by Ci He, C2H6. As a further example, He could be taken alone for elongation CxH& or CxH(i+y) and (Ci+X)Hywherein X or Y = 1 to 16.

[0093] In some embodiments, the method of creating an amphipathic polypeptide (AP) sequence comprises: a) obtaining a sequence having the formula of C i+yHi+xwherein C i+yrepresents the number of cationic amino acids forming a cationic motif on one end terminus of the AP, wherein Hi+Xrepresents the number of hydrophobic amino acids forming a hydrophobic motif on the other end terminus of the AP; and wherein x and y are the same or different and equal to any number between 0 and 16; b) varying y or adding one cationic amino acid at either end of the cationic motif of the AP to create a cationic AP extension product while x remains unchanged results in linear APs, which can become cyclic by joining the cationic end terminus and the hydrophobic end terminus or by adding a cysteine residue at each end terminus to join the two end termini via a disulfide bridge; and / or c) varying x or adding one hydrophobic amino acid at either end of the hydrophobic motif of the AP to create a hydrophobic AP extension product while y remains unchanged results in linear APs, which can become cyclic by joining the cationic end terminus and the hydrophobic end terminus or by adding a cysteine residue at each end terminus to join the two end termini via a disulfide bridge.

[0094] In some embodiments, the method further comprises: d) determining antimicrobial activity, anticancer activity, and / or toxicity of each of the group of cyclized polypeptides of step c); and e) creating an amphipathic polypeptide comprising the one or more polypeptide sequences associated with an expected antimicrobial activity and / or expected anticancer activity.

[0095] In other embodiments, the method further comprises: d) determining antimicrobial activity, anticancer activity, and / or toxicity of each of the group of cyclized polypeptides of step c); andDocket No. 10504-103W01 e) selecting one or more of the group of cyclized polypeptides of step d) having a higher antimicrobial activity, a higher anticancer activity, and / or a lower toxicity as compared to other cyclized polypeptides in the group and thereby selecting for an amphipathic polypeptide sequence.

[0096] As discussed above, included herein are embodiments wherein each cationic amino acid is selected from arginine, lysine, or histidine. In those or other aspects, each hydrophobic amino acid is selected from alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, or glycine. In some embodiments, each hydrophobic amino acid is selected from isoleucine, leucine, phenylalanine, or valine. In still other embodiments, each hydrophobic amino acid is tryptophan or phenylalanine.

[0097] In some embodiments “x” of Ci+xHi and / or CiHi+xis equal to or between about 0 and 16, equal to or between about 2 and 14, equal to or between about 4 and 12, equal to or between about 6 and 10, equal to or about 8. In some embodiments, “x” of Ci+xHi and / or CiHi+xis equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. In other or further embodiments, “y” of Ci+yHi+xand / or Ci+xHi+yis equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

[0098] Antibacterial activity assays are known to persons of skill in the art and are described in Klousnitzer, Jessie, et al. "Comparative Properties of Helical and Linear Amphipathicity of Peptides Composed of Arginine, Tryptophan, and Valine." Antibiotics 13.10 (2024): 954, incorporated herein for all purposes. In some embodiments, the antibacterial activity assay comprises minor modifications of a standard growth inhibition assay endorsed by the Clinical and Laboratory Standards Institute (CLSI). Bacterial inocula for testing can be prepared from exponential growth phase by diluting overnight cultures at 1 :100 with fresh Tryptic soy broth (TSB) (Millipore Sigma, USA, Cat. No. 22092-500G) and incubated for an additional 3-4 h. Bacteria are spun at 3000x g for 10 min and the pellet is resuspended in phosphate- buffered saline (PBS) (Millipore Sigma, St Louis, MO, USA, Cat. No. P4417) to determine bacterial turbidity using a densitometer (Den-IB, Grant Instruments, Beaver Falls, PA, USA) at 0.5 McFarland (unit of bacterial density) corresponding to 108 CFU / mL. The prepared bacteria are incubated with each of the AP peptides to be tested at a maximum test concentration of 16 pM and serially diluted in MHB2 and RPMI 1640 (Corning, NY, USA, Cat. No. 15-040-CM) at a 1:1 ratio. The plates are kept in an 8-drawer incubator for 18 h at 37 °C. A robotic system takes a plate from the drawer every hour and feeds a plate reader, which records the kinetics of bacterial growth atDocket No. 10504-103W01570 nm to examine growth inhibition in real time (BioTek Instruments, Winooski, VT, USA). The minimum inhibitory concentration or MIC, as defined herein elsewhere, is the minimum peptide concentration that prevented bacterial growth, and it is objectively indicated by a flat (horizontal) line in the bacterial growth kinetic graph. Most assays are completed in duplicate. To assay for anti-biofilm activities, the 96-well plate from the growth inhibition assay is incubated for an additional 6 h (24 h total). The biomass is detected with crystal violet using a plate reader at A570.

