Stapled antimicrobial peptides and uses thereof

Abstract

This disclosure relates to buforin II peptides and stapled buforin II peptides, compositions comprising the same, and methods for using such buforin II peptides or stapled peptides in the treatment of a bacterial infection, a fungal infection, or cancer in a subject (e.g., plant, animal, e.g., human) in need thereof.). The disclosure also relates to methods of making such stapled peptides.

Description

Attorney Docket No. 00530-0422W01 / DFCI 3328.W01 WO STAPLED ANTIMICROBIAL PEPTIDES AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of U.S. Provisional Appl. No.63 / 749,325, filed January 24, 2025, the contents of which are incorporated by reference herein in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 21, 2026, is named 00530-0422WOl_SL.xml and is 168,510 bytes in size.TECHNICAL FIELD

[0003] This disclosure relates to stapled peptides based on the antimicrobial peptide buforin II and methods for using such stapled peptides to treat bacterial infections, fungal infections, and cancer.SUMMARY

[0004] The present disclosure is based, at least in part, on the identification of stapled peptides based on buforin 11 having anti-fungal activity, anti-bacterial activity, anti-cancer activity, or a combination thereof. These stapled peptides have clinical applications in treating bacterial infections, fungal infections, and cancer.

[0005] Provided herein is a stapled peptide comprising the structure of formula (I):Formula (I)or a pharmaceutically acceptable salt thereof, wherein:each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, aiylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted, optionally wherein each Ri and R2 is a methyl;each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted, optionally wherein R3 is Cx alkylene, Cx alkenylene, or Cx alkynylene, optionally wherein R3 is (CH2)3-CH=CH-(CH2)3; andwherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 3 to 6 substitutions relative to the sequence TRSSRAGLQWPVGRVHRLLRK (SEQ ID NO: 1),wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 is with a lysine at position 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, or 20 of the sequence of SEQ ID NO: 1, andwherein two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at (i) positions 12 and 16 of the sequence of SEQ ID NO: 1, or (ii) positions 13 and 17 of the sequence of SEQ ID NO: 1;wherein [Xaa]xis (i) positions 13-15 of the sequence of SEQ ID NO: 1, optionally comprising one or two amino acid substitutions, or (ii) positions 14-16 of the sequence of SEQ ID NO:1, optionally comprising one or two amino acid substitutions; and wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, exhibits an antimicrobial effect against at least one microbe, optionally wherein the at least one microbe is E. coli, or wherein the stapled peptide, or the pharmaceutically acceptable salt thereof exhibits an anticancer effect. In some instances, Ri is methyl, R3 is (CH2)3-CH=CH-(CH2)3, and R2is methyl.

[0006] In some instances, two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 are at positions 12 and 16 of the sequence of SEQ ID NO:1, wherein [Xaa]xcorresponds to positions 13-15 of the sequence of SEQ ID NO: 1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 1, 15, or 19 of the sequence of SEQ ID NO:1.

[0007] In some instances, two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 13 and 17 of the sequence of SEQ ID NO: 1, wherein[Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO:1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 is with a lysine at position 3, 5, 6, 7, 8, 11, 12, 18, 19, or 20 of the sequence of SEQ ID NO:1.

[0008] In some instances, two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 are at positions 13 and 17 of the sequence of SEQ ID NO:1, wherein [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 1, 3, 6, or 9 of the sequence of SEQ ID NO:1.

[0009] In some instances, two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 13 and 17 of the sequence of SEQ ID NO: 1, wherein [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1, and wherein one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 9 of the sequence of SEQ ID NO: 1.

[0010] In some instances, (a) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:63), [Xaa]xconsists of the sequence GRV, and [Xaa]yconsists of the sequence RLLRK (SEQ ID NO:85); (b) [Xaa]wconsists of the sequence TRSSRAGLQWP (SEQ ID NO:64), [Xaa]xconsists of the sequence GRK, and [Xaa]yconsists of the sequence RLLRK (SEQ ID NO:86); (c) [Xaa]wconsists of the sequence TRSSRAGLQWP (SEQ ID NO:65), [Xaa]xconsists of the sequence GRV, and [Xaa]yconsists of the sequence RLKRK (SEQ ID NO:87); (d) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:66), [Xaa]xconsists of the sequence GRK, and [Xaa]yconsists of the sequence RLLRK (SEQ ID NO:88); (e) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:67), [Xaa]xconsists of the sequence GRV, and [Xaa]yconsists of the sequence RLKRK (SEQ ID NO:89); (f) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:68), [Xaa]xconsists of the sequence GRK, and [Xaa]yconsists of the sequence RLKRK (SEQ ID NOVO); (g) [Xaa]wconsists of the sequence TRSSRAGLQWP (SEQ ID NO:69), [Xaa]xconsists of the sequence GRK, and [Xaa]yconsists of the sequence RLKRK (SEQ ID NO:91); (h) [Xaa]wconsists of the sequence KRSSRAGLQWPV (SEQ ID NO:70), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:92); (i) [Xaa]wconsists of the sequenceTRKSRAGLQWPV (SEQ ID N0:71), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:93); (j) [Xaa]wconsists of the sequence TRSSKAGLQWPV (SEQ ID NO:72), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:94); (k) [Xaa]wconsists of the sequence TRSSRKGLQWPV (SEQ ID NO:73), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:95); (1) [Xaa]wconsists of the sequence TRSSRAKLQWPV (SEQ ID NO:74), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:96); (m) [Xaa]wconsists of the sequence TRSSRAGKQWPV (SEQ ID NO:75), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:97); (n) [Xaa]wconsists of the sequence TRSSRAGLKWPV (SEQ ID NO:76), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:98); (o) [Xaa]wconsists of the sequence TRSSRAGLQWKV (SEQ ID NO:77), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:99); (p) [Xaa]wconsists of the sequence TRSSRAGLQWPK (SEQ ID NO:78), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO: 100); (q) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:79), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence KLRK (SEQ ID NO: 101); (r) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:80), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LKRK (SEQ ID NO: 102); (s) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:81), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLKK (SEQ ID NO: 103); (t) [Xaa]wconsists of the sequence KRSSRAGLKWPV (SEQ ID NO:82), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO: 104); (u) [Xaa]wconsists of the sequence TRKSRAGLKWPV (SEQ ID NO:83), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO: 105); or (v) [Xaa]wconsists of the sequence TRSSRKGLKWPV (SEQ ID NO:84), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO: 106).

[0011] In some instances, (a) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:66), [Xaa]xconsists of the sequence GRK, and [Xaa]yconsists of thesequence RLLRK (SEQ ID NO:88); (b) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:67), [Xaa]xconsists of the sequence GRV, and [Xaa]yconsists of the sequence RLKRK (SEQ ID NO:89); (c) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO: 68), [Xaa]xconsists of the sequence GRK, and[Xaa]yconsists of the sequence RLKRK (SEQ ID NO: 90); (d) [Xaa]wconsists of the sequence ERSSRAGLQWP (SEQ ID NO:69),[Xaa]xconsists of the sequence GRK, and [Xaa]yconsists of the sequence RLKRK (SEQ ID NO:91); (e) [Xaa]wconsists of the sequence TRKSRAGLQWPV (SEQ ID NO:71), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:93); (f) [Xaa]wconsists of the sequence TRSSKAGLQWPV (SEQ ID NO:72), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:94); (g) [Xaa]wconsists of the sequence TRSSRKGLQWPV (SEQ ID NO:73), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:95); (h) [Xaa]wconsists of the sequence TRSSRAKLQWPV (SEQ ID NO:74), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:96); (i) [Xaa]wconsists of the sequence TRSSRAGKQWPV (SEQ ID NO:75), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:97); (j) [Xaa]wconsists of the sequence TRSSRAGLQWKV (SEQ ID NO:77), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:99); (k) [Xaa]wconsists of the sequence TRSSRAGLQWPK (SEQ ID NO:78), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO: 100); (1) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:79), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence KLRK (SEQ ID NO: 101); (m) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:80), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LKRK (SEQ ID NO: 102); or (n) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:81), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLKK (SEQ ID NO: 103). In some instances, (a) [Xaa]wconsists of the sequence KRSSRAGLQWPV (SEQ ID NO:70), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:92); (b) [Xaa]wconsists of the sequence TRKSRAGLQWPV (SEQ ID NO:71), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:93); (c) [Xaa]wconsists of the sequence TRSSRKGLQWPV (SEQ ID NO:73), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:95); (d) [Xaa]wconsists of the sequence TRSSRAGLKWPV (SEQ ID NO:76), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:98); (e) [Xaa]wconsists of the sequence KRSSRAGLKWPV (SEQ ID NO:82), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO: 104); (f) [Xaa]wconsists of the sequence TRKSRAGLKWPV (SEQ ID NO:83), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO: 105); or (g) [Xaa]wconsists of the sequence TRSSRKGLKWPV (SEQ ID NO:84), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO: 106).

[0012] In some instances, [Xaa]wconsists of the sequence TRSSRAGLKWPV (SEQ ID NO: 76), [Xaa]xconsists of the sequence RVH, and [Xaa]yconsists of the sequence LLRK (SEQ ID NO:98).

[0013] Also provided herein is a stapled peptide comprising the sequence of any one of SEQ ID NOs:20, 33, 36, 38-42, 44, 46-50, 52, 53, and 57-62, except for 0, 1, 2 or 3 amino acid substitutions relative to the sequence of any one of SEQ ID NOs:20, 33, 36, 38-42, 44, 46-50, 52, 53, and 57-62, respectively, or a pharmaceutically acceptable salt thereof, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:20, the amino acid substitutions are not at any of positions 1, 12, and 16 of SEQ ID NO:20, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:33, the amino acid substitutions are not at any of positions 12, 15, and 16 of SEQ ID NO:33, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO: 36, the amino acid substitutions are not at any of positions 12, 16, and 19 of SEQ ID NO:36, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:38, the amino acid substitutions are not at any of positions 1, 12, 15, and 16 of SEQ ID NO:38, wherein if stapled peptide or the pharmaceutically acceptable salt thereofcomprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:39, the amino acid substitutions are not at any of positions 1, 12, 16, and 19 of SEQ ID NO:39, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:40, the amino acid substitutions are not at any of positions 1, 12, 15, 16, and 19 of SEQ ID NO:40, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:41, the amino acid substitutions are not at any of positions 12, 15, 16, and 19 of SEQ ID NO:41, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:42, the amino acid substitutions are not at any of positions 1, 13, and 17 of SEQ ID NO:42, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:44, the amino acid substitutions are not at any of positions 3, 13, and 17 of SEQ ID NO:44, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:46, the amino acid substitutions are not at any of positions 5, 13, and 17 of SEQ ID NO:46, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:47, the amino acid substitutions are not at any of positions 6, 13, and 17 of SEQ ID NO:47, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:48, the amino acid substitutions are not at any of positions 7, 13, and 17 of SEQ ID NO:48, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:49, the amino acid substitutions are not at any of positions 8, 13, and 17 of SEQ ID NO:49, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:50, the amino acid substitutions are not at any of positions 9, 13, and 17 of SEQ ID NO:50, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ IDNO:52, the amino acid substitutions are not at any of positions 11, 13, and 17 of SEQ ID NO:52, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:53, the amino acid substitutions are not at any of positions 12, 13, and 17 of SEQ ID NO:53, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:57, the amino acid substitutions are not at any of positions 13, 17, and 18 of SEQ ID NO: 57, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:58, the amino acid substitutions are not at any of positions 13, 17, and 19 of SEQ ID NO: 58, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:59, the amino acid substitutions are not at any of positions 13, 17, and 20 of SEQ ID NO:59, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:60, the amino acid substitutions are not at any of positions 1, 9, 13, and 17 of SEQ ID NO:60, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:61, the amino acid substitutions are not at any of positions 3, 9, 13, and 17 of SEQ ID NO:61, wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:62, the amino acid substitutions are not at any of positions 6, 9, 13, and 17 of SEQ ID NO:62, wherein positions 12 and 16 of any one of SEQ ID NOs: 20, 33, 36, 38-41 are cross-linked to each other, wherein positions 13 and 17 of any one of SEQ ID NOs: 42, 44, 46-50, 52, 53, and 57-62 are cross-linked to each other, and wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, exhibits an antimicrobial effect against at least one microbe, optionally wherein the at least one microbe is E. coli, or wherein the stapled peptide, or the pharmaceutically acceptable salt thereof exhibits an anticancer effect.

[0014] In some instances, stapled peptide, or the pharmaceutically acceptable salt thereof, comprises the sequence of any one of SEQ ID NOs:38-41, 44, 46-49, 52, 53, and 57-59, except for the 0, 1, 2 or 3 amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 38-41, 44, 46-49, 52, 53, and 57-59, respectively.

[0015] In some instances, stapled peptide, or the pharmaceutically acceptable salt thereof, comprises the sequence of any one of SEQ ID NOs:42, 44, 47, 50, and 60-62, except for the 0, 1, 2 or 3 amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 42, 44, 47, 50, and 60-62, respectively.

[0016] In some instances, stapled peptide, or the pharmaceutically acceptable salt thereof, comprises the sequence of SEQ ID NO: 50, except for the 0, 1, 2 or 3 amino acid substitutions relative to the sequence of SEQ ID NO:50.

[0017] Also provided herein is a pharmaceutical composition comprising any one of the foregoing stapled peptides, or the pharmaceutically acceptable salt thereof; and a pharmaceutically acceptable carrier.

[0018] Also provided herein is a method of treating a fungal infection in a subject in need thereof, the method comprising administering to the subject any one of the foregoing stapled peptides, or the pharmaceutically acceptable salt thereof. In some instances, the subject is a human. In some instances, the fungal infection is a Chytridiomycota fungal infection. In some instances, the fungal infection is a Zygomycota fungal infection. In some instances, the fungal infection is an Ascomycota fungal infection. In some instances, the fungal infection is a Basidiomycota fungal infection. In some instances, the fungal infection is a Glomeromycota fungal infection. In some instances, the fungal infection is a C. albicans, a C. auris, or a C. neoformans fungal infection.

[0019] Also provided herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising administering to the subject any one of the foregoing stapled peptides, or the pharmaceutically acceptable salt thereof, optionally wherein the subject is a human. In some instances, the bacterial infection is a gramnegative bacterial infection. In some instances, the bacterial infection is an A. baumannii,E. coli, P. aeruginosa, S. aureus, or B. cereus bacterial infection. Tn some instances, the method further comprises administering to the subject an antibiotic, optionally wherein the antibiotic is selected from the group consisting of a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof, optionally wherein the P-lactam is selected from ampicillin, ceftazidime, cefotaxime, or a combination thereof, the fluoroquinolone is ciprofloxacin, the aminoglycoside is selected from gentamicin, tobramycin, or a combination thereof, the carbapenem is meropenem, or a combination thereof.

[0020] Also provided herein is a method of treating a multi-drug resistant bacterial infection in a subject in need thereof, the method comprising administering to the subject any one of the foregoing stapled peptides, or the pharmaceutically acceptable salt thereof, optionally wherein the subject is a human. In some instances, the bacterial infection is a gram-negative bacterial infection. In some instances, wherein the multi-drug resistant bacterial infection is an A. baumannii, E. coli, or K. pneumonia bacterial infection. In some instances, the multi-drug resistant bacterial infection is a bacterial infection resistant to treatment with a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof. In some instances, the P-lactam is selected from ampicillin, ceftazidime, cefotaxime, or a combination thereof, the fluoroquinolone is ciprofloxacin, the aminoglycoside is selected from gentamicin, tobramycin, or a combination thereof, the carbapenem is meropenem, or a combination thereof.

[0021] Also provided herein is a method of synergistically killing bacteria in a subject in need thereof, the comprising administering to the subject any one of the foregoing stapled peptides, or the pharmaceutically acceptable salt thereof, optionally wherein the subject is a human. In some instances, the bacteria are gram-negative bacteria. In some instances, the bacteria are multi-drug resistant. In some instances, the bacteria are A. baumannii, E. coli, or K. pneumonia. In some instances, the bacteria are resistant to treatment with a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof. In some instances, the P-lactam is selected from ampicillin, ceftazidime, cefotaxime, or a combination thereof, the fluoroquinolone isciprofloxacin, the aminoglycoside is selected from gentamicin, tobramycin, or a combination thereof, the carbapenem is meropenem, or a combination thereof.