[0099] The growth inhibition assay can be modified as a kinetic bacterial killing assay to determine if the peptides are active in the absence of a major carbon source and the rate at which they act. Kinetic bacterial killing assays are known to those of skill in the art and are described in Klousnitzer, Jessie, et al. "Comparative Properties of Helical and Linear Amphipathicity of Peptides Composed of Arginine, Tryptophan, and Valine." Antibiotics 13.10 (2024): 954, incorporated herein for all purposes. For example, bacterial inocula is prepared as it was for the growth inhibition assay, but it is added to a non-carbon-containing source (PBS in our experiments) treated with 4 pM peptide. At different time points ranging from 30 s to 2 h, the samples are serially diluted, drip plated onto broth agar medium and incubated at 37 °C overnight. The colony forming unit / mL or CFU / mL is enumerated after incubation.

[0100] In some embodiments, the AP polypeptides produced by the methods described herein have increased stability (i.e., less proteolytic degradation) compared to their counterpart helical polypeptide sequences having identical amino acid composition. Proteolytic degradation assays can be performed according to methods known to persons of skill in the art. For example, the neutrophil elastase can be dissolved in 200 mmol / L Tris buffer, pH 8.8, and used at a molar ratio of 1:50 with an AP polypeptide in 200 mM ammonium bicarbonate pH 8.0 for 1 h or 4 h. The control experiment incubates polypeptides alone in 200 mmol / L ammonium bicarbonate for 1 h or 4 h. Upon completion of incubations, peptides are serially diluted to test MIC. 50% (v / v) MHB2 can be added to the plates for reference. MIC can then be determined.

[0101] The toxicity of AP polypeptides produced by the methods described herein can be examined using human red blood cells (RBCs). RBC toxicity studies are known to those of skill in the art and are described in Mitra, Saheli, et al. "Cyclization of Two Antimicrobial Peptides Improves Their Activity." ACS omega 10.9 (2025): 9728-9740, incorporated herein by reference for all purposes. For example, RBCs can be separated by histopaque differential centrifugation using blood obtained from a blood bank. ForDocket No. 10504-103W01 an RBC lysis assay, isolated RBCs are resuspended in PBS (e.g., at 5% concentration). The AP polypeptides can be serially diluted twofold in 100 uL of PBS before adding 100 uL of 5% RBC to a final dilution of 2.5% RBC to ensure that the A570 of hemoglobin does not saturate a plate reader. In parallel, RBCs are osmotically burst with water at increasing concentrations to generate a standard curve of RBC lysis. The assay can be independent conducted by several individuals to ensure reproducibility.

[0102] As discussed above, end-to-end cyclizing means the joining of the cationic end and the hydrophobic end of the amphipathic polypeptide for a monocyclic AP. End-to- end cyclizing can be achieved by any suitable method including disulfide bonding (cysteine bridge) and end-to-end amidation. Achieving two (2) or more S-S bridges (Cysteine at the ends in inside the sequence of the AP) will result in a bicyclic or multicyclic AP.

[0103] Also included herein are methods of identifying a cationic and / or hydrophobic motif sequence within an amphipathic polypeptide. A “motif sequence” is any sequence that demonstrates a functionality either alone or in conjunction with another motif sequence. The functionality can be, for example, an antimicrobial activity or an anticancer activity. The disclosure also includes creating amphipathic polypeptides comprising one or more cationic and / or hydrophobic motif sequences.