[0022] Also provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject administering to the subject any one of the foregoing stapled peptides, or the pharmaceutically acceptable salt thereof, optionally wherein the subject is a human, and optionally wherein the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0023] Also provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a stapled peptide, or a pharmaceutically acceptable salt thereof, wherein the stapled peptide comprises:(a) the structure of formula (I):Formula (I)wherein: each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted, optionally wherein each Ri and R2 is a methyl; each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted, optionally wherein R3 is Cs alkylene, Cs alkenylene, or Cs alkynylene, optionally wherein R3 is (CH2)3-CH=CH-(CH2)3; each Xaa is independently an amino acid; each w and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and each x is 3 or 6; and wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 2 to 6 substitutions relative to the sequence of TRSSRAGLQWPVGRVHRLLRK (SEQ ID NO: 1), wherein (a) 2 of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are substitutions with non-natural amino acids, the side chains of which are cross-linked to form R3, optionally wherein the2 of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 with non-natural amino acids, the side chains of which are cross-linked to form R3 are at positions 13 and 17 of the sequence of SEQ ID NO: 1 and [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1; or (b) 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1, wherein two of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are with stapling amino acids that are cross-linked to each other; and optionally wherein the subject is a human, and optionally wherein the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0024] Also provided herein is a method of making a stapled peptide, the method comprising: (a) providing a peptide comprising the sequence of SEQ ID NO: 1 with 3 to 6 substitutions, wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 is with a lysine at position 1, 3, 5, 6, 7, 8, 9, 11, 12, 18, 19, or 20 of the sequence of SEQ ID NO: 1, and wherein two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are with stapling amino acids at (i) positions 12 and 16 of the sequence of SEQ ID NO: 1, or (ii) positions 13 and 17 of the sequence of SEQ ID NO: 1; and (b) cross-linking the peptide thereby making the stapled peptide, optionally wherein the stapling amino acids are (S)-2-(4’-pentenyl)alanine, optionally wherein the crosslinking is by a ruthenium catalyzed metathesis reaction, and optionally wherein the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition.

[0025] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating a fungal infection in a subject (e.g., human, plant, animal, insect (e.g., bee, silkworm), aquatic species (e.g., fish, mollusk), bird, amphibian, reptile), and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human. In some instances, the fungal infection is a Chytridiomycota fungal infection. In some instances, the fungal infection is a Zygomycota fungal infection. In some instances, the fungal infection is an Ascomycota fungal infection. In some instances, the fungal infection is a Basidiomycota fungal infection. In some instances, the fungal infection is a Glomeromycota fungal infection. In some instances, the fungal infection is a C. albicans, a C. auris, or a C. neoformans fungal infection.

[0026] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating a bacterial infection in a subject (e.g., human, plant, animal), and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human. In some instances, the bacterial infection is a gram-negative bacterial infection. In some instances, the bacterial infection is an A. baumannii, E. coli, P. aeruginosa, S. aureus, or B. cereus ctexi X infection.

[0027] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating cancer in a subject (e.g., an animal, e.g., a human), and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0028] Also provided herein is a pharmaceutical composition comprising: (a) a means for selectively killing gram-negative bacteria in a subject (e.g., human, plant, animal), and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human. In some instances, the bacterial infection is a gram-negative bacterial infection. In some instances, the bacterial infection is an A. baumannii, E. coli, P. aeruginosa, S. aureus, or B. cereus bacterial infection.

[0029] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, an osteosarcoma, or other liquid or solid tumor cells in a subject (e.g., human, animal), including for example by a non-membrane-lytic mechanism of action, and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human.BRIEF DESCRIPTION OF DRAWINGS

[0030] FIG. 1 includes a cartoon showing the production of Buforin I in the gastric lumen of an Asian toad Bufo Gargarizans (top), the sequence of Buforin I and Buforin II (which is derived from Buforin I) (middle), and a cartoon of the unique structure of Buforin II represented by an N-terminal unstructured domain, a proline hinge, and a helical C-terminus (bottom). The sequence of Buforin I is SEQ ID NO: 107. Thesequence of Buforin II is SEQ ID NO: 108. Figure discloses SEQ ID NOS 107-108 and 1, respectively, in order of appearance.

[0031] FIG. 2A shows the sequences of the Buforin II F10W mutant and i, i+4 stapled Buforin II (F10W) peptides. The figure also shows the lysine scans of Buf(i+4)12 and Buf(i+4)13. The lysine and stapling position are in bold. SEQ ID NOs:l-19 (top left, from top to bottom; wherein the Xs are cross-linked to each other), SEQ ID NOs: 14 and 20-37 (bottom left, from top to bottom; wherein the Xs are cross-linked to each other), and SEQ ID NOs: 15 and 42-59 (bottom right, from top to bottom; wherein the Xs are cross-linked to each other). For SEQ ID NOs: 2-59, Xi is (S)-2-(4’-pentenyl)alanine cross-linked to X2 is (S)-2-(4’-pentenyl)alanine (see, FIG. 2B).

[0032] FIG. 2B is a cartoon depicting a helical peptide comprising a (S)-2-(4’-pentenyl)alanine at position i and a (S)-2-(4’-pentenyl)alanine at position i+4 (left side) and the resulting stapled peptide in which the (S)-2-(4’-pentenyl)alanines at positions i, i+4 are cross-linked.

[0033] FIG. 3 lists the minimum inhibitory concentration (MIC) (in pM) and % toxicity (red blood cells (RBC), human umbilical vein endothelial cells (HUVEC), and renal proximal tubule epithelial cells (RPTEC) at 10 pM for the indicated peptides (SEQ ID NOs: 1-19, from top to bottom).

[0034] FIG. 4 lists the MIC (in pM) and % toxicity (RBC, HUVEC and RPTEC) at 10 pM for the indicated peptides (SEQ ID NOs: 14 and 20-37, from top to bottom).

[0035] FIG. 5A-5B lists the MIC (in pM) and % toxicity (RBC, HUVEC and RPTEC) at 10 pM for the indicated single lysine scanning peptides (SEQ ID NOs: 15 and 42-59, from top to bottom, FIG. 5A) and select double lysine mutants that demonstrate further improved potencies (SEQ ID NOs:60 and 61, from top to bottom, FIG. 5B).

[0036] FIG. 6 shows the scanning electron microscopy images of E. coli and A. baumcmnii after exposure to MIC concentrations of Buf WT (SEQ ID NO: 1), Buf(i+4)12 (SEQ ID NO: 14), and Buf(i+4)13 (SEQ ID NO: 15) for 2 hours (scale bar: 3 pm).

[0037] FIGs. 7A-7B are graphs showing the inner membrane permeabilization of A. baumannii treated with the indicated peptides or polymyxin (Poly) at the indicatedconcentrations for 30 minute (FIG. 7A) or 180 minute (FIG. 7B) exposure times, as measured by fluorescence intensity of propidium iodide in baumannii at the 535 / 615 excitation / emission wavelength. For each condition, the bars from left to right represent: 2x MIC, lx MIC, 0.5x MIC, 0.25x MIC, 0.125x MIC, 0.06x MIC, 0.03x MIC, 0.02x MIC, O.Olx MIC, and Ox MIC. Buf WT (SEQ ID NO:1), Buf(i+4)12 (SEQ ID NO: 14), Buf(i+4)13 (SEQ ID NO: 15).

[0038] FIGs. 7C-7D are graphs showing the inner membrane permeabilization of A. baumannii treated with Buf(i+4)13 (SEQ ID NO: 15) or polymyxin (Poly) at the indicated concentrations for 30 minute (FIG. 7C) or 180 minute (FIG. 7D) exposure times, as measured by fluorescence intensity of propidium iodide in A. baumannii at the 535 / 615 excitation / emission wavelength. For each condition, the bars from left to right represent: 8x MIC, 4x MIC, 2x MIC, lx MIC, 0.5x MIC, 0.25x MIC, 0.125x MIC, 0.06x MIC, and 0.03xMIC, and Ox MIC.

[0039] FIGs. 8A-8C is a series of graphs showing the kill curves of Buf WT (SEQ ID NO:1) (FIG. 8A), Buf(i+4)12 (SEQ ID NO: 14) (FIG. 8B), or Buf(i+4)13 (SEQ ID NO: 15) (FIG. 8C) upon treating A. baumannii at MIC concentration for up to 6 hours with samples taken at the specified time points on the x axis.

[0040] FIG. 9 is a graph showing the comparative proteolytic inactivation of unstapled (Buf WT (SEQ ID NO: 1) vs. stapled Buforin II (Buf(i+4)12 (SEQ ID NO: 14) or Buf(i+4)13 (SEQ ID NO: 15) after exposure to the indicated proteinase K, with stapling conferring resistance to proteinase K-based inactivation. Bars for each condition are: 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, 0.0063 mg / mL, 0.0031 mg / mL, 0.0016 mg / mL, 0.0008 mg / mL, 0.0004 mg / mL, 0.0002 mg / mL, and 0 mg / mL, from left to right.

[0041] FIGs. 10A-10B are graphs showing the inner membrane permeabilization of A. baumannii treated with the indicated lysine mutant peptides or Magainin II (Mag2) at the indicated concentrations for 30 minute (FIG. 10A) or 180 minute (FIG. 10B) exposure times, as measured by fluorescence intensity of propidium iodide in A. baumannii at the 535 / 615 excitation / emission wavelength. For each condition, the barsfrom left to right represent: 4x MIC, 2x MIC, lx MIC, 0.5x MIC, 0.25x MIC, Ox MIC. Buf(i+4)13 T1K (SEQ ID NO:42), Buf(i+4)13 S3K (SEQ ID NO:44), Buf(i+4)13 S4K (SEQ ID NO:45), Buf(i+4)13 A6K (SEQ ID NO:47), Buf(i+4)13 Q9K (SEQ ID NO:50), Buf(i+4)13 P11K (SEQ ID NO:52), and Mag2 (SEQ ID NO:109).

[0042] FIG. 11 shows the scanning electron microscopy images of A. baumannii after exposure to PBS or MIC concentrations of Buf WT (SEQ ID NO: 1), Buf(i+4)12 (SEQ ID NO: 14), and Buf(i+4)13 Q9K (SEQ ID NO:50) for 2 hours (scale bar: 3 pm for SEM and 500 nm for TEM).

[0043] FIG. 12 is a graph showing the comparative proteolytic inactivation of unstapled (Buf WT (SEQ ID NO: 1) vs. stapled Buforin II (Buf(i+4)13 Q9K (SEQ ID NO:50) after exposure to the proteinase K, with stapling conferring marked resistance to proteinase K-based inactivation. Bars for each condition are: 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, 0.006 mg / mL, 0.003 mg / mL, 0.0015 mg / mL, and 0.0008 mg / mL, from left to right.

[0044] FIG. 13 shows the cytotoxicity dose response curve for human RBC, HUVEC, and RPTEC cells upon treatment with Buf(i+4)13 Q9K (SEQ ID NO:50), with little to no adverse effect at doses that are between 1-3 orders of magnitude higher than antibacterial MICs. Peptide starting concentration was 200 pM with a 2-fold serial dilution.

[0045] FIG. 14 shows time-course confocal microscopy of A. baumannii treated with FITC -labelled Buf(i+4)13 (Q9K) (SEQ ID NO:50) at 2* MIC for 0, 60, and 180 minutes. Bacterial membranes were stained with FM4-64 (red). FITC fluorescence indicates peptide translocation across the membrane.

[0046] FIGS. 15A-C show that exogenous bacterial DNA reduces antibacterial activity (FIG. 15A) and that A. baumannii DNA (FIG. 15B) and E. coli DNA (FIG. 15C) cause dose-dependent quenching of DNA-binding activity of the indicated peptides, as assessed by ethidium bromide displacement assays, supporting a DNA-binding mechanism for Buf(i+4)13 Q9K (SEQ ID NO:50).

[0047] FIGS. 16A-B show that Buf(i+4)13 (Q9K) (SEQ ID NO:50), but not Buf WT, eradicates matured, baumannii biofilms at 8 / MIC, (FIG. 16A). This activity is further enhanced upon co-treatment with MIC levels of polymyxin B or ciprofloxacin, with complete biofilm clearance observed at 2* MIC (FIG. 16B).

[0048] FIGS. 17A-B show checkerboard assays performed against baumannii upon treatment with varying doses of Buf(i+4)13 Q9K (SEQ ID NO:50) combined with polymyxin B (FIG. 17A) or ciprofloxacin (FIG. 17B), revealing synergistic activity.

[0049] FIGS. 18A-B show checkerboard assays performed against A. baumannii upon treatment with varying doses of Buf(i+4)13 Q9K (SEQ ID NO:50) combined with indolicidin (FIG. 18A) showing synergistic activity or gentamicin (FIG. 18B) showing potential antagonism.

[0050] FIG. 19 shows transcriptomic changes in A. baumannii upon treatment with Buf(i+4)13 (SEQ ID NO: 15) for 30 minutes versus vehicle control.

[0051] FIG. 20 shows transcriptomic changes in A. baumannii upon treatment with Buf(i+4)13 Q9K (SEQ ID NO:50) for 30 minutes versus vehicle control.

[0052] FIG. 21 shows transcriptomic changes in A. baumannii upon treatment with Buf(i+4)13 Q9K (SEQ ID NO:50) for 90 minutes versus vehicle control.

[0053] FIG. 22 shows transcriptomic changes in A. baumannii upon treatment with Buf(i+4)13 Q9K (SEQ ID NO:50) for 120 minutes versus vehicle control.

[0054] FIG. 23 shows a 30-day resistance acquisition study upon treatment of A. baumannii with Buf WT, the indicated stapled Buforin peptides, and polymyxin B.

[0055] FIG. 24 shows pathway-level enrichment analysis of they!, baumannii proteome in response to Buf(i+4)13 (Q9K) (SEQ ID NO: 50) exposure. Gene Ontology (GO) terms for Biological Process (top) and Molecular Function (middle), along with KEGG pathway gene set enrichment analysis (GSEA, bottom) were computed at day 4 (left) and day 30 (right) of treatment.

[0056] FIGS. 25A-B show MIC values of Buf WT and Buf(i+4)13 (Q9K) (SEQ ID NO:50) against drug-resistant Gram-negative clinical isolates from the U.S. Centers forDisease Control and Prevention (CDC) (FIG. 25A), including colistin-resistant . baumannii and MCR-1 plasmid-positive E. coli and MDR Gram-negative clinical isolates from the Massachusetts General Hospital (FIG.25B). Antibiotic susceptibility designations are as follows: S, susceptible; I, intermediate; R, resistant. Antibiotic abbreviations: Amp, ampicillin; Ceftaz, ceftazidime; CTX, ceftriaxone; Cipro, ciprofloxacin; Cst, colistin; Gent, gentamicin; Mero, meropenem; Tob, tobramycin, Doxy, doxycycline and TMP / SMX, trimethoprim-sulfamethoxazole. ^Denotes MCR-1 plasmid carrier.

[0057] FIGS. 26A-C demonstrate in vivo proof-of-concept for the efficacy of Buf(i+4)13 Q9K (SEQ ID NO:50) in a peritonitis-sepsis model, in which C57BL / 6J mice, rendered neutropenic with two doses of cyclophosphamide, were then challenged with a 108CFU dose of baumannii (ATCC 19606) via intraperitoneal (IP) injection, followed by treatment with vehicle or Buf(i+4)13 Q9K (5 mg / kg dose; SEQ ID NO:50) at 2, 24, and 48 hours post-infection (FIG. 26A). Kaplan Meier analysis shows that all vehicle treated animals died by day 2 after infection, whereas all Buf(i+4)13 Q9K were cured (FIG. 26B). Assessment of peritoneal fluid at time of death (vehicle treated mice) or termination of the experiment (day 10 post-infection), showed that Buf(i+4)13 Q9K eradicated bacteria from peritoneal fluid (FIG. 26C, left side: vehicle; right side:Buf(i+4)13 (Q9K)).

[0058] FIG. 27 lists the MIC (in pM) for C. albicans , C. auris, and C. neoformans and % toxicity (RBC, HUVEC and RPTEC) at 10 pM for the indicated library of i, i+4 staple scanning peptides (SEQ ID NOs: 1-19, from top to bottom). Amphotericin B (Amp B) served as a positive control.

[0059] FIG. 28 lists the MIC (in pM) for C. albicans, C. Auris and C. neoformans and % toxicity (RBC, HUVEC and RPTEC) at 10 pM for the indicated lysine scan of the Buf (i+4)l 2 stapled peptide (SEQ ID NOs: 14 and 20-37, from top to bottom). Amp B served as a positive control.