[0104] It should be understood that the foregoing relates to preferred embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention. The invention is further illustrated by the following examples, which are not to be construed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof, which, after reading the description herein, may suggest themselves to those skilled in the art without departing from the spirit of the present invention and / or the scope of the appended claims. All patents, patent applications, and publications referenced herein are incorporated by reference in their entirety for all purposes.

[0105] EXAMPLES

[0106] Example 1. Therapeutic potential of amphipathic peptides

[0107] The emergence of bacterial multidrug resistance (MDR) has threatened to reverse the medical achievements credited to antibiotics. Ribosomally-synthesized (classical) cationic antimicrobial peptides (AMPs) kill bacteria regardless of resistanceDocket No. 10504-103W01 to traditional antibiotics and, thus, could be an effective part of a comprehensive series of countermeasures to combat antibiotic resistance (AR). Despite these promising properties, widespread clinical use of classical AMPs is yet to occur.

[0108] An iterative rational framework, which is informed by the amphipathic properties of classical AMPs, is described herein for engineering amphipathic polypeptides (APs) with enhanced systemic efficacy against MDR bacteria. Helical amphipathicity, the segregation of the cationic (C) and hydrophobic (H) domains only when APs fold into a helical structure, is primarily formed by hydrogen bonds while the two motifs appear scrambled to amphipathicity in the primary sequence (e.g., H- WRV12). Many persons skilled in the art have primarily studied this type of amphipathicity for AP design, and it is the basis of APs such as LL37 (SEQ ID NO: 106) and WLBU2 (SEQ ID NO: 107). By contrast, the term “linear amphipathicity” is defined herein as the segregation of C and H residues into separate motifs directly formed by peptide bonds or covalence when the C or H residues are aligned side by side in the primary sequence (e.g., L-WRV12). Conventionally, the role of linear amphipathicity in AP functions remains unclear and markedly underexplored in AP design.

[0109] Linear amphipathicity can be partially illustrated by amphipathic cell penetrating peptides (CPPs; e.g., Pep-1 KETWWETWWTEWSQPKKKRKV, SEQ ID NO: 101), which represent another structural class of cationic amphipathic APs that can transfer molecular cargoes into host cells. It is known that classical AMPs may fold into any type of secondary structures such as helical (e.g., LL37 (SEQ ID NO: 106), FIG. 2), extended helices (e.g., indolicidin) (SEQ ID NO: 108), [(-sheets (the defensins), or a combination thereof (e.g., al-purothionin). While not wishing to be bound by any one theory, and while helical amphipathicity has been validated and secondary structure is important, to approach AP design exclusively from the standpoint of a particular secondary structure is to miss the significance of the concept of cationic amphipathicity as the essential determinant of activity and to severely limit the therapeutic potential of this diverse class of peptides. Further, because AMPs are proteinaceous, clinical development may require structural modification of lead candidates to enhance pharmacological properties. As helical amphipathicity depends on the secondary structure, it is more susceptible to disruption upon modification than linear amphipathicity, which solely depends on the primary structure. In addition, optimized helical peptides generally carry a higher risk for host toxicity.Docket No. 10504-103W01

[0110] The present disclosure unexpectedly demonstrates that linear amphipathic polypeptides (L-APs) are a diverse class of novel agents with systemic applications for different types of communicable diseases and other disorders alike.

[0111] Example 2. Premise of AP design

[0112] Because both C and H motifs are important for target recognition and killing, functionally, the C and H motifs are different from a classical binding- and-effec tor domains model. However, there is no doubt the C and H motifs do not have equal effects on AP functions. Yet, a previously described framework helical-AP design focused on equal numbers of C and H residues, which masks the functional role of each motif. Thus, AP structural optimization is limited unless the roles of these two motifs are understood or decoupled. As amphipathicity is the key to AP function, to decouple each function of the C or H motif, it is necessary to consider one motif only relative to the other. In this context, AP structural optimization requires elucidation of the key roles of the C and H motifs in AP functions. This can only be accomplished if the 2 motifs are decoupled in relation to bacterial (or other target, e.g., cancer cells) recognition and killing as well as host toxicity, with the inclusion of arginine (Arg, R) and lysine (Lys, K) and the typically hydrophobic amino acids (W, F, L, I, P, and V). Thus, keeping one motif constant while varying the other (FIG. 2) is the most effective strategy to decouple the 2 motifs for functional specificities.