[0060] FIG. 29 lists the MIC (in pM) for C. albicans and % toxicity (RBC, HUVEC and RPTEC) at 10 pM for the indicated lysine scan of the Buf (i+4) 13 stapled peptide (SEQ ID NOs: 15 and 42-59, from top to bottom). Amp B served as a positive control.

[0061] FIG. 30 lists the MIC (in pM) for C. albicans, C. auris, and C. neoformans and % toxicity (RBC, HUVEC and RPTEC) at 10 pM for the indicated single, double, and triple lysine-mutant Buf(i+4)12 peptides (SEQ ID NOs:20 and 38-41, from top to bottom).

[0062] FIG. 31 lists the MIC (in pM) for fungal strains C. albicans, C. auris, and C. neoformans, and bacterial strains A. baumannii, E. coli, and P aeruginosa for the indicated double and triple lysine-mutant Buf(i+4)12 peptides (SEQ ID NOs:38-41, from top to bottom), highlighting the relative specificity of select lysine-mutant peptides for fungi over bacteria.

[0063] FIG. 32 shows the SEM images of C. albicans, A. baumannii and HUVEC after exposure to MIC concentrations of amphotericin B, Buf(i+4)12 T1K (SEQ ID NO:2), Buf(i+4)12 T1K,V15K (SEQ ID NO:38), and Buf(i+4)12 V15K,L19K (SEQ ID NO:41) for 3 hours (scale bar: C. albicans 3 pm, A. baumannii 1 pm, HUVEC 10 pm).

[0064] FIG. 33 shows the inner membrane permeabilization of peptides at 4x, 2x, lx, 0.5x, 0.25x MIC concentrations at 1-hour and 3-hour exposure times, as measured by fluorescence intensity of PI in C. albicans at the 535 / 615 excitation / emission wavelength.

[0065] FIG. 34 shows the antifungal activity MIC (in pM) of Buf(i+4)12 V15K,L19K (SEQ ID NO:41) against C. albicans in the presence of added membrane lipid components ergosterol, cholesterol, chitin, 0-glucan, phosphatidylcholine, phosphatidylethanolamine. Whereas amphotericin B activity is impeded by ergosterol (and minimally by cholesterol), Buf(i+4)12 V15K,L19K (SEQ ID NO:41) is selectively inhibited by 0-glucan. Of note, the comparatively weaker activity of Buforin WT is unaffected by added lipid components.

[0066] FIG. 35 shows the contrasting dose-dependent effects of ergosterol and 0-glucan on the MICs of amphotericin B and Buf(i+4)12 V15K,L19K (SEQ ID NO:41) against C. albicans. Left: Amp B is top line. Right: Amp B is bottom line.

[0067] FIGS. 36A-C show the cytoxicity dose-response curve of RBC (FIG. 36A), HUVEC (FIG. 36B), and RPTEC (FIG. 36C) cells upon treatment with the indicated stapled Buforin peptides, revealing for example the striking lack of cytotoxic effect of Buf(i+4)12 (V15K, L19K) (SEQ ID NO:41); across all three human cell types. Peptide starting concentration was 100 pM with a 2-fold serial dilution.

[0068] FIGS. 37A-C show the MIC changes in Amp B (FIG.37A), Buf WT (FIG.37B) and Buf(i+4)12 V15K, L19K (SEQ ID NO:41) (FIG. 37C) upon treatment of C. albicans in the presence of exogenous fungal DNA. The arrows show maximal biofdm eradication for Amp B occurring at the same dose (0.78 pM, FIG. 37A), at slightly different doses for BUF WT (6.25 pM without DNA, 12.5 pM with DNA, FIG.37B), and striking potency and exogenous fungal DNA effect for C. albicans (0.39 pM without DNA, 3.13 pM with DNA, FIG.37C).

[0069] FIGS. 38A-B show the dose-responsive effect of Buf WT, Buf(i+4)12 VI 5K, L19K (SEQ ID NO:41) and Amp B on eradicating mature biofdms of C. albicans (FIG.38A) and C. neoformans (FIG.38B).

[0070] FIG. 39 shows proteolytic stability studies as determined by the influence of exposing Buf(i+4)12 VI 5K, L19K (SEQ ID NO:41) and Buf WT to proteinase K (starting protease concentrations of 0.1 mg / ml with 2-fold serial dilutions) on anti-C. albicans activity.

[0071] FIG. 40 lists the % lysis of the indicated cancer cells (OCI-AML3, K4562, MV4;11, U937, SJSA-1, A549) or non-transformed mammalian cells (RBC, HUVEC, RPTEC), as measured by LDH release assay for all cells other than RBCs, which are assessed by absorbance assay, upon treatment with 10 pM of the indicated peptides (SEQ ID NOs: 1-19, from top to bottom).

[0072] FIGS. 41A-D are graphs showing percent cellular lysis (cytotoxicity) as measured by LDH release assay and percent cell viability as measured by Cell Titer Gio assay upon treatment of OCLAML3 cancer cells at the indicated doses of the indicated peptides (WT Buforin II (SEQ ID NO:1), Buf(i+4)12 (SEQ ID NO: 14), Buf(i+4)13 (SEQ ID NO:15), or Buf(i+4)13) Q9K (SEQ ID NO:50) for 90 minutes (FIG. 41A, FIG. 41B)and 24 hours (FIG. 41C, FIG. 41D) Bars for each condition, from left to right: 20 pM, 10 pM, and 5 pM, respectively.

[0073] FIGS. 42A-B are graphs showing (FIG. 42A) percent cellular lysis (cytotoxicity) as measured by LDH release assay and (FIG. 42B) percent cell viability as measured by Cell Titer Gio assay upon treatment of EOL1, SUDHL8, or OCI-AML3 cells at the indicated doses of Buf(i+4)13 (SEQ ID NO: 15) for 24 hours.

[0074] FIGS. 43A-E are graphs showing the dose-responsive effect of Buf(i+4)13 (SEQ ID NO: 15) treatment on viability of a collection of over 900 characterized cancer cell lines (FIG. 43A), the relative susceptibility of cancer cells to Buf(i+4)13 (SEQ ID NO: 15) by cancer subtype (x axis, effect size; y axis, -loglO q value) (FIG. 43B), comparative susceptibility of ovarian, skin, bowel, and lymphoid cancer cell subtypes (ovary and skin, relatively high susceptibility; bowel and lymphoid, comparatively lower susceptibility) (FIG. 43C), and dose-responsive effect of Buf(i+4)13 (SEQ ID NO: 15) on the viability of ovarian (FIG. 43D) and skin (FIG. 43E) cancer subtypes.

[0075] FIGS. 44A-B show a scatter plot of drugs that correlate or anti -correlate with the cancer cell susceptibility profile of Buf(i+4)13 (SEQ ID NO: 15) (FIG. 44A) and a table of compounds that have the highest correlation with the susceptibility profile of Buf(i+4)13 (SEQ ID NO: 15), the majority of which are DNA-targeting agents (FIG. 44B)DETAILED DESCRIPTION

[0076] The present disclosure is based, inter alia, on the discovery that stapled buforin 11 peptides have clinical application in treating cancer. The present disclosure is also based, inter alia, on the discovery that stapled buforin II peptides having distinct and specified mutations, such as lysine substitutions and inserted all-hydrocarbon staples, have clinical applications in treating bacterial infections, fungal infections, and cancer. Accordingly, the present disclosure provides stapled peptides and compositions (e g., pharmaceutical compositions) comprising the stapled peptides that are useful for treating a bacterial infection, treating a fungal infection, and treating cancer. Also provided hereinare methods of treating a bacterial infection, methods of treating a fungal infection, and methods of treating cancer. Also provided herein are methods of producing the stapled peptides described herein.BUFORIN II

[0077] Buforin II is a histone-derived antimicrobial peptide (AMP), which was isolated from the stomach tissue of the Asian toad, Bufo bufo garagrizans (Park et al., 2000, PNAS, 97 (15): 8245-8250). The amino acid sequence of buforin II is TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO: 108). Unlike other amphibian AMPs like magainin II, buforin II does not disrupt the integrity of bacterial membranes. Instead, buforin II translocates into the bacterial cytosol, where it is able to bind to DNA and inhibit transcription (Yi et al., 1996, FEBS Letters, 398 (1): 87-90). Nevertheless, the generally poor stability and low potency of intracellular targeting AMPs such as buforin II have reduced interest in further development of buforin II and / or other intracellular targeting AMPs as active pharmaceutical agents and have led to their being generally relegated to use as research reagents.

[0078] Provided herein are buforin II peptides comprising one or more (e.g., 1, 2, 3, 4, 5) lysine substitutions (relative to SEQ ID NO:1) at positions selected from 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, and 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the buforin II peptide comprises a lysine substitution (relative to SEQ ID NO:1) at position 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, and 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the buforin II peptide comprises two lysine substitutions (relative to SEQ ID NO: 1), wherein the two lysine substitutions are at positions selected from 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, and 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the buforin 11 peptide comprises three lysine substitutions (relative to SEQ ID NO: 1), wherein the three lysine substitutions are at positions selected from 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, and 20 (numbered according to the sequence of SEQ ID NO: 1 ). In some instances, the buforin II peptide comprises a lysine at each of positions 1 and 15 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the buforin II peptide comprises a lysineat each of positions 1 and 19 (numbered according to the sequence of SEQ ID NO: 1 ). In some instances, the buforin II peptide comprises a lysine at each of positions 1, 15, and 19 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the buforin II peptide comprises a lysine at each of positions 15 and 19 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the buforin II peptide comprises a lysine at each of positions 1 and 9 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the buforin II peptide comprises a lysine at each of positions 3 and 9 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the buforin II peptide comprises a lysine at each of positions 6 and 9 (numbered according to the sequence of SEQ ID NO: 1).

[0079] In some instances, the buforin II peptide comprising the lysine substitution(s) further comprises 1, 2, 3, 4, or 5 additional substitutions. The skilled artisan will understand that the 1, 23, 4, or 5 additional substitutions are not at the location of the lysine substitution. For example, in some instances, the buforin II peptide comprising a lysine substitution at each of positions 1 and 15 of the sequence of SEQ ID NO: 1 and comprising 1, 2, 3, 4, or 5 additional substitutions has a lysine substitution at each of positions 1 and 14 of the sequence of SEQ ID NO: 1 and 1, 2, 3, 4, or 5 additional substitutions at positions other than positions 1 and 15 of the sequence of SEQ ID NO: 1. In some instances, the 1, 2, 3, 4, or 5 additional substitutions are conservative amino acid substitutions. In some instances, the 1, 2, 3, 4, or 5 additional substitutions are not conservative amino acid substitutions. In some instances, the 1, 2, 3, 4, or 5 additional substitutions are a combination of conservative amino acid substitutions and nonconservative amino acid substitutions.

[0080] In some instances, the buforin II peptide comprising the lysine substitution(s) is 21 to 30 amino acids in length (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids in length). In some instances, the buforin II peptide comprising the lysine substitution(s) is 21 amino acids in length. In some instances, the buforin II peptide comprising the lysine substitution(s) is 22 amino acids in length.STAPLED PEPTIDES

[0081] Disclosed herein are stapled peptides. Tn some instances, the stapled peptide is abuforin II peptide described herein comprising an i, i+4 staple. Exemplary stapled buforin II peptides are described in Table 1 and Table 2. Exemplary stapled buforin II peptides of Formula (I) are described in Table 3.

[0082] Table 1 provides exemplary stapled peptides based on buforin II (F10W). In some instances, the stapled peptide comprises or consists of a stapled peptide of Table 1.In some instances, the stapled peptide comprises or consists of a stapled peptide of Table 1 except that it comprises one or more (e.g., 1, 2, 3, 4, 5) substitutions (relative to the stapled peptide of Table 1). The skilled artisan will understand that the one or more (e.g., 1, 2, 3, 4, or 5) substitutions, when present, are not at the location of the cross-link in the peptide of Table 1 (i.e., position Xi and X2). For example, in some instances, the stapled peptide comprises or consists of the stapled peptide of SEQ ID NO: 14 with one or more (e.g., 1, 2, 3, 4, or 5) substitutions relative to SEQ ID NO: 14, wherein the one or more substitutions are not at positions 12 and 16 of SEQ ID NO: 14. In some instances, one or more of the substitutions is with a lysine at position(s) selected from 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, of 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the one or more substitutions are conservative amino acid substitutions. In some instances, the one or more substitutions are not conservative amino acid substitutions. In some instances, the one or more substitutions are a combination of conservative amino acid substitutions and non-conservative amino acid substitutions. In some instances, the stapled peptide comprises or consists of a stapled peptide of Table 1 except that it comprises one or more (e.g., 1, 2, 3, 4, 5) lysine substitutions (relative to the stapled peptide of Table 1) at positions selected from 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, and 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the stapled peptide comprises a lysine substitution (relative to the stapled peptide of Table 1) at position 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, or 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the stapled peptide comprises two lysine substitutions (relative to the stapled peptide of Table 1), wherein the two lysine substitutions are at positions selected from 1 , 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, and 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the stapledpeptide comprises three lysine substitutions (relative to the stapled peptide of Table 1), wherein the three lysine substitutions are at positions selected from 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, and 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the stapled peptide comprises a lysine at each of positions 1 and 15 (numbered according to the sequence of SEQ ID NO:1). In some instances, the stapled peptide comprises a lysine at each of positions 1 and 19 (numbered according to the sequence of SEQ ID NO:1). In some instances, the stapled peptide comprises a lysine at each of positions 1, 15, and 19 (numbered according to the sequence of SEQ ID NO:1). In some instances, the stapled peptide comprises a lysine at each of positions 15 and 19 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the stapled peptide comprises a lysine at each of positions 1 and 9 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the stapled peptide comprises a lysine at each of positions 3 and 9 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the stapled peptide comprises a lysine at each of positions 6 and 9 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the stapled peptide is 21 to 30 amino acids in length (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids in length). In some instances, the stapled peptide comprises or consists of the sequence of SEQ ID NO: 14. In some instances, the stapled peptide comprises or consists of the sequence of SEQ ID NO: 15. In some instances, the stapled peptide is 21 amino acids in length. In some instances, the stapled peptide is 22 amino acids in length. In some instances, the stapled peptide exhibits an antimicrobial effect against at least one microbe, optionally wherein the at least one microbe is E. coll, or wherein the stapled peptide exhibits an anticancer effect.

[0083] Table 2 provides exemplary stapled peptides with lysine substitution(s). In some instances, the stapled peptide comprises or consists of a stapled peptide of Table 2.

[0084] In some instances, the stapled peptide comprises or consists of a stapled peptide of Table 2 except that it comprises one or more (e.g., 1, 2, 3, 4, 5) substitutions (relative to the stapled peptide of Table 2). The skilled artisan will understand that the one or more (e.g., 1, 2, 3, 4, or 5) substitutions, when present, are not at the location of the cross-link in the peptide of Table 2 (i.e., position Xi and X2) and are not present at thelysine substitution (relative to SEQ ID NO: 1). For example, in some instances, the stapled peptide comprises or consists of the stapled peptide of SEQ ID NO:50 with one or more (e.g., 1, 2, 3, 4, or 5) substitutions relative to SEQ ID NO:50, wherein the one or more substitutions are not at positions 9, 13, and 17 of SEQ ID NO:50. In some instances, one or more of the substitutions is with a lysine at position(s) selected from 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, and 20 (numbered according to the sequence of SEQ ID NO: 1). In some instances, the one or more substitutions are conservative amino acid substitutions. In some instances, the one or more substitutions are not conservative amino acid substitutions. In some instances, the one or more substitutions are a combination of conservative amino acid substitutions and non-conservative amino acid substitutions. In some instances, the stapled peptide comprises or consists of a stapled peptide of Table 2 except that it comprises one or more (e.g., 1, 2, 3, 4, 5) additional lysine substitutions (relative to the stapled peptide of Table 2) at positions selected from 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, of 20 (numbered according to the sequence of SEQ ID NO:1). In some instances, the stapled peptide is 21 to 30 amino acids in length (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 amino acids in length). In some instances, the stapled peptide is 21 amino acids in length. In some instances, the stapled peptide is 22 amino acids in length. In some instances, the stapled peptide exhibits an antimicrobial effect against at least one microbe, optionally wherein the at least one microbe is E. coli, or wherein the stapled peptide exhibits an anticancer effect.

[0085] In some instances, stapled peptide comprises or consists of the sequence of any one of SEQ ID NO:38 (Buf(i+4)12 (T1K, V15K)), SEQ ID NO:39 (Buf(i+4)12 (T1K, L19K)), SEQ ID NO:40 (Buf(i+4)12 (T1K, V15K, L19K)), SEQ ID NO:41 (Buf(i+4)12 (V15K, L19K)), SEQ ID NO:44 (Buf(i+4)13 (S3K)), SEQ ID NO:46 (Buf(i+4)13 (R5K)), SEQ ID NO:47 (Buf(i+4)13 (A6K)), SEQ ID NO:48 (Buf(i+4)13 (G7K)), SEQ ID NO:49 (Buf(i+4)13 (L8K)), SEQ ID NO:52 (Buf(i+4)13 (Pl IK)) SEQ ID NO: 53 (Buf(i+4)13 (V12K)), SEQ ID NO: 57 (Buf(i+4)13 (L18K)), SEQ ID NO: 58 (Buf(i+4)13 (L19K)), and SEQ ID NO:59 (Buf(i+4)13 (R20K)), or a pharmaceutically acceptable salt thereof.

[0086] In some instances, stapled peptide comprises or consists of the sequence of any one of SEQ ID NO:41 (Buf(i+4)12 (V15K, L19K)), SEQ ID NO: 38 (Buf(i+4)12 (T1K, V15K)), SEQ ID NO:39 (Buf(i+4)12 (T1K, L19K)), and SEQ ID NO:40 (Buf(i+4)12 (T1K, V15K, L19K)), or a pharmaceutically acceptable salt thereof.

[0087] In some instances, stapled peptide comprises or consists of the sequence of any one of SEQ ID NO:42 (Buf(i+4)13 (T1K)), SEQ ID NO:44 (Buf(i+4)13 (S3K)), SEQ ID NO:47 (Buf(i+4)13 (A6K)), SEQ ID NO:50 (Buf(i+4)13 (Q9K)), SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), SEQ ID NO:61 (Buf(i+4)13 (S3K, Q9K)), and SEQ ID NO:62 (Buf(i+4)13 (A6K, Q9K)), or a pharmaceutically acceptable salt thereof.

[0088] In some instances, stapled peptide comprises or consists of the sequence of SEQ ID NO:50 (Buf(i+4)13 (Q9K)), SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), or SEQ ID NO:61 (Buf(i+4)13 (S3K, Q9K)), or a pharmaceutically acceptable salt thereof.

[0089] In some instances, stapled peptide comprises or consists of the sequence of SEQ ID NO:5 (Buf(i+4)3), SEQ ID NO:18 (Buf(i+4)16), or SEQ ID NO:50 (Buf(i+4)13 (Q9K)), or a pharmaceutically acceptable salt thereof.

[0090] In some instances, position 10 of a stapled peptide described herein (numbered according to SEQ ID NO: 1) is any aromatic amino acid (e.g., F or W). In some instances, position 10 of a stapled peptide described herein (numbered according to SEQ ID NO:1) is an F (phenylalanine). In some instances, position of a stapled peptide described herein (numbered according to SEQ ID NO: 1) is a W (tryptophan).

[0091] In some instances, the stapling amino acids Xi and X2 in Table 1 and Table 2 are replaced with stapling amino acids capable of making the / , z+4 cross-link. “Peptide stapling” is a term coined from a synthetic methodology wherein two olefin-containing side-chains (e.g., cross-linkable side chains) present in a peptide chain are covalently joined e.g., “stapled together”) using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, e.g., Blackwell etal., J. Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). The structural-stabilization may be by, e.g., stapling the peptide (see, e.g., Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated by reference herein in its entirety). In some instances,the cross-linked stapling amino acids Xi and X2 in Table 1 and Table 2 are each independently replaced with an a, a-disubstituted non-natural amino acid with olefinic side chain, wherein Xi is cross-linked to X2.

[0092] Table 1. Exemplary stapled Buforin II (F10W) peptides. Xi is (S)-2-(4’-pentenyl)alanine cross-linked to X2 is (S)-2-(4’-pentenyl)alanine (see, FIG. 2B).

[0093] Table 2. Exemplary stapled Buforin II (Fl 0W) peptides with lysine substitution(s). Xi is (S)-2-(4’-pentenyl)alanine cross-linked to X2 is (S)-2-(4’-pentenyl)alanine (see, FIG. 2B).

[0094] In some instances, the stapled peptide comprises the structure of formula (I):Formula (I)or a pharmaceutically acceptable salt thereof, wherein: each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted, optionally wherein each Ri and R2 is a methyl; each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted, optionally wherein R3 is Cs alkylene, Cs alkenylene, or Cs alkynylene, optionally wherein R3 is (CH2)3-CH=CH-(CH2)3; and wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 3 to 6 substitutions relative to the sequence TRSSRAGLQWPVGRVHRLLRK (SEQ ID NO: 1), wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 is with a lysine at position 1, 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, or 20 of the sequence of SEQ ID NO: 1, and wherein two of the 3 to 6 substitutions relative to thesequence of SEQ ID NO: 1 are at (i) positions 12 and 16 of the sequence of SEQ ID NO: 1, or (ii) positions 13 and 17 of the sequence of SEQ ID NO: 1; wherein [Xaa]xis (i) positions 13-15 of the sequence of SEQ ID NO: 1, optionally comprising one or two amino acid substitutions, or (ii) positions 14-16 of the sequence of SEQ ID NO: 1, optionally comprising one or two amino acid substitutions; and wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, exhibits an antimicrobial effect against at least one microbe, optionally wherein the at least one microbe is E. coli, or wherein the stapled peptide, or the pharmaceutically acceptable salt thereof exhibits an anticancer effect. In some instances, Ri is methyl, R3 is (CH2)3-CH=CH-(CH2)3, and R2 is methyl. In some instances, two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 12 and 16 of the sequence of SEQ ID NO: 1, wherein [Xaa]xcorresponds to positions 13-15 of the sequence of SEQ ID NO: 1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 is with a lysine at position 1, 15, or 19 of the sequence of SEQ ID NO:1. In some instances, two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 13 and 17 of the sequence of SEQ ID NO:1, wherein [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 3, 5, 6, 7, 8, 11, 12, 18, 19, or 20 of the sequence of SEQ ID NO:1. In some instances, two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 13 and 17 of the sequence of SEQ ID NO: 1, wherein [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 1, 3, 6, or 9 of the sequence of SEQ ID NO:1. In some instances, two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 13 and 17 of the sequence of SEQ ID NO: 1, wherein [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1, and wherein one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 9 of the sequence of SEQ ID NO: 1.

[0095] In some instances, each of the [Xaa]wof Formula (I), the [Xaa]xof Formula (I), and the [Xaa]yof Formula (I) is as described for any one of constructs 1-22 of Table3. For example, for a structurally-stabilized peptide comprising the [Xaa]w, the [Xaa]x, and the [Xaa]yof construct 1 of Table 3, the [Xaa]w, the [Xaa]x, and the [Xaa]yare: KRSSRAGLQWP (SEQ ID NO:63), GRV, and RLLRK (SEQ ID NO:85), respectively.

[0096] Table 3

[0097] In some instances, the stapled peptide comprises the structure of formula (I):Formula (I)wherein: each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted orunsubstituted, optionally wherein each Ri and R2 is a methyl; each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted, optionally wherein R3 is Cs alkylene, Cs alkenylene, or Cs alkynylene, optionally wherein R3 is (CH2)3-CH=CH-(CH2)3; each Xaa is independently an amino acid; each w and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and each x is 3 or 6; and wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 2 to 6 substitutions relative to the sequence of TRSSRAGLQWPVGRVHRLLRK (SEQ ID NO : 1), wherein (a) 2 of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are substitutions with non-natural amino acids, the side chains of which are cross-linked to form R3, optionally wherein the 2 of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 with non-natural amino acids, the side chains of which are cross-linked to form R3 are at positions 13 and 17 of the sequence of SEQ ID NO:1 and [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1; or (b) 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1, wherein two of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are with stapling amino acids that are cross-linked to each other.

[0098] In some instances, the stapled peptide has a free amine at the N-terminus. In some instances, the stapled peptide has a free carboxylate at the C-terminus. In some instances, the stapled peptide has a free amine at the N-terminus and a free carboxylate at the C-terminus.

[0099] In some instances, the stapled peptide isN-terminal acetylated and / or C-terminal amidated. N-terminal acetylation and C-terminal amidation create modified proteins that mimic the native protein by reducing the overall charge of a peptide, thus increasing the metabolic stability of peptides and the ability to resist enzymatic degradation. N-terminal acetylation is a post-translational modification in which an acetyl group is appended to the N-terminal amino group of a peptide, altering the charge, hydrophobicity, and size of the N-terminus. N-terminal acetylation is catalyzed by N“-acetyltransferases, which accept acetyl-CoA as the donor for the transfer of the activated acetyl moiety to the Na-terminus of the protein (Linster et ctl., J Exp Bot. 2018 Aug 31;69(19):4555-4568). In some embodiments, an acetylation reaction includesdeprotection of an Fmoc group, followed by reaction with an esterification agent (e.g, neat acetic anhydride) and an organic compound (e.g., N,N-Diisopropylethylamine (DIPEA)). C-terminal amidation is a post-translational modification to include an amide group at the C-terminus. In some embodiments, the modified amino acid is followed by a glycine, which provides the amide group. During C-terminal amidation, the glycine is oxidized to form alpha-hydroxy -glycine. The oxidized glycine cleaves into the C-terminally ami dated peptide and an N-glyoxylated peptide. In some instances, for peptide synthesis the synthesis is started with RINK-AMIDE resin, which renders the most C-terminal residue “amidated”.

[0100] In some instances, the stapled peptide comprises or consists of a stapled peptide described in the working examples or figures herein.

[0101] In some instances, the stapled peptide is a pharmaceutically acceptable salt of the stapled peptide. In some instances, the pharmaceutically acceptable salt includes a salt comprising hydrochloride, sodium, sulfate, acetate, phosphate or diphosphate, chloride, potassium, maleate, calcium, citrate, mesylate, nitrate, tartrate, aluminum, gluconate, and any combination thereof.PHARMACEUTICAL COMPOSITIONS

[0102] One or more of any of the buforin II peptides and stapled peptides described herein can be formulated for use as or in pharmaceutical compositions. The pharmaceutical compositions may be used in the methods of treatment described herein. In some instances, the pharmaceutical composition comprises a buforin II peptide described herein and a pharmaceutically acceptable carrier. In some instances, the pharmaceutical composition comprises a stapled peptide described herein and a pharmaceutically acceptable carrier. In certain instances, the pharmaceutical composition comprises a stapled peptide comprising or consisting of an amino acid sequence that is identical to an amino acid sequence set forth in Table 1 or Table 2 and a pharmaceutically acceptable carrier. In certain instances, the pharmaceutical composition comprises a stapled peptide comprising or consisting of Formula (I) (see Table 3) and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can beformulated or adapted for administration to a subject via any route, e.g., any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA’s CDER Data Standards Manual, version number 004 (which is available at fda.give / cder / dsm / DRG / drg00301.htm). For example, pharmaceutical compositions can be formulated or adapted for administration by inhalation (e.g., oral and / or nasal inhalation (e.g., via nebulizer or spray)), injection (e.g., intravenously, intraarterial, subdermally, intraperitoneally, intramuscularly, and / or subcutaneously); and / or for oral administration, transmucosal administration, and / or topical administration (including topical (e.g., nasal) sprays, eye drops, and / or solutions).

[0103] In some instances, pharmaceutical compositions can include an effective amount of one or more buforin II peptides or stapled peptides described herein. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of the described agent (e.g., the peptide or stapled peptide) or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., treatment of bacterial infection, treatment of fungal infection, or treatment of cancer).

[0104] Pharmaceutical compositions of this disclosure can include one or more peptides or stapled peptides described herein and any pharmaceutically acceptable carrier and / or vehicle. In some instances, pharmaceutical compositions can further include one or more additional therapeutic agents in amounts effective for achieving a modulation of disease (e.g., bacterial infection, fungal infection, or cancer) or disease symptoms.

[0105] Pharmaceutically acceptable carriers and adjuvants include carriers and adjuvants that may be administered to a patient or a subject from another species provided herein, together with a compound of this disclosure (e.g., a peptide or stapled peptide), and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the compound.

[0106] In some instances, the pharmaceutical compositions of this disclosure include one or more of acetate, citrate and / or maleate. In some instances, the pharmaceutical compositions can include water or phosphate buffer saline (PBS). In some instance, the pharmaceutical compositions can include chitosan.

[0107] The pharmaceutical compositions disclosed herein can include one or more pharmaceutically acceptable salts. In some instances, the pharmaceutically acceptable salts include salts comprising hydrochloride, sodium, sulfate, acetate, phosphate or diphosphate, chloride, potassium, maleate, calcium, citrate, mesylate, nitrate, tartrate, aluminum, gluconate, and any combination thereof.

[0108] The pharmaceutical compositions of this disclosure may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. In some cases, the pH of the formulation may be adjusted with pharmaceutically acceptable acids, bases or buffers to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intra-cutaneous, intravenous, intra-muscular, intra-articular, intra-arterial, intra-synovial, intra-sternal, intrathecal, intra-lesional and intra-cranial injection or infusion techniques.

[0109] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating a fungal infection in subject (e.g., human, plant, animal, insect (e.g., bee, silkworm), aquatic species (e.g., fish, mollusk), bird, amphibian, reptile), and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human. In some instances, the fungal infection is a Chytridiomycota fungal infection. In some instances, the fungal infection is a Zygomycota fungal infection. In some instances, the fungal infection is an Ascomycota fungal infection. In some instances, the fungal infection is a Basidiomycota fungal infection. In some instances, the fungal infection is a Glomeromycota fungal infection. In some instances, the fungal infection is a C. albicans, a C. auris, or a C. neoformans fungal infection.

[0110] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating a bacterial infection in a subject (e.g., human, plant, animal), and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human. In someinstances, the bacterial infection is a gram-negative bacterial infection. In some instances, the bacterial infection is an A. baumannii, E. coli, P. aeruginosa, S. aureus, or B. cereus bacterial infection.

[0111] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating cancer in a subject (e.g., human), and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0112] Also provided herein is a pharmaceutical composition comprising: (a) a means for selectively killing gram-negative bacteria in a subject (e.g., human, plant, animal), and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human. In some instances, the bacterial infection is a gram-negative bacterial infection. In some instances, the bacterial infection is an A. baumannii, E. coli, P. aeruginosa, S. aureus, or B. cereus bacterial infection.

[0113] Also provided herein is a pharmaceutical composition comprising: (a) a means for treating ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, an osteosarcoma, or other liquid or solid tumor cells in a subject (e.g., human, animal), including for example by a non-membrane-lytic mechanism of action, and (b) a pharmaceutically acceptable carrier. In some instances, the subject is a human.METHODS OF MAKING STAPLED PEPTIDES

[0114] Methods of synthesizing the peptides and stapled peptides described herein are known in the art. Nevertheless, the following exemplary method and the exemplary method(s) described in the working examples herein can be used. It will be appreciated that the various steps can be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the peptides and stapled peptides described herein are known in the art and include, e.g., those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3d. Ed., lohn Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0115] Methods of performing different types of stapling are well known in the art (see, e.g., Lactam stapling'. Shepherd et al., J. Am. Chem. Soc., 127:2974-2983 (2005); Triazole stapling. Kawamoto et al., J. Med. ('hem... 55:1137-1146 (2011); f / 1-cycloaddition stapling: Madden et al., Bioorg. Med. Chem. Lett., 21: 1472-1475 (2011); Disulfide stapling: Jackson et al., Am. Chem. Soc., 113 :9391-9392 (1991); Oxime stapling: Haney et al., Chem. Commun., 47:10915-10917 (2011); Thioether stapling: Brunel and Dawson, Chem. Commun., 552-2554 (2005); Photoswitchahle stapling: J. R. Kumita et al., Proc. Natl. Acad. Sci. U. S. A., 97:3803-3808 (2000); Double-click stapling: Lau et al., Chem. Sci., 5:1804-1809 (2014); Bis-lactam stapling: J. C. Phelan et al.,, J. Am. Chem. Soc., 119:455-460 (1997); and Bis-arylation stapling: A. M. Spokoyny etal., J. Am. Chem. Soc., 135:5946-5949 (2013)). I

[0116] The peptides and stapled peptides of this invention can be made by chemical synthesis methods, which are well known to the ordinarily skilled artisan. See, e.g., Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence, peptides and stapled peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the 0.-NH2 protected by either t-Boc or Fmoc chemistry using side chain protected amino acids on, e.g., an Applied Biosystems Peptide Synthesizer Model 430A or 431.