[0113] The other important premise of the innovative framework as described herein is the paradigm shift from the exclusive focus on helical amphipathicity to the recognition of the importance of linear amphipathicity. While not wishing to be bound by any one theory, the examination of linear amphipathicity exponentially unleashes the therapeutic potential of APs as a diverse class of drugs that are safer and more amenable to cyclization from L-AP (linear sequence) to C-AP (cyclic) and other types of modifications than their helical counterparts. These advantages expand AP pharmacological properties and, therefore, the potential of APs as described herein for systemic applications. Thus, the expected outcome of this innovation is not a single best drug but a more effective class of therapeutics associated with multiple targets.

[0114] Different research groups tend to define engineered APs based on their structural preferences, which are largely skewed toward helical amphipathicity of APs. As a result, there have been intensive efforts to develop WLBU2-like peptides as the only typical APs. Helical amphipathicity, however, is formed and stabilized byDocket No. 10504-103WG1 hydrogen bonds in the secondary structure, and linear amphipathicity by covalence in the primary structure. While a-helix is thought to be generally a more stable form of secondary structure, linear amphipathicity does not depend on the secondary structure to exist; instead, it is defined by the primary structure. As a result, linear amphipathicity is much less susceptible to disruption when a lead AP requires modification. For instance, head-to-tail cyclization tends to affect the folding of a peptide and reduce the activity of helical APs, as has been seen with the cyclization of WLBU2 (SEQ ID NO: 107) and E35. By contrast, L-AP cyclization maintains a length-dependent increase in activity until an optimal activity is reached. The most remarkable effect of cyclization can be illustrated by the C-RL series (FIG. 3) and longest L- / C-AP, the L- / C-WRV16, with markedly enhanced activity of the cyclized AP (FIGs. 3 and 6). These L- / C-AMPs display negligible RBC lysis and WBC toxicity at all test concentrations up to 32 pM.

[0115] The L-RL series, which display mean MICs>16pM against ESKAPE isolates, is another remarkable example of the impact of cyclization. This is a partial RL series of varying charge that were deduced from the design framework shown in FIG. 2 by simply replacing H with L (Leu) and C (referring to a Cationic residue, not cysteine) with R (Arg) while the number of L residues (6) remains constant to determine activity as a function of charge. When disulfide bridge-mediated cyclization was introduced (end to end, FIGs. 2-3) of LRL AP, the resulting CRL ASP displayed broad C / H- dependent spectrum of activity against 24 MDR test strains from the CDC, except for P. aeruginosa (FIG. 2B). CRL11 (charge +6) (SEQ ID NO: 3) and CRL12 (+7) (SEQ ID NO: 4) achieved the lowest MICs, although CRL11 has the highest selectivity index (SI20, >32 / mean MIC) using RBC lysis assay (FIG. 5) and is therefore, in some embodiments, worthy of selection over CRL 12.

[0116] This is a typical example of how APs are selected for advancement, although this is a partial series of only 4 AP. The impact of cyclization can be further illustrated by the enhanced activity vs. 30 linezolidresistant MRSA strains (FIG. 6) in test medium consisting of RPMI (70%) and MHB2 (30%). Thus, in addition to stability, cyclization clearly enhances activity. The parallel development of the L- / C-AP will significantly enhance understanding of the advantages and limitations of the H-AP and unleash the full therapeutic potential of AP.Docket No. 10504-103WG1

[0117] Example 3. Large-scale design of L-AP libraries

[0118] The framework for designing APs as described herein consists of using a template of minimal length, charge, and specific amino acids that can segregate into C and H motifs. In one aspect, the rationale for the systematic design is as follows:(i) The C and H motifs are the essential contributors to AP activity;(ii) The two (2) motifs do not necessarily contribute equally to each AP function; therefore, they need to be functionally decoupled;(iii) The types of amino acid composition affect bacterial target specificity and host toxicity;(iv) There is an optimal proportion of each motif relative to the other, designated C / H, which depends on the bacterial target specificity.