[0117] One manner of making of the peptides and stapled peptides described herein is using solid phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups.

[0118] Longer peptides can be made by conjoining individual synthetic peptides using native chemical ligation. Alternatively, the longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed. Next, a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods. The peptides can be made in a high-throughput, combinatorial fashion, e.g., using a high-throughput multiple channel combinatorial synthesizer available from Advanced Chemtech.

[0119] One or more peptide bonds can be replaced, e.g., to increase physiological stability of the stapled peptide, by: a retro-inverso bonds (C(O)-NH); a reduced amide bond (NH-CH2); a thiomethylene bond (S-CH2 or CH2-S); an oxomethylene bond (O-CH2 or CH2-O); an ethylene bond (CH2-CH2); a thioamide bond (C(S)-NH); a transolefin bond (CH=CH); a fluoro-substituted trans-olefin bond (CF=CH); a ketomethylene bond (C(O)-CHR) or CHR-C(O), wherein R is H or CH3; and a fluoro-ketomethylene bond (C(O)-CFR or CFR-C(O), wherein R is H, F, or CH3.

[0120] The peptides and stapled peptides can be further modified by one or more of: acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, lipidation (e.g., myristoylation, palmitoylation, cholesterol modification), phosphorylation (Ser, Tyr, or Thr), stearoyl ati on, succinylation, and sulfurylation. In some instances, a peptide or stapled peptide described herein is N-terminal acetylated. In some instances, a peptide or stapled peptide described herein is N-terminal acetylated and C-terminal amidated. In some instances, a peptide or stapled peptide described herein is C-terminal amidated.

[0121] Methods of producing stapled peptides using a, a-di substituted non-natural amino acids are known in the art (see, e.g., Bird et al., Methods EnzymoL, 446:369, 2008; Bird et al, Current Protocols in Chemical Biology, 2011; US Publication No. 2020-0352899, each of which is incorporated by reference herein in its entirety), a, a-disubstituted non-natural amino acids containing olefinic side chains of varying length can be synthesized by known methods (see, e.g., Williams et al., J. Am. Chem.Soc.113:9276, 1991; Schafmeister et al., J. Am. Chem Soc. 122:5891, 2000; Bird et al., Methods EnzymoL, 446:369, 2008; Bird et al, Current Protocols in Chemical Biology, 2011). For stapled peptides where an i linked to i+ 7 staple is used (two turns of the helix stabilized), either one Ss amino acid ((S)-a-(4'-pentenyl)alanine) and one Rs amino acid ((R)-a-(7'-octenyl)alanine) can be used, or one Ss amino acid ((S)-a-(7'-octenyl)alanine)) and one Rs amino acid ((R)-a-(4'-pentenyl)alanine) can be used. For stapled peptides where an z linked to i I 4 staple is used (one turn of the helix stabilized), either two Ss amino acids ((S)-a-(4'-pentenyl)alanine) can be used, or two Rs amino acids ((R)-a-(4'-pentenyl)alanine) can be used. The starting chiral auxiliary confers the R- or S- alkyl-stereoisomer. Also, 8-iodooctene can be used in place of 5 -iodopentene. Inhibitors can be synthesized on a solid support using solid-phase peptide synthesis (SPPS) on MB HA resin (see, e.g., WO 2010 / 148335).

[0122] Fmoc-protected a-amino acids (other than the olefinic amino acids Fmoc- ?s-OH, FmocNs-OH, Fmoc-As-OH, and Fmoc-bs-OH), 2-(6-chloro-l- / / -benzotriazole-l-yl)-l,l,3,3-tetramethylaminium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available from, e.g., Novabiochem (San Diego, CA).Dimethylformamide (DMF), A-methyl-2-pyrrolidinone (NMP), N,N-di isopropyl ethyl amine (DIEA), trifluoroacetic acid (TFA), 1,2-di chloroethane (DCE), fluorescein isothiocyanate (FITC), and piperidine are commercially available from, e.g., Sigma-Aldrich. Olefinic amino acid synthesis is reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0123] In some instances, the peptides and stapled peptides are synthesized according to methods described in the working examples herein.

[0124] In some instances, a method of making a stapled peptide described herein comprises: (a) provided a peptide comprising an amino acid sequence of the stapled peptide (prior to cross-linking), and (b) cross-linking the peptide (e.g., by a ring-closing metathesis (RCM reaction) thereby making the stapled peptide. In some instances, the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition.

[0125] In some instances, provided herein is a method of making a stapled peptide, the method comprising: (a) providing a peptide comprising the sequence of SEQ ID NO: 1 with 3 to 6 substitutions, wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 1 , 3, 5, 6, 7, 8, 9, 11, 12, 18, 19, or 20 of the sequence of SEQ ID NO: 1, and wherein two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are with stapling amino acids at (i) positions 12 and 16 of the sequence of SEQ ID NO: 1, or (ii) positions 13 and 17 of the sequence of SEQ ID NO: 1; and (b) cross-linking the peptide thereby making the stapled peptide. In some instances, the stapling amino acids are (S)-2-(4’-pentenyl)alanine. In some instances, the cross-linking is by a ruthenium catalyzed metathesis reaction, and optionally wherein the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition.

[0126] In some instances, the peptides and stapled peptides can include a detectable label. A label includes a moiety that has at least one element, isotope, or functional group incorporated into the moiety which enables detection of the peptide to which the label is attached. Labels can be directly attached (i.e., via a bond) or can be attached by a linker. Labels can be attached to peptide or stapled peptide described herein at any position that does not interfere with the biological activity (or characteristic of the inventive stapled peptide or peptide that is being detected. Known labels include Biotin or Fluorescein, e.g., attached by Fluorescein Isothiocyanate (FITC).

[0127] Again, methods suitable for obtaining (e.g., synthesizing), stapling, and purifying the peptides and stapled peptides disclosed herein are also known in the art (see, e.g., Bird et. al., Methods in Enzymology 446:369-386 (2008); Bird et al, Current Protocols in Chemical Biology 2011; Walensky et al., Science 305: 1466-1470 (2004);Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); U.S. Patent Application Publication No. 2010 / 0168388; and U.S. Patent No. 7,723,468, each of which are hereby incorporated by reference in their entirety).

[0128] In some embodiments, the peptides are isolated. In some embodiments, the stapled peptides are substantially free of non-stapled peptide contaminants or are isolated. Methods for purifying peptides and stapled peptides include, for example, synthesizing the peptide on a solid-phase support (and, for stapled peptides, performing the cyclization). Next, the solid-phase support may be isolated and suspended in a solution of a solvent such as DMSO, DMSO / dichloromethane mixture, or DMSO / NMP mixture. The DMSO / dichloromethane or DMSO / NMP mixture may comprise about 30%, 40%, 50%, or 60% DMSO. In a specific embodiment, a 50% / 50% DMSO / NMP solution is used. The solution may be incubated for a period of 1, 6, 12, or 24 hours, following which the resin may be washed, for example with dichloromethane or NMP. In one embodiment, the resin is washed with NMP. Shaking and bubbling an inert gas into the solution may be performed.

[0129] Properties of the peptides and stapled peptides described herein can be assayed, for example, using the methods described below and in the working examples.

[0130] Assays to determine a-helicity, melting temperature (Tm), in vitro protease resistance, and in vivo protease resistance are known in the art (see, e.g., US Patent Application Publication No. 2020-0352899, which is incorporated herein in its entirety). To assess the membrane tolerability to a peptide or stapled peptide described herein, membrane interaction studies (e.g., as described in the working examples herein) can be performed. To assess the ability of a peptide or stapled peptide described herein to kill bacterial cells, bacterial killing kinetics and / or minimum inhibitory concentration assays (e.g., as described in the working examples herein) can be performed. To assess the ability of a peptide or stapled peptide described herein to kill fungal cells, antifungal activity testing and / or minimum inhibitory concentration assays (e.g., as described in the working examples herein) can be performed.

[0131] To measure the cell penetrability of the peptides or stapled peptides described herein, intact cells are incubated with fluoresceinated stapled peptides (10 pM) for 4 hours in serum-free media or in media supplemented with human serum at 37°C, washed twice with media and incubated with trypsin (0.25%) for 10 minutes at 37°C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics KineticScan® HCS Reader.

[0132] To determine the suitability of the peptides, stapled peptides, or pharmaceutical compositions described herein for treatment of humans, clinical trials can be performed. For example, patients having a cancer or suspected of having a cancer requiring chemotherapy are selected and separated into treatment and one or more control groups, wherein the treatment group is administered a peptide, stapled peptide, or pharmaceutical composition of the invention, while the control groups receive a placebo or a known cytoprotective drug. The treatment safety and efficacy of the peptide, stapled peptide, or pharmaceutical compositions of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors, such as prevention of symptoms, time to resolution of symptoms, and / or time to a decrease in the number, severity, or frequency of one or more symptoms of the disease. In some embodiments, subject administered a peptide, stapled peptide, or pharmaceutical composition of the invention can have a reduced number of symptoms of the disease as compared to a subject in a control group receiving a placebo.

[0133] As another example, patients are separated into treatment and one or more control groups, wherein the treatment group is administered a peptide, stapled peptide, or pharmaceutical composition of the invention, while the control groups receive a placebo or a known antibacterial or antifungal drug. The treatment safety and efficacy of the peptide, stapled peptide, or pharmaceutical compositions of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors, such as prevention of symptoms, time to resolution of symptoms, and / or time to a decrease in the number, severity, or frequency of one or more symptoms of the disease. In some embodiments, subject administered a peptide, stapled peptide, or pharmaceuticalcomposition of the invention can have a reduced number of symptoms of the disease as compared to a subject in a control group receiving a placebo.METHODS OF USE

[0134] The disclosure features methods of using any of the peptides or stapled peptides (or compositions, e.g., pharmaceutical compositions, comprising the same) described herein for the treatment of a fungal infection, a bacterial infection, or a cancer in a subject (e.g., plant, animal, e.g., human) in need thereof. The disclosure also features uses of any of the peptides or stapled peptides (or compositions, e.g., pharmaceutical compositions, comprising the same) described herein in preparation of a medicament for treating a fungal infection, a bacterial infection, or a cancer in a subject (e g., plant, animal, e g., human) in need thereof. In some instances, the treating comprises alleviating, inhibiting, or ameliorating the disease (e.g., cancer) or infection (e.g., bacterial or fungal) from which the subject (e.g., plant, animal, e.g., human) is suffering. In some instances, the subject is an animal. In some instances, the subject is a mammal such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey or human). In some instances, the subject is a domesticated animal (e.g., a dog or cat). In some instances, the subject is a human. In some instances, the subject is a plant. In some instances, the subject is an insect (e.g., bee, silkworm). In some instances, the subject is an aquatic species (e.g., fish, mollusk). In some instances, the subject is a bird. In some instances, the subject is an amphibian. In some instances, the subject is a reptile.

[0135] Thus, provided herein is a method of treating a fungal infection in a subject in need thereof, the method comprising administering to the subject a peptide (or a pharmaceutically acceptable salt thereof) or a stapled peptide (or a pharmaceutically acceptable salt thereof) (e.g., a therapeutically effective amount thereof) described herein. In some instances, the subject is a human. In some instances, the subject is a plant. In some instances, the subject is an animal. In some instances, the subject is an insect (e.g., bee, silkworm). In some instances, the subject is an aquatic species (e.g., fish, mollusk). In some instances, the subject is a bird. In some instances, the subject is an amphibian. In some instances, the subject is a reptile. In some instances, the fungal infection is aChytridiomycota fungal infection. Tn some instances, the fungal infection is a Zygomycota fungal infection. In some instances, the fungal infection is an Ascomycota fungal infection. In some instances, the fungal infection is a Basidiomycota fungal infection. In some instances, the fungal infection is a Glomeromycota fungal infection. In some instances, the fungal infection is a C. albicans infection. In some instances, the fungal infection is a C. auns infection. In some instances, the fungal infection is a C. neoformans infection. In some instances, the method comprises administering to the subject a stapled peptide comprising or consisting of the sequence of any one of SEQ ID NO:38 (Buf(i+4)12 (T1K, V15K)), SEQ ID NO:39 (Buf(i+4)12 (T1K, L19K)), SEQ ID NO:40 (Buf(i+4)12 (T1K, V15K, L19K)), SEQ ID NO:41 (Buf(i+4)12 (V15K, L19K)), SEQ ID NO:44 (Buf(i+4)13 (S3K)), SEQ ID NO:46 (Buf(i+4)13 (R5K)), SEQ ID NO:47 (Buf(i+4)13 (A6K)), SEQ ID NO:48 (Buf(i+4)13 (G7K)), SEQ ID NO:49 (Buf(i+4)13 (L8K)), SEQ ID NO:52 (Buf(i+4)13 (Pl IK)) SEQ ID NO:53 (Buf(i+4)13 (V12K)), SEQ ID NO:57 (Buf(i+4)13 (L18K)), SEQ ID NO:58 (Buf(i+4) 13 (L19K)), and SEQ ID NO:59 (Buf(i+4)13 (R20K)), or a pharmaceutically acceptable salt thereof. In some instances, the stapled peptide or a pharmaceutically acceptable salt thereof comprises a Phe (F) at position 10 of SEQ ID NO: 1 (e.g., comprises the sequence of SEQ ID NO:41 (Buf(i+4)12 (V15K, L19K)), except with a Phe (F) at position 10 of SEQ ID NO:41).

[0136] In some instances, the method of treating a fungal infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:41 (Buf(i+4)12 (V15K, L19K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0137] In some instances, the method of treating a fungal infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO: 38 (Buf(i+4)12 (T1K, VI 5K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0138] In some instances, the method of treating a fungal infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:39 (Buf(i+4)12 (T1K, L19K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0139] In some instances, the method of treating a fungal infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:40 (Buf(i+4)12 (T1K, V15K, L19K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0140] Also provided herein is a method of treating a bacterial infection in a subject in need thereof, the method comprising administering to the subject a peptide (or a pharmaceutically acceptable salt thereof) or a stapled peptide (or a pharmaceutically acceptable salt thereof) (e.g., a therapeutically effective amount thereof) described herein. In some instances, the subject is a plant. In some instances, the subject is an animal. In some instances, the subject is a human. In some instances, the bacterial infection is a gram-positive bacterial infection. In some instances, the bacterial infection is a gramnegative bacterial infection. In some instances, the bacterial infection is an A. baumannii infection. In some instances, the bacterial infection is an E. coli infection. In some instances, the bacterial infection is a P. aeruginosa infection. In some instances, the bacterial infection is an S. aureus infection. In some instances, the bacterial infection is an B. cereus infection. In some instances, the method comprises administering to the subject a stapled peptide comprising or consisting of the sequence of any one of SEQ ID NO:42 (Buf(i+4)13 (T1K)), SEQ ID NO:44 (Buf(i+4)13 (S3K)), SEQ ID NO:47 (Buf(i+4)13 (A6K)), SEQ ID NO:50 (Buf(i+4)13 (Q9K)), SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), SEQ ID NO:61 (Buf(i+4)13 (S3K, Q9K)), and SEQ ID NO:62 (Buf(i+4)13 (A6K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the stapled peptide or a pharmaceutically acceptable salt thereof comprises a Phe (F) at position 10 of SEQ ID NO: 1 (e.g., comprises the sequence of SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), except with a Phe (F) at position 10 of SEQ ID NO: 60).

[0141] In some instances, the method of treating a bacterial infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:50 (Buf(i+4)13 (Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0142] In some instances, the method of treating a bacterial infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0143] In some instances, the method of treating a bacterial infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:61 (Buf(i+4)13 (S3K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0144] In some instances, the method of treating a bacterial infection further comprises administering to the subject an antibiotic (e.g., concurrently, or within 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 1 hour, 2 hours, 1 day, 2 days, 1 week or the administration of the peptide or stapled peptide). In some instances, the antibiotic is selected from the group consisting of a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof. In some instances, the P-lactam is ampicillin, ceftazidime, cefotaxime, or a combination thereof. In some instances, the fluoroquinolone is ciprofloxacin. In some instances, the aminoglycoside is gentamicin, tobramycin, or a combination thereof. In some instances, the carbapenem is meropenem.