[0119] Thus, to be potent and safe, one AP cannot be designed to fight all infections. While not wishing to be bound by any one theory, the dissection of each motif at a time by incremental addition of 1 residue will correlate with each AP function and define a C / H-based minimum optimal length or MOL for antibacterial selectivity and therapeutic efficacy, depending on the bacterial species. This hypothesis is based on the data of the L-WRV and CRL series (FIGs. 2-3, 5-6).

[0120] In addition, end-to-end cyclization of linear amphipathic peptides clearly enhances activity of an L-AP while it would likely inactivate a helical amphipathic AP as previously observed. Thus, the overall objective is not to identify a single best peptide that kills almost all bacterial targets. Rather, it is to elucidate the determinants of AP function to unlock the key to their target- specific optimization in response to anticipated and long-term clinical challenges. The framework as described herein comprises a long-term “mining” of AP as a diverse class of therapeutics as opposed to a quick approach for short-term gain of function with limited impact, which has been largely adopted for 4 decades of AP research.

[0121] Thus, preliminary evidence for activity of L / C-AP against specific bacterial organisms forms the basis for extending comparative analysis of the L- / C-AP to a more systematic combination of cationic (C = R or K) and hydrophobic (H = W, F, L, I, P, or V) amino acids (FIG. 1).

[0122] AP libraries as described herein are designed as binary (2 amino acids, C = R or K and H = W, F, V, P, L, or I) or ternary (3 amino acids: C + aromatic W or F + either V, P, L, or I) just as in the RWV AP using the following steps (FIG. 1):(i) Each library will begin with a template of 8 residues (4C and 4H);Docket No. 10504-103W01(ii) Unlike the original H-AP that are extended by the simultaneous addition of 1C + 1H, only 1 C or 1 H (not both) will be added at a time, which keeps one domain (C or H) constant to functionally “titrate” each motif relative to the other. This approach allows a more effective uncoupling of the effects of H and C domains determined by the ratios of C-to-H residues;(iii) Sequences of L- / C-AP will be compared to corresponding lead H-AP as well;(iv) L-AP can undergo regular end-to-end cyclization (FIGs. 1 -3), which may also be disulfide bridge-mediated;(v) because shorter peptides are sought for heightened potency and reduced host toxicity, length will be limited to 8-18 (at least initially) residues and charge to +4-10; and(vi) AP selected for safety and potency will be compared to their analogs of unconventional peptides / peptoids for stability using strategic modifications outlined in FIG. 1 and illustrated in FIG. 7.|0123 | Design of analogs of lead peptides. In addition to the dissection of the amphipathic structures, further modifications can be performed on lead AP candidates with high SI20 [(maximum test concentration causing <20% toxicity or TC20) / MIC] and therapeutic index or TI (MTD / MED or systemic minimum effective therapeutic dose) using commercially available unatural amino acids (FIG. 7), amino acid mimics [e.g., [3-Trp, P-Phe or peptoid (FIG. 1 C)], or even polymers mimicking the side chains, which may reduce susceptibility to proteases.

[0124] AP libraries are typically commercially produced by Genscript (NJ, USA), and a peptide core facility will be utilized for the more technically challenging synthesis of cyclic peptides and peptoids / peptide mimics. Cyclization is performed under standard conditions with free carboxyl and amino termini under basic environment in dimethylformamide (DMF) overnight. Excess solvents are removed by roto evaporator and extracted by ether precipitation. The crude cyclized peptides are purified by RP- HPLC. Another important strategy is Denantiomerization, which has been validated. Thus, L- / C-AP lead compounds along with their putatively more stable analogs can be compared for selection to advance to pre-clinical and clinical development.