[0145] Also provided herein is a method of treating a treating a multi-drug resistant bacterial infection in a subject in need thereof, the method comprising administering to the subject a peptide (or a pharmaceutically acceptable salt thereof) or a stapled peptide (or a pharmaceutically acceptable salt thereof) (e.g., a therapeutically effective amountthereof) described herein. In some instances, the subject is a plant. Tn some instances, the subject is an animal. In some instances, the subject is a human. In some instances, the multi-drug resistant bacterial infection is a gram-positive bacterial infection. In some instances, the multi-drug resistant bacterial infection is a gram-negative bacterial infection. In some instances, the multi-drug resistant bacterial infection is an A. baumannii infection. In some instances, the multi-drug resistant bacterial infection is an E. coli infection. In some instances, the multi-drug resistant bacterial infection is a / < aeruginosa infection. In some instances, the multi-drug resistant bacterial infection is an S. aureus infection. In some instances, the multi-drug resistant bacterial infection is an B. cereus infection. In some instances, the multi-drug resistant bacterial infection is a bacterial infection resistant to treatment with a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof. In some instances, the P-lactam is ampicillin, ceftazidime, cefotaxime, or a combination thereof. In some instances, the fluoroquinolone is ciprofloxacin. In some instances, the aminoglycoside is gentamicin, tobramycin, or a combination thereof. In some instances, the carbapenem is meropenem. In some instances, the method comprises administering to the subject a stapled peptide comprising or consisting of the sequence of any one of SEQ ID NO:42 (Buf(i+4)13 (T1K)), SEQ ID NO:44 (Buf(i+4)13 (S3K)), SEQ ID NO:47 (Buf(i+4)13 (A6K)), SEQ ID NO:50 (Buf(i+4)13 (Q9K)), SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), SEQ ID NO:61 (Buf(i+4)13 (S3K, Q9K)), and SEQ ID NO:62 (Buf(i+4)13 (A6K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the stapled peptide or a pharmaceutically acceptable salt thereof comprises a Phe (F) at position 10 of SEQ ID NO: 1 (e.g., comprises the sequence of SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), except with a Phe (F) at position 10 of SEQ ID NO:60).

[0146] In some instances, the method of treating a multi-drug resistant bacterial infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:50 (Buf(i+4)13 (Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0147] In some instances, the method of treating a multi-drug resistant bacterial infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0148] In some instances, the method of treating a multi-drug resistant bacterial infection in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:61 (Buf(i+4)13 (S3K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0149] Also provided herein is a method of synergistically killing bacteria in a subject in need thereof, the method comprising administering to the subject a peptide (or a pharmaceutically acceptable salt thereof) or a stapled peptide (or a pharmaceutically acceptable salt thereof) (e.g., a therapeutically effective amount thereof) described herein. In some instances, the subject is a plant. In some instances, the subject is an animal. In some instances, the subject is a human. In some instances, the bacteria are gram-positive bacteria. In some instances, the bacteria are gram-negative bacteria. In some instances, the bacteria are A. baumannii. In some instances, the bacteria are E. coli. In some instances, the bacteria are P. aeruginosa. In some instances, the bacteria are S. aureus. In some instances, the bacteria are B. cereus. . In some instances, the bacteria are multidrug resistant. In some instances, the multi-drug resistant bacteria are resistant to treatment with a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof. In some instances, the P-lactam is ampicillin, ceftazidime, cefotaxime, or a combination thereof. In some instances, the fluoroquinolone is ciprofloxacin. In some instances, the aminoglycoside is gentamicin, tobramycin, or a combination thereof. In some instances, the carbapenem is meropenem. In some instances, the method comprises administering to the subject a stapled peptide comprising or consisting of the sequence of any one of SEQ ID NO:42 (Buf(i+4)13 (T1K)), SEQ ID NO:44 (Buf(i+4)13 (S3K)), SEQ ID NO:47 (Buf(i+4)13 (A6K)), SEQ ID NO:50 (Buf(i+4)13 (Q9K)), SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), SEQ ID NO:61(Buf(i+4)13 (S3K, Q9K)), and SEQ TD NO:62 (Buf(i+4)13 (A6K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the stapled peptide or a pharmaceutically acceptable salt thereof comprises a Phe (F) at position 10 of SEQ ID NO:1 (e.g., comprises the sequence of SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), except with a Phe (F) at position 10 of SEQ ID NO:60).

[0150] In some instances, the method of synergistically killing bacteria in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:50 (Buf(i+4)13 (Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0151] In some instances, the method of synergistically killing bacteria in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:60 (Buf(i+4)13 (T1K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0152] In some instances, the method of synergistically killing bacteria in a subject in need thereof comprises administering to the subject a therapeutically effective amount of a stapled peptide comprising or consisting of the sequence of SEQ ID NO:61 (Buf(i+4)13 (S3K, Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human.

[0153] Also provided herein is a method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a peptide (or a pharmaceutically acceptable salt thereof) or a stapled peptide (or a pharmaceutically acceptable salt thereof) (e.g., a therapeutically effective amount thereof) described herein. In some instances, the subject is a human. In some instances, the cancer is a cancer with susceptibility to treatment with a therapeutic agent that specifically targets DNA or other nucleic acids. In some instances, the cancer is a cancer with a susceptibility to treatment with a therapeutic agent that damages nucleic acid (e g., DNA) and / or interferes with a regulatory mechanism responsible for surveillance and repair of DNA and RNA. In someinstances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, osteosarcoma, a bowel cancer, a colon cancer, or a lymphoid cancer. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0154] In some instances, the method of treating a cancer in a subject in need thereof comprises administering to the subject a stapled peptide or a pharmaceutically acceptable salt thereof, wherein the stapled peptide comprises or consists of the structure of formula (I):Formula (I)wherein: each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted; each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; each Xaa is independently an amino acid; each w and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; and each x is 3 or 6, and wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 2 to 6 substitutions relative to the sequence of TRSSRAGLQWPVGRVHRLLRK (SEQ ID NO: 1), wherein 2 of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are substitutions with non-natural amino acids, the side chains of which are cross-linked to form R3. In some instances, each Ri and R2 is a methyl. In some instances, R3 is Cs alkylene, Cs alkenylene, or Cs alkynylene. In some instances, R3 is (CH2)3-CH=CH-(CH2)3. In some instances, the 2 of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 with non-natural amino acids, the side chains of which are cross-linked to form R3 are at positions 13 and 17 of the sequence of SEQ ID NO: 1 and [Xaa]xcorresponds to positions 14-16 of the sequence ofSEQ ID NO: 1. In some instances, the stapled peptide or a pharmaceutically acceptable salt thereof comprises a Phe (F) at position 10 of SEQ ID NO: 1. In some instances, the subject is a human. In some instances, the cancer is a cancer with an upregulated DNA repair system. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, osteosarcoma, a bowel cancer, a colon cancer, or a lymphoid cancer. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0155] In some instances, the method of treating a cancer in a subject in need thereof comprises administering to the subject a stapled peptide comprising or consisting of 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1, wherein two of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are with stapling amino acids that are cross-linked to each other. In some instances, the subject is a human. In some instances, the cancer is a cancer with an upregulated DNA repair system. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, osteosarcoma, a bowel cancer, a colon cancer, or a lymphoid cancer. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0156] In some instances, the method of treating a cancer in a subject in need thereof comprises administering to the subject a stapled peptide comprising or consisting of the sequence of SEQ ID NO:5 (Buf(i+4)3), or a pharmaceutically acceptable salt thereof. In some instances, the method of treating a cancer in a subject in need thereof comprises administering to the subject a stapled peptide comprising or consisting of the sequence of SEQ ID NO:18 (Buf(i+4)16), or a pharmaceutically acceptable salt thereof. In some instances, the subject is a human. In some instances, the cancer is a cancer with an upregulated DNA repair system. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, osteosarcoma, a bowel cancer, a colon cancer, or a lymphoid cancer. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0157] In some instances, the method of treating a cancer in a subject in need thereof comprises administering to the subject a stapled peptide comprising or consisting of the sequence of SEQ ID NO:50 (Buf(i+4)13 (Q9K)), or a pharmaceutically acceptable salt thereof. In some instances, the stapled peptide or a pharmaceutically acceptable salt thereof comprises a Phe (F) at position 10 of SEQ ID NO: 1 (e.g., comprises the sequence of SEQ ID NO:50 (Buf(i+4)13 (Q9K)), except with a Phe (F) at position 10 of SEQ ID NO:50). In some instances, the subject is a human. In some instances, the cancer is a cancer with an upregulated DNA repair system. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, osteosarcoma, a bowel cancer, a colon cancer, or a lymphoid cancer. In some instances, the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

[0158] In general, methods include selecting a subject and administering to the subject an effective amount of one or more of the peptides (or pharmaceutically acceptable salts thereof) or stapled peptides (or pharmaceutically acceptable salts thereof) described herein, e.g., in or as a pharmaceutical composition, and optionally repeating administration as required for the treatment of the bacterial infection, the fungal infection, or the cancer and can be administered orally, intranasally, intravenously, subcutaneously, intramuscularly, or topically, including skin, nasal, sinus, ocular, oropharynx, respiratory tree, and lung administration. In some instances, the administration is by a topical respiratory application which includes application to the nasal mucosa, sinus mucosa, oropharyngeal mucosa, or respiratory tree, including the lungs. In some instances, topical application includes application to the skin or eyes. A subject can be selected for treatment of a bacterial infection, or a fungal infection based on, e.g., determining that the subject is at risk to acquire or has a bacterial infection or a fungal infection, respectively. A subject can be selected for treatment of a cancer based on, e.g., determining that the subject has a cancer.

[0159] Specific dosage and treatment regimens for any particular patient or subject will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the disease, condition orsymptoms, the patient’s or subject’s disposition to the disease, condition or symptoms, and the judgment of the treating physician or veterinarian.

[0160] An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a peptide or stapled peptide ( / .< ., an effective dosage) depends on the therapeutic compounds selected. The compositions can be administered from one or more times per day to one or more times per week, including once every other day. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the risk to acquire or severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. For example, effective amounts can be administered at least once.EXAMPLESEXAMPLE 1. GENERATION OF STAPLED BUFORIN II PEPTIDES

[0161] Structure and sequences

[0162] Buforin II is a 21 -amino acid peptide derived from Buforin I that efficiently crosses the lipid bilayer of bacterial membranes and is believed to bind to nucleic acids without causing significant membrane perturbations. Buforin II possesses a unique structure comprised of an organized alpha-helical shaped C-terminal region and a disordered N-terminal region separated by Prol 1 (numbered according to SEQ ID NO: 1), which creates a kink in the peptide (FIG. 1). The presence of Prol 1 distorts the helix and concentrates the basic amino acid residues in a limited amphipathic region, which is believed to facilitate peptide translocation. In the peptides tested throughout the examples, the PhelO adjacent to the Prol 1 in wild type buforin II (SEQ ID NO: 108) (both numbered according to SEQ ID NO: 1) was replaced with tryptophan for both faciledetermination of peptide concentration by UV spectroscopy. SEQ ID NO: 1 is the buforin II sequence with a Trp (W) at position 10.

[0163] A library of alternatively stapled i, i+4 stapled Buforin II peptides (FIG.2) was generated to identify constructs bearing structural and compositional features that could potentially discern between bacterial and mammalian membranes, maintain the membrane translocation properties of Buforin II, and exert selective antibacterial activity without causing mammalian cell toxicity.EXAMPLE 2. ANTIBACTERIAL STAPLED BUFORIN II PEPTIDES

[0164] Minimum inhibitory concentration (MIC) vs. Toxicity

[0165] Buforin II is reported to exhibit broad spectrum gram-negative selective killing and the control Buf WT peptide (SEQ ID NO: 1) indeed showed selective, but weak, gram-negative killing (FIG.3). To further enhance the potency, selectivity, and, thus, the therapeutic window, Buforin II stapled peptides (FIG. 2) were screened for antibacterial activity and toxicity profile against human red blood cells (RBCs), endothelial human umbilical vein endothelial cells (HUVECs) and kidney renal proximal tubule epithelial cells (RPTECs) (FIG. 3). Select i, i+4 stapled peptides significantly improved gram-negative killing, especially against baumannii. Stapled analogs Buf(i+4)3 (SEQ ID NO:5) and Buf(i+4)13 (SEQ IDNO:15) showed A. baumannii killing at 1.6 pM concentration with no evidence of hemolysis or cytotoxicity (FIG. 3). In contrast, Buf(i+4)9 (SEQ ID NO: 11), Buf(i+4)11 (SEQ ID NO: 13), and Buf(i+4)12 (SEQ ID NO: 14) showed potent A. baumannii killing but were notably cytotoxic to mammalian cells (FIG.3). Some stapled peptides, such as Buf(i+4)0 (SEQ ID NO:2), Buf(i+4)6 (SEQ ID NO:8), Buf(i+4)8 (SEQ ID NO: 10), and Buf(i+4)15 (SEQ ID NO: 17), showed little to no enhancement of antibacterial activity as compared to the WT Buf peptide (SEQ ID NO: 1) (FIG.3). Stapling around Prol 1 (numbered according to SEQ ID NO:1), such as in the case of Buf(i+4)7 to Buf(i+4)11 (SEQ ID NOs:9-13), significantly enhanced the bacterial killing but also resulted in non-selective killing activity, and thus higher toxicity (FIG. 3).

[0166] A lysine mutation scanning library was generated using Buf(i+4)12 (SEQ ID NO: 14) and Buf(i+4)13 (SEQ ID NO: 15) as the templates (FIG. 2). Buf(i+4)12 (SEQ ID NO: 14) showed pan -bacterial killing in the context of non-specific cytotoxic activity toward HUVEC and RPTEC cells, with relatively less cytotoxicity (hemolysis) of RBCs (FIG. 3). In contrast, Buf(i+4)13 (SEQ ID NO: 15) showed more selective bacterial killing among strains (favoring gram-negative species) and was uniformly non-hemolytic and non-cytotoxic (FIG. 3). As shown in FIG. 4, several analogs from the Buf(i+4)12 Lys scan, such as Buf(i+4)12 T1K (SEQ ID NO:20), Buf(i+4)12 S3K (SEQ ID NO:22), Buf(i+4)12 A6K (SEQ ID NO:25), Buf(i+4)12 G7K (SEQ ID NO:26), Buf(i+4)12 Q9K (SEQ ID NO: 28), Buf(i+4)12 Pl IK (SEQ ID NO: 30), and Buf(i+4)12 G13K (SEQ ID NO:31), showed improved bacterial killing with moderate hemolysis (except for Buf(i+4)12 T1K (SEQ ID NO:20), which exhibited relatively increased hemolysis), yet higher cytotoxicity. Buf(i+4)12 V15K (SEQ ID NO:33) and Buf(i+4)12 L19K (SEQ ID NO:36) had a relatively better therapeutic window with maintenance of E. coli activity but relative loss of A. baumannii and P. aeruginosa activity as a result (FIG. 4).

[0167] The Lys mutational scan of Buf(i+4)13 (SEQ ID NO: 15) yielded many more potent and bacterial-selective analogs (FIG. 5A). Notably, all of the analogs showed selective gram-negative killing with improved potency against A. baumannii and E. coli (FIG. 5A). Buf(i+4)13 T1K (SEQ ID NO:42), Buf(i+4)13 S3K (SEQ ID NO:44), Buf(i+4)13 S4K (SEQ ID NO:45), Buf(i+4)13 A6K (SEQ ID NO:47), Buf(i+4)13 Q9K (SEQ ID NO:50), and Buf(i+4)13 Pl IK (SEQ ID NO:52) all showed potent bacterial killing (FIG. 5A). Buf(i+4)13 Pl IK (SEQ ID NO:52) substitution was an exception, with pan-bacterial killing, again demonstrating that Prol 1 substitution significantly alters the properties of the stapled peptide (FIG. 5A).

[0168] Single Lys mutants from the Buf(i+4)13 Lys scan library were combined to generate multiple Lys mutations within a stapled peptide (FIG. 5B). For example, to improve the activity against P. aeruginosa, the sequence was modified to include additional Lys mutations: whereas Buf(i+4)13 Q9K showed exceptional potency, Buf(i+4)13 T1K and S3K substitutions demonstrated improved Pseudomonas killing (FIG. 5B). Thus, Buf (i+4)13 (T1K, Q9K) (SEQ ID NO:60) and Buf (i+4)13 (S3K, Q9K)(SEQ ID N0:61) were generated to synergize attributes and accomplish more potent broader spectrum gram-negative killing (FIG. 5B).