[0125] Potency as a function of C / H can be achieved with shorter peptides (FIGs. 1-2), resulting in reduced optimal charge, production cost, and host toxicity. The CRL series indicate that the novel AP may be selective against several bacterial species whileDocket No. 10504-103W01 activity may not be optimal against others (e.g., P. aeruginosa, FIG. 2). Advanced in vitro activity will demonstrate that the C and H domains can be effectively uncoupled (C / H >1, <1, or = 1). The total number of initial peptides of the libraries described in FIG. 1-2 is estimated to surpass 1000, although it cannot be accurately specified in advance because of the iterative nature of our framework. An important observation is the decreasing trend in RBC lytic effects from the 24-mer SAAP-148 (SEQ ID NO: 112), WLBU2 (SEQ ID NO: 107), and H-RWV16 (E35) to the negligible effects of the L / C-AP (FIG. 8) at their MOL, underscoring the importance of our comparative approach. There are no observed lytic effects of L- AP and C-AP derived by end-to-end cyclization, except for some of the cysteine-mediated cyclic peptides. Both MICs and cytotoxicity are required for peptide selection, as shown in FIGs. 2 and 8. The novel peptides also show no evidence of instability in human plasma. AP were compared for functional stability by examining time-dependent antibacterial activity (post-exposure activity, PEA) after incubating peptides in human plasma (FIG. 9). The peptides retained activity, and in some cases, activity appeared to be enhanced after incubation in plasma.

[0126] Example 4. In vivo efficacyTo determine the target applications for the emerging lead peptides, it is important to validate the selection of lead compounds with in vivo testing. As indicated in FIG. 10, the L-AP polypeptide L-WRV12 and the cysteine-based CRL11 (SEQ ID NO: 3) displayed remarkable therapeutic effects when injected systemically in mice preinfected with linezolid-resistant staphylococcus.

[0127] Example 5. Overall impact

[0128] In one aspect, hospitalized patients having MDR or XDR-related bacterial sepsis or life-threatening pneumonia will be treated with saline formulations of an AP as described herein (or in combination with antibiotics) intravenously.

[0129] In another aspect, the generated libraries as described herein are used to treat types of infections, including but not limited to infections caused by fungi or viruses. In another aspect, the generated libraries as described herein are used to treat cancer.

Claims

1. Docket No. 10504-103W01CLAIMS1. A method of creating an amphipathic polypeptide (AP) sequence comprising: a) obtaining an AP comprising a sequence having the formula of C1H1 wherein C is a cationic amino acid, H is a hydrophobic amino acid, and wherein the polypeptide has a cationic amino acid end and a hydrophobic amino acid end; b) adding one cationic amino acid at the cationic amino acid end of the AP to create a cationic AP extension product, and cyclically adding one same or different cationic amino acid at the cationic amino acid end of each cationic AP extension product to create a group of cationic extension polypeptides having the formula of Ci+xHi, wherein x is equal to or between 1 and 16; and / or c) adding one hydrophobic amino acid at the hydrophobic amino acid end of the AP to create a hydrophobic AP extension product, and cyclically adding one same or different hydrophobic amino acid at the hydrophobic amino acid end of each hydrophobic AP extension product to create a group of hydrophobic extension polypeptides having the formula of CiHi+x, wherein x is equal to or between 1 and 16; and d) adding one cationic amino acid at the cationic amino acid end of one or more of the cationic extension polypeptides; and / or e) adding one hydrophobic amino acid at the hydrophobic amino acid end of one or more of the hydrophobic extension polypeptides; and f) end-to-end cyclizing each of the group of polypeptides of step b), step c), step d) and / or step e) to create a group of cyclized polypeptides.

2. The method of claim 1, further comprising: g) determining antimicrobial activity, anticancer activity and / or toxicity of each of the group of polypeptides of step b), step c), step d) and / or step e) and of each of the group of cyclized polypeptides of step f); and h) creating an amphipathic polypeptide comprising the one or more polypeptide sequences associated with an expected antimicrobial activity and / or expected anticancer activity.