[0169] Membrane interaction studies

[0170] Helical peptides can bind to the bacterial membrane, followed by permeabilization and bacterial cell lysis. To assess the impact of the Buf(i+4)12 (SEQ ID NO: 14) and Buf(i+4)13 (SEQ ID NO: 15) peptides on membranes, scanning electron microscopy (SEM) of treated bacteria was performed. As shown in FIG. 6, Buf(i+4)12 (SEQ ID NO: 14) treated cells demonstrated significant membrane damage and perturbations when compared to the untreated cells, which were structurally well-defined and had intact membranes. In contrast, Buf WT (SEQ ID NO:1) and Buf(i+4)13 (SEQ ID NO: 15) treated cells maintained the membrane morphology of the control, untreated cells. Moreover, propidium iodide (PI) assay revealed that Buf(i+4)12 (SEQ ID NO: 14) showed a dose-dependent increase in membrane permeability (FIGS. 7A-7D), comparable to polymyxin treated cells, a positive control for this assay. Conversely, no rise in fluorescent signal upon treatment with Buf WT (SEQ ID NO: 1) and Buf(i+4)13 (SEQ ID NO: 15) was evident at 30 minutes (FIG. 7A) or 180 minutes (FIG. 7B).Membrane permeability of Buf(i+4)13 (SEQ ID NO: 15) at significantly higher concentrations, such as 8x MIC at 30 minutes (FIG. 7C) and 180 minutes (FIG. 7D), was further assayed. Membrane tolerability to Buf(i+4)13 (SEQ ID NO: 15) at high concentration and prolonged exposure was observed (FIGs. 7C-7D).

[0171] Bacterial killing kinetics

[0172] A bactericidal kinetics study of Buf WT (SEQ ID NO : 1 (-treated 4. baumannii at the MIC concentration showed delayed bacterial killing, with no colonies observed at 3 hours (FIG. 8A). Buf(i+4)12 (SEQ ID NO: 14) treated A. baumannii displayed a rapid reduction in bacterial growth, with complete eradication of colonies at 0.5 hour (FIG. 8B), a common feature of other membrane lytic peptides such as LL-37 and magainin. Buf(i+4)13 (SEQ ID NO: 15) showed intermediate killing kinetics, with a striking reduction of the colonies observed by 1 hour post treatment (FIG. 8C).

[0173] Stability to proteinase K

[0174] The effect of different concentrations of proteinase K on the antimicrobial activity of peptides was determined (FIG. 9). Buf WT (SEQ ID NO: 1) was highly sensitive to proteinase K, with complete degradation / inactivation of the peptide at the lowest enzyme concentration of 0.0002 mg / ml. Buf(i+4)13 (SEQ ID NO:15) demonstrated a degree of resistance to the degradation, with concentrations of 0.0125 mg / ml proteinase K or higher required to inactivate the stapled peptide. In the case of Buf(i+4)12 (SEQ ID NO: 14), the distinct staple positioning conferred complete protection from proteinase K degradation / inactivation. Thus, maximal protection from proteolysis by hydrocarbon stapling did not necessarily correlate with maximal and selective biological activity.

[0175] RNAseq

[0176] A. baumannii cells were treated with vehicle or Buforin (i+4) 13 (SEQ ID NO: 15) and RNA was isolated from the bacteria over time for RNAseq analysis. Buforin (i+4) 13 (SEQ ID NO: 15) treatment increased a series of RNA transcripts relevant to general and oxidative stress responses, systems involved in osmotic regulation, and compensatory systems that break down and repair the cell wall in A. baumannii (Table 4). The upregulation of pathways that involve amino sugar and nucleotide sugar metabolism is consistent with the substrate needs for peptidoglycan biosynthesis and cell wall repair. The oxidative stress responses included multiple enzyme systems involved in detoxifying ROS, repairing S-S bonds in cytoplasmic and periplasmic locations, and electron handling systems that support these and other primary systems induced to repair ROS-mediated damage of proteins, DNA, etc. Notably, Buf(i+4)13 (SEQ ID NO: 15) also affected the proton gradient machinery, consistent with impairment of cellular systems associated with electron transport and ATP synthase in treated versus control cells. The control cells were metabolically active, generating energy and proteins via the ribosome / translation machinery. A notable downregulation upon Buf(i+4)13 (SEQ ID NO: 15) treatment involved cell adhesion and bacterial secretion systems, the 2 pilus systems, and the type 6 secretion system. The adhesion and secretion systems are important for survival, including in the context of clinical infection. Buf(i+4)13 (SEQ ID NO: 15) treatment suppressed the adhesion and secretion systems, which are needed forenvironmental adaptation and survival. These data demonstrate that Buf(i+4)13 (SEQ ID NO: 15) treatment induces specific transcriptional signatures, informing a defined and selective mechanism of action.

[0177] Table 4 shows that Buf(i+4)13 (SEQ ID NO: 15) treatment selectively influenced key processes relevant to A. baumannii survival and homeostasis. %Enriched = % of genes significantly upregulated in the indicated system under the indicated condition, q value = false discovery rate. Systems with a q value <0.05 are shown.

[0178] Features of the Optimized Buf(i+4)13 Q9K (SEQ ID NO: 50)

[0179] Lysine scanning of Buf(i+4)13 (SEQ ID NO: 15) revealed that Buf(i+4)13 Q9K (SEQ ID NO: 50) exhibited potent anti-bacterial activity while maximally sparing mammalian cells. Comparative membrane permeabilization analyses revealed that Buf(i+4)13 Q9K (SEQ ID NO: 50), in contrast to a sampling of other lysine mutants, demonstrated non-lytic behavior even after extended treatment times (up to 180 minutes) and at concentrations up to 4 times the MIC, indicating a significantly lower propensity for membrane disruption compared to the other lysine mutants and lytic controls (Pl IK mutant and Mag2) (FIG. 10A-10B).

[0180] Scanning electron microscopy (SEM) further revealed that cells treated with Buf(i+4)13 Q9K showed no apparent changes in morphological features and membranes were intact with no signs of lytic damage, comparable to the vehicle and Buf WT control cells (FIG. 11). In sharp contrast, lytic damage and membrane abrasion was observed after Buf(i+4)12 treatment (FIG. 11). Consistent with the SEM results, the TEM characterization showed obvious membrane damage with Buf(i+4)12 treatment as evident by cellular leakage and lower intensity staining of the organelles, whereas the vehicle control, Buf WT and Buf(i+4)13 Q9K treatment showed intact cells and intense staining of the organelles (FIG. 11).

[0181] To investigate the stability of the peptides under proteolytic conditions, A. baumannii was incubated with the MIC of Buf WT or Buf(i+4)13 Q9K peptides in the presence of varying concentrations of Proteinase K (a top dose 0.4 mg / mL, 2-fold, 10-point serial dilution) for 1 hour. The relative survival rates of A. baumannii were then determined to assess the peptides' bactericidal activity post-incubation. The results demonstrated that Buf WT was highly susceptible to proteolytic degradation, as bactericidal activity was almost entirely lost at the lowest enzyme concentration of 0.001 mg / mL (FIG. 12). In contrast, Buf(i+4)13 Q9K exhibited a significantly higher resistance to proteolytic degradation by maintaining bactericidal function even at a higher Proteinase K concentration of 0.05 mg / mL (FIG. 12).

[0182] Treatment of A. baumannii cells with vehicle or Buforin (i+4) 13 Q9K (SEQ ID NO:50), followed by RNA isolation from the bacteria over time and RNAseq analyses, revealed that, in contrast to Buf(i+4)13 (SEQ ID NO: 15), Buforin (i+4)13 Q9K(SEQ ID NO:50) treatment had no evident effect on modulating transcripts relevant to cell wall damage and repair, and instead demonstrated slowed growth compared to vehicle (Table 5).

[0183] Table 5 shows that Buf(i+4)13 Q9K (SEQ ID NO:50) treatment selectively influenced key processes relevant to A. baumannii growth and homeostasis relative to vehicle treated cells at 30 min. %Enriched = % of genes significantly upregulated in the indicated system under the indicated condition, q value = false discovery rate. Systems with a q value <0.05 are shown.

[0184] Mitigation of mammalian cell toxicity by Buf(i+4)13 Q9K.

[0185] Buf(i+4)13 Q9K exhibited no cytotoxic effect on RBCs, RPTEC, or HUVEC cells at 50 pM (50x and 30x MIC for A. baumannii and E. coli, respectively), with cytotoxicity levels maintained below 25% even up to 200 pM (200x and 125x MIC for A. baumannii and E. coli, respectively), indicative of a robust specificity window (FIG. 13).

[0186] Cellular uptake of FITC-Buf(i+4)13 Q9K by l. baumannii.

[0187] Confocal microscopy tracked the cellular localization of FITC-conjugated Buf(i+4)13 Q9K in treated bacteria overtime. Imaging at 0, 60, and 180 minutes revealed progressive cytoplasmic accumulation of the peptide, while membrane integrity, as visualized by FM4-64 dye, remained intact (FIG. 14).

[0188] Buf(i+4)13 Q9K exhibits strong DNA binding as reflected by functional sequestration by bacterial genomic DNA

[0189] Antibacterial activity of Buf(i+4)13 Q9K was attenuated in the presence of exogenous A. baumannii DNA, indicative of peptide sequestration by the added DNA when compared to peptide alone (FIG. 15A). An ethidium bromide (EtBr) displacement assay was performed using genomic DNA isolated from A. baumannii and E. coli.Whereas EtBr intercalates between DNA base pairs, yielding enhanced fluorescence upon binding, displacement by a competing ligand leads to fluorescence quenching.Incubation with Buf(i+4)13 Q9K led to a concentration-dependent reduction in fluorescence intensity, indicative of effective displacement of EtBr from the DNA duplex by peptide binding of base-pair sites (FIG. 15B). Comparable results were observed with DNA from E. coli (FIG. 15C). Quantitatively, Buf II and its stapled analogues, Buf(i+4)12, Buf(i+4)13, and Buf(i+4)13 Q9K, exhibited markedly greater DNA-binding capacity than Mag II and polymyxin B, which showed minimal displacement activity.[00190J Buf(i+4)13 Q9K eradicates matured, baumannii biofilms

[0191] To assess the therapeutic potential of Buf(i+4)13 Q9K beyond planktonic killing, its efficacy in eradicating matured, baumannii biofilms, which pose a major clinical challenge due to their persistence and formidable barrier to bacterial clearance, often resulting in chronic or recurrent infections, was tested. Buf(i+4)13 Q9K dose-responsively decreased biofilm mass, whereas Buf II was ineffective even at 8x MIC (FIG. 16A). Notably, co-admini strati on of Buf(i+4)13 Q9K with polymyxin B or ciprofloxacin resulted in complete biofilm clearance at only 2x MIC (FIG. 16B), underscoring Buf(i+4)13 Q9K’s capacity to disrupt biofilms and its potential for synergistic activity in combination with other antibiotics or therapeutics.

[0192] Synergistic anti-bacterial activity of Buf(i+4)13 Q9K in combination with other antibiotics

[0193] Given the enhanced biofilm clearance observed with Buf(i+4)13 Q9K and antibiotic co-treatment, it was tested whether the stapled peptide could similarly potentiate antibacterial activity in planktonic cultures. Using checkerboard microdilution assays against A baumannii, Buf(i+4)13 Q9K demonstrated synergy with both polymyxin B and ciprofloxacin, yielding fractional inhibitory concentration indices (FICI) of 0.225 and 0.292, respectively (FIG. 17A, FIG. 17B). Synergy was also observed with the antimicrobial peptide indolicidin (FICI = 0.113; FIG. 18A). In contrast, combination with gentamicin yielded potential antagonism (FICI > 0.5; FIG.18B) These data highlight the benefit of selective combinatorial activity of Buf(i+4)13 Q9K and support its role as a potentiator of membrane- and / or nucleic acid-targeting antimicrobials.

[0194] A. baumannii's transcriptional responses to Buf(i+4)13 Q9K treatment inform bactericidal mechanisms

[0195] To investigate the mechanism by which the optimized Buf(i+4)13 Q9K peptide induces bactericidal activity, A. baumannii's transcriptional responses after exposure to Buf(i+4)13 Q9K and its progenitor peptide Buf(i+4)13 were evaluated. Systems-level enrichment analyses of responses in Buf(i+4) 13 -treated baumannii revealed profound disruptions relative to vehicle-treated bacteria, as reflected by the upregulation of multiple pathways required for cell wall turnover and repair, including cell wall acylases, peptidoglycan synthases, inner and outer membrane lipoproteins, as well as operons involved in biofilm and LPS biosynthesis (e g., O-antigen, lipid A biosynthesis and export) (FIG. 19). Oxidative stress responses were strongly induced, including catalases, peroxidases, superoxide dismutases, hemerythrins, disulfide bond and methionine sulfoxide repair systems, and aquaporin Z and mechanosensitive channels. General responses to membrane and cell wall stress (e.g., DegS, OmpW), in addition to protein degradation pathways and multiple periplasmic / lipoprotein chaperones, were also increased. Among the most highly upregulated genes in Buf(i+4)13-treated cells was 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (423-fold increase), a key enzyme in the methyl-erythritol phosphate pathway for isoprenoid biosynthesis, which contributes to lipid production and electron transport. Trehalose metabolism genes were also induced, supporting production of osmoprotectants that help preserve membrane and surface structure integrity. Concurrently, nucleo-sugar and amino-sugar metabolism pathways were upregulated, including genes involved in the uptake and processing of N-acetylglucosamine and UDP-glucose, which contribute to peptidoglycan, LPS biosynthesis, and biofdm formation.

[0196] With respect to transcriptional suppression, Buf(i+4) 13 -exposed cells showed reductions in protein synthesis machinery among ribosomal structural components, tRNAs, and associated translational machinery, and induction of stress response proteins associated with suppression of protein synthesis including ribosome hibernation factor Hpf and ribosome-associated inhibitor A, both previously linked to antibiotic exposure. Notably, exposed cells demonstrated a marked reduction in expression of membrane-associated energy systems, including the NADH-ubi quinone electron transport chain and F-type ATP synthase. In contrast, vehicle-treated control cells expressed P- and type IV pili, and A. baumcmni type VI secretion system — systems associated with host pathogenesis — and showed relative induction of genes for amino acid and TCA cycle metabolism, supporting energy generation via NADH-ubiquinone and ATP synthase.

[0197] Intriguingly, Buf(i+4)13 Q9K induced distinct disruption patterns at 30 minutes as reflected by a profound shutdown of protein synthesis and metabolism, without induction of cell wall or membrane repair pathways (FIG. 20). DegS and OmpW stress proteins were induced, in addition to other stress responses linked to temperature and pH shock, as well as ribosome hibernation and silencing factors Hpf and RsfA. Bacteriophage locus 5 genes were also activated and included structural capsid and tail proteins. As with Buf(i+4) 13 -exposed cells, Buf(i+4)13 Q9K suppressed expression of adhesive organelles and the type 6 secretion system (T6SS).

[0198] As A. baumannii exposure toBuf(i+4)13 Q9K progressed, by 90 minutes, induction of antibiotic resistance genes, including those associated with resistance to cationic peptides (e.g., SbmA, bacteriocin resistance peptidase C39), tetracycline, macrolide, other drug efflux systems, a class D P-lactamase, and putative AdeT aminoglycoside-modifying enzymes was observed (FIG. 21). Intriguingly, Buf(i+4)13 Q9K-treated cells also induced DNA mismatch repair systems, including the G:T / U mismatch glycosylase, excision-repair enzymes, UmuCD error-prone lesion-bypass DNA polymerase IV, RecA, and the RecX regulator. Despite these responses, energy metabolism remained suppressed, with only benzoate degradation pathways showing upregulation, including 3-oxoadipate CoA-transferase and 3-carboxy-cis, cis-muconate cycloisomerase. Control cells, by comparison, showed specific induction of genes associated with pantothenate / CoA biosynthesis, the NADH-ubiquinone electron transport chain and ATP synthase, consistent with active growth. By 120 minutes, Buf(i+4)13 Q9K-exposed cells continued to repress central metabolic and energetic pathways and showed only up-regulation of phage locus 5 transcripts (FIG. 22). In contrast, control cells upregulated genes related to phenylalanine and porphyrin metabolism, NADH-ubiquinone electron transport, and virulence factors including type IV pili, T6SS, andsiderophores such as bacterioferritin and acinetobactin — the latter synthesized by a non-ribosomal peptide synthetase incorporating phenylalanine-derived intermediates and recognized as a key siderophore in sterile-site infections.