3. A method of selecting for an AP sequence comprising:Docket No. 10504-103W01 a) obtaining an AP comprising a sequence having the formula of C1H1 wherein C is a cationic amino acid, H is a hydrophobic amino acid, and wherein the polypeptide has a cationic amino acid end and a hydrophobic amino acid end; b) adding one cationic amino acid at the cationic amino acid end of the AP to create a cationic AP extension product, and cyclically adding one same or different cationic amino acid at the cationic amino acid end of each cationic AP extension product to create a group of cationic extension polypeptides having the formula of Ci+xHi, wherein x is equal to or between 1 and 16; and / or c) adding one hydrophobic amino acid at the hydrophobic amino acid end of the AP to create a hydrophobic AP extension product, and cyclically adding one same or different hydrophobic amino acid at the hydrophobic amino acid end of each hydrophobic AP extension product to create a group of hydrophobic extension polypeptides having the formula of CiHi+x, wherein x is equal to or between 1 and 16; and d) adding one hydrophobic amino acid at the hydrophobic amino acid end of one or more of the cationic AP extension polypeptides to create a second group of cationic extension polypeptides having the formula of Ci+xHi+ywherein x and y are the same or different and equal to or between 1 and 16; and / or e) adding one cationic amino acid at the cationic amino acid end of one or more of the hydrophobic extension polypeptides to create a second group of hydrophobic extension polypeptides having the formula of Ci+yHi+xwherein x and y are the same or different and equal to or between 1 and 16; and f) end-to-end cyclizing each of the group of polypeptides of step b), step c), step d) and / or step e) to create a group of cyclized polypeptides; and g) determining antimicrobial activity, anticancer activity and / or toxicity of each of the group of polypeptides of step b), step c), step d) and / or step e) and of each of the group of cyclized polypeptides of step f).

4. The method of claim 3, further comprising: h) selecting one or more of the group of cyclized polypeptides of step g) having a higher antimicrobial activity, a higher anticancer activity and / or a lower toxicity as compared to other cyclized polypeptides in the group and thereby selecting for an amphipathic polypeptide sequence.Docket No. 10504-103W015. A method of creating an AP sequence comprising: a) obtaining a sequence having the formula of Ci+yHi+Xwherein Ci+yrepresents the number of cationic amino acids forming a cationic motif on one end of the AP, wherein Hi+Xrepresents the number of hydrophobic amino acids forming a hydrophobic motif on the other end of the AP; and wherein x and y are the same or different and equal to any number between 0 and 16; b) varying y or adding one cationic amino acid at either end of the cationic motif of the AP to create a cationic AP extension product while x remains unchanged results in linear APs, which can become cyclic by joining the cationic end terminus and the hydrophobic end terminus or by adding a cysteine residue at each end terminus to join the 2 end termini via a disulfide bridge; and / or c) varying x or adding one hydrophobic amino acid at either end of the hydrophobic motif of the AP to create a hydrophobic AP extension product while y remains unchanged results in linear APs, which can become cyclic by joining the cationic end terminus and the hydrophobic end terminus or by adding a cysteine residue at each end terminus to join the 2 end termini via a disulfide bridge.

6. The method of any one of claims 1-5, wherein x is equal to or between 2 and 14.

7. The method of any one of claims 1-5, wherein x is equal to or between 4 and 12.

8. The method of any one of claims 1-5, wherein x is equal to or between 6 and 10.

9. The method of any one of claims 1-8, wherein each C amino acid is selected from the group consisting of arginine, lysine, and histidine.

10. The method of any one of claims 1-9, wherein each H amino acid is selected from the group consisting of alanine, isoleucine, leucine, tryptophan, phenylalanine, valine, proline, and glycine.

11. The method of any one of claims 1-9, wherein each H amino acid is selected from the group consisting of isoleucine, leucine, phenylalanine, and valine.Docket No. 10504-103W0112. The method of any one of claims 1-9, wherein each H amino acid is selected from the group consisting of tryptophan and phenylalanine.

13. The method of any one of claims 1-12 wherein one or more of the C and / or H amino acids is a D amino acid.

14. The method of any one of claims 1-9, wherein one or more of the C and / or H amino acids is an a-peptide, an a-peptoid, or a peptoid.

15. The method of any one of claims 1-14, wherein the end-to-end cyclizing is through cysteine bridging.

16. The method of any one of claims 1-14, wherein the end-to-end cyclizing is through end- to-end amidation.

17. The method of any one of claims 1-16, wherein the AP sequence comprises a bi-cyclic or a multi-cyclic sequence.

18. The method of any one of claims 1-17, wherein the antimicrobial activity is against S. aureus.

19. The method of claim 18, wherein the S. aureus is methicillin resistant.

20. The method of any one of claims 1-17, wherein the antimicrobial activity is against E. faecalis.

21. The method of any one of claims 1-17, wherein the antimicrobial activity is against E. coli.

22. The method of any one of claims 1-17, wherein the antimicrobial activity is against A. baumannii.

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