[0199] Taken together, these results provide new insights into the distinct mechanisms of action by which Buf(i+4)13 and Buf(i+4)13 Q9K exert bactericidal effects in 4. baumannii. Buf(i+4)13 rapidly disrupts the bacterial surface, cell wall, and energy -generating systems, consistent with — at least in part — a lytic mode of action. In contrast, Buf(i+4)13 Q9K, a rationally selected analog optimized for enhanced antibacterial activity with reduced nonspecific membrane lysis, clearly favors a mechanism of membrane translocation and intracellular engagement that includes DNA binding as opposed to specific membrane disruption.

[0200] Buf(i+4)13 Q9K delayed the onset and magnitude of acquired resistance by A. baumannii

[0201] Given the challenges posed by antimicrobial resistance (AMR), the potential for acquired genetic resistance to Buf(i+4)13 Q9K was evaluated using a 30-day serial passage assay in A. baumannii at sub-MIC concentrations, in comparison to Buf II, Buf(i+4)12, or polymyxin B. Polymyxin B exhibited the fastest emergence of resistance, with MICs increasing up to 2,500-fold after just nine passages (FIG. 23). Rapid resistance also developed to Buf II, with MICs rising 16-fold during the same period. In contrast, Buf(i+4)13 Q9K exhibited delayed resistance with serial passage and exposure, showing only a gradual 16-fold MIC increase over 30 days. Buf(i+4)12 demonstrated the slowest development of resistance, highlighting the capacity of select membrane-lytic peptides to avoid resistance in A. baumannii.

[0202] Quantitative proteomics reveals the mechanisms of delayed resistance

[0203] To probe the mechanistic basis of resistance to Buf(i+4)13 Q9K, quantitative proteomic profiling on strains serially exposed to each agent at days 4 and 30, representing early and late adaptive stages to sub-MIC peptide exposure, was performed. Compared to vehicle-treated controls, 461 and 906 proteins were differentially expressed at days 4 and 30, respectively. Early responses, as examined by gene ontology (GO) andgene set enrichment (GSEA) analyses, included upregulation of DNA-related pathways, such as viral genome integration, DNA recombination, replication, repair, and transcription, as well as activation of two-component signal transduction systems (FIG.24). Downregulated processes included translation (ribosome biogenesis), oxidative phosphorylation, and ABC (ATP-binding cassette) transporters. By day 30, DNA recombination and two-component signaling pathways remained upregulated, suggesting continued activation of DNA repair-driven genome remodeling and transcriptional adaptation to peptide-induced stresses, while suppression of ribosomal, electron transport, and ABC transporter proteins also persisted, consistent with the disruptions in cellular metabolism and growth.

[0204] Buf(i+4)13 Q9K is effective against multidrug resistant clinical isolates.

[0205] To assess the breadth of Buf(i+4)13 Q9K’s antibacterial activity, the stapled peptide was tested against a diverse panel of clinical isolates, including 38 A. baumannii strains with varying resistance phenotypes, two MCR-1+ E. coli strains obtained from the US Centers for Disease Control and Prevention (CDC) (FIG. 25A), and additional multidrug-resistant (MDR) Gram-negative isolates obtained from the Massachusetts General Hospital (MGH) (FIG. 25B). Among the A. baumannii isolates, the majority (35 of 38) were resistant to multiple antibiotic classes, including 0-lactams (ampicillin, ceftazidime, cefotaxime), fluoroquinolones (ciprofloxacin), aminoglycosides (gentamicin, tobramycin), and carbapenems (meropenem). While many retained sensitivity only to colistin, several isolates displayed intermediate or full resistance to colistin. Buf II showed only modest activity against these highly resistant strains, with MICs ranging from 25 to 100 pM, whereas Buf(i+4)13 Q9K demonstrated potent bactericidal activity, with MIC values ranging from 0.4 to 1.6 pM. Buf(i+4)13 Q9K also retained full activity against two A. coli strains harboring the mobilized colistin resistance gene (MCR-1), with MICs of 1.6 pM, again far outperforming Buf II (MIC >100 pM). In vitro efficacy against four MDR isolates of E. coli and K. pneumoniae reinforced the broad-spectrum efficacy of Buf(i+4)13 Q9K against Gram-negative pathogens. In this subset of clinical isolates, MICs ranged from 1.6 to 6.3 pM, whereas Buf II remained inactive (MIC >100 pM) against all strains. These data underscore the robust activity ofBuf(i+4)13 Q9K against highly drug-resistant clinical isolates, including ones with resistance to the cationic peptide colistin. Buf(i+4)13 Q9K's enhanced potency relative to Buf II and retained activity in the presence of MCR-1 resistance genes further highlight its therapeutic promise as a next-generation antimicrobial.

[0206] Given the promising in vitro antibacterial activity and selectivity of Buf(i+4)13 Q9K (SEQ ID NO:50), the investigation was extended to assess in vivo efficacy using a mouse peritonitis model. C57BL / 6J mice were rendered neutropenic with two doses of cyclophosphamide, followed by challenge with a 108CFU dose of A. baumannii (ATCC 19606) via intraperitoneal (IP) injection on Day 0 (FIG. 26A), which resulted in 100% mortality within 48 hours in the untreated saline control group (FIG.26B). Buf(i+4)13 Q9K achieved a 100% survival / cure rate (10 day experimental duration post-infection) when administered intraperitoneally at a dose of 5 mg / kg, at 2 hours, 24 hours, and 48 hours after infection (FIG. 26B ). Correspondingly, Buf(i+4)13 Q9K sterilized the peritoneal fluid (FIG. 26C).

[0207] Methods used in Examples 1 and 2

[0208] Solid-phase peptide synthesis: Peptides were generated using Fmoc-based solid-phase peptide synthesis. To generate a single i, i+4 staple, two chiral (S)-pentenyl alanine (S5) non-natural amino acids were inserted at sequential i, i+4 positions along the length of the peptide template. The staple was formed by olefin metathesis using the Grubbs first-generation ruthenium catalyst in dichloromethane. The peptides were then cleaved from the resin using a TFA cleavage cocktail. The peptides are C-terminal amidated as a result of cleaving from the amide resin. The cleavage solution was filtered and concentrated under nitrogen and the crude peptides were precipitated using an excess of cold hexane: diethyl ether mixture. After centrifugation, the pellet was dissolved in water and lyophilized to obtain the crude peptide, which was then purified by LC-MS and quantified by amino acid analysis.

[0209] Minimum Inhibitory Concentration (MIC) determination: A. baumannii E. coli, P. aeruginosa, S. aureus and A cereus strains were sourced from ATCC, USA. The strains were stored as glycerol stocks at -80°C and -20°C. For routine use, bacteria wereplated on agar plates containing Mueller Hinton Broth (MHB), previously modified by anion exchange (Q Sepharose fast flow) to ensure peptide solubility. The assays were performed in 96 well polypropylene plates following standard literature procedures. Briefly, the overnight cultures were grown at 37°C in modified MHB media until the turbidity of 0.08-0.13 at 625 nm (1 x 108CFU / mL) was obtained. Peptide stock solution was prepared in modified MHB media and was then further diluted to achieve a range of concentrations. The antimicrobial assay was performed by adding diluted cultures (1 x 106CFU / ml) to the peptide solution in each well of the plates. The plates, which also contained suitable growth and sterility controls, were then incubated at 37°C for 24 hours to determine MIC. Streptomycin and polymyxin were used as positive controls for Grampositive and Gram-negative bacteria, respectively. Optical density measurements at 600 nm for determination of MIC were performed using a spectrophotometer plate reader. MIC is reported as the concentration at which no visible growth was observed. Three biological replicate experiments, each performed with three technical replicates, were used to determine the average MIC values.

[0210] Cytotoxicity assay: Adherent HUVEC and RPTEC cells were maintained in the appropriate media, confirmed mycoplasma free (My coAlert kit), and plated at 2xl04cells / mL in 96-well format. Following treatment with a screening 10 pM dose of peptide for 90 min, plates were centrifuged, 50 pL of media transferred to a new plate, incubated with 50 pL LDH reagent for 10 minutes, and absorbance recorded at 490 nm on a SpectraMax M5 microplate reader. Vehicle and 1% Triton X-100 served as negative and positive controls, respectively.

[0211] Hemolysis: Whole human blood cells (Innovative Research) were centrifuged at lOOOxg for 5 minutes to remove buffy coat and isolate RBCs. RBCs (pellet) were washed thrice with PBS and resuspended in PBS to a final concentration of 1% v / v. The cell suspension was then added to a peptide solution prepared in PBS to achieve a final concentration of 10 pM in 96-well plates, which were then incubated for 1 hour at 37 °C without agitation. The plates were centrifuged at 3500xg for 10 min, supernatant (100 pL) transferred to new plates, and absorbance measured at 540 nm. Vehicle and Triton X-100 were used for controls, as above. Percentage hemolysis was calculated as(absorbance of peptide-treated minus vehicle-treated) x 100 / (absorbance of Triton X-100-treated minus vehicle-treated).

[0212] Inner membrane permeability assay: Bacterial cell membrane permeability was measured using the fluorescent dye propidium iodide (PI) in a black 96-well polystyrene plate. Briefly, mid-logarithmic growth-phase cultures of A. baumannii in modified MHB were washed with PBS and diluted in PBS to l x 106CFU / ml. PI at 5 pM final concentration was incubated with bacterial suspension, subsequently treated with the peptides at various concentrations for 30 minutes and 180 minutes. The cell membrane permeability was measured at the indicated time points using a Tecan microplate reader with 535 nm / 615 nm excitation / emission wavelengths.

[0213] Scanning Electron Microscopy imaging: Overnight grown cultures of A. baumannii and E. coli were incubated at 37°C in fresh MHB media and allowed to reach an ODeoo of 0.3. Peptides in modified MHB media (2x MIC) were mixed with an equal volume of bacterial suspension in an Eppendorf tube and incubated at 37°C for 2 hours. Vehicle control sample was prepared under the same conditions. Samples were then centrifuged at 6000 rpm for 5 minutes, the supernatant was removed, and pellet washed twice with PBS. The cell pellet was carefully submerged in glutaraldehyde solution (2.5% in PBS) and fixed at 4°C overnight. The fixative solution was then carefully removed, the pellet washed with PBS two times, water two times, and then subjected to dehydration with ethanol in gradient fashion (25%, 50%, 75%, 90% and 100%). Samples were dried using a critical point dryer, mounted on the carbon coated stubs, and sputter coated before imaging under a field emission scanning electron microscope (Hitachi S-4700).

[0214] Transmission Electron Microscopy imaging: overnight grown cultures of A. baumannii were processed, treated with peptides and fixed as described for SEM.Samples were post-fixed with 1% osmium tetroxi de / 1.5% potassium ferrocyanide for 60 min at room temperature. After extensive washing with distilled water, samples were incubated for 60 minutes with 1% uranyl acetate. After washing with distilled water, samples were dehydrated with ethanol in gradient fashion (25%, 50%, 75%, 90% and 100%) and embedded in epon mixed propylene oxide (1:1) resin overnight at 4°C.Ultrathin sections were prepared using a glass knife (ultramicrotome), post stained with uranyl acetate, and imaged using a JEOL 1200EX equipped with an AMT XR111 (11 megapixel) CCD camera.

[0215] Protease resistance study: Protease resistance of the peptides was assessed by A. baumannii growth inhibition assay. The two-fold serially diluted concentration of proteinase K (from 0.1 to 0.00002 mg / mL) in PBS buffer were incubated with peptides at MIC final concentration for 1 hour at 37°C in a 96-well plate. After treatment of peptides with the enzyme, bacterial cultures adjusted to 1 x 106CFU / ml were added, and the plates incubated overnight. The plate was read at OD 600 nm to determine bacterial growth inhibition.

[0216] Killing kinetics: A. baumannii bacterial culture from the exponential phase was adjusted to 1 x 106CFU / ml in modified MHB. The cultures were then treated with peptides (final MIC concentration) and incubated at 37°C for 0, 0.5, 1, 3, and 6 hours (Buf WT and Buf(i+4)13) and 0, 0.08, 0.25, 0.5, 1, and 6 hours (Buf(i+4)12). At the specified time intervals, samples were serially diluted ten-fold and plated on MHB-agar plates. The colonies were counted after overnight incubation to determine CFU / ml. An untreated inoculum group was used as the negative control.

[0217] RNAseq profiling: A. baumannii bacterial culture from the exponential phase was adjusted to 1 x 108CFU / ml and treated with vehicle or the MIC concentration of Buf(i+4)13 or Buf(i+4)13 Q9K for 0 minutes and 30 minutes. After treatment for the specified time, the A. baumannii culture was immediately pelleted at 5,000 x g for 5 minutes at 4°C, resuspended in 1 ml IX DNA / RNA Shield (Zymo Research, Irvine, CA), and stored at -80°C. RNA was extracted from 800 pl of preserved ATCC 19606 A. baumannii using the Quick-RNA MiniPrep Plus Kit (Zymo Research, Irvine, CA) and lysed with IX DNA / RNA Shield at full speed for 15 minutes on a Vortex Genie 2 with a microcentrifuge adapter (Scientific Industries, Bohemia, NY). The RNA was eluted in 50 pl nuclease-free water and 40 pl was purified with the RNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA). RNA quality was assessed using the Agilent RNA 6000 Pico kit (Agilent Technologies, Waldbronn, Bermany) with samples having an average RNA integrity value of 9.2. cDNA libraries were generated with the Zymo-Seq RiboFreeTotal RNA Library Kit with a 3.5 hour depletion incubation and 16-cycle library amplification. The individual libraries were diluted to 4 nM to generate an equimolar pool for sequencing. A 12 pM pooled library was sequenced with the MiSeq v3 150-cycle kit.

[0218] Localization of FITC-labeled peptide in bacteria: Mid-log phase bacterial cultures (ODeoo = 0.2) grown in MHB were resuspended in PBS containing FITC-labeled Buf(i+4)13 (Q9K) at a final concentration of lx MIC. Following incubation for 0, 60, and 180 min at 37 °C, samples were washed with PBS and stained with 10 mg / mL FM4-64 membrane dye for 15 min in the dark. After washing with PBS, 10 mL of stained cells were spotted onto microscope slides mounted with a 2% agarose pad and imaged using a Spinning Disk Nikon Ti2 confocal laser microscope (Yokogawa CSU-W1, with sequential scanning of FITC (488 nm excitation, 500-530 nm emission) and FM4-64 (543 nm excitation, 555-655 nm emission).

[0219] Ethidium bromide displacement assay: DNA binding was assessed using a fluorescence-based ethidium bromide (EtBr) displacement assay with genomic DNA from A baumannii (ATCC 19606) and 7 / . coli (ATCC 25922), purified using the Promega Wizard Genomic DNA Purification Kit. Genomic DNA was diluted to 20 mg / mL in HEPES buffer (10 mM, pH 7.4). EtBr was added to the DNA solution at a final concentration of 4 pM to prepare a 2x EtBr-DNA mixture. The mixture was then incubated at room temperature for 15 min to allow for EtBr intercalation. Peptides were serially diluted in HEPES buffer (0.04-10 pM) in black 96-well microplates, and 50 pL of the EB-DNA complex was added to 50 pL of each peptide dilution. After a 15-min incubation, fluorescence was measured using a Tecan Spark plate reader (535 nm excitation, 615 nm emission). Controls included ErBr alone, EtBr+DNA, and peptide alone.

[0220] Bacterial biofilm eradication assay: To form mature biofilms, 100 pL of fl. baumannii (ATCC 19606) cultures (grown overnight) were adjusted to IxlO6CFU / mL in modified MHB and loaded into polystyrene 96-well plates, followed by incubation at 37 °C for 48 h. The wells were gently washed twice with sterile distilled water to removeplanktonic cells, and fresh MHB containing serially diluted peptides was added for an additional 24-h treatment. In parallel, biofilm eradication assays were also conducted in the presence of MIC levels of polymyxin B and ciprofloxacin under identical conditions. After treatment, the wells were washed, stained with 0.1% crystal violet for 15 min, and excess dye removed by washing. Stained biofilms were solubilized in 30% glacial acetic acid and absorbance at 560 nm was measured using a SpectraMax M5 microplate reader. Biofilm eradication was calculated by comparing absorbance of peptide-treated versus vehicle-treated wells.

[0221] Antibiotic synergy testing: To evaluate the synergistic potential of the Buf(i+4)13 Q9K in combination with the antibiotics, polymyxin B, ciprofloxacin, Indolicidin and gentamicin, A. baumannii cells were treated and prepared as described for the MIC assay. Drug dilution plates were prepared separately for each peptide-drug combination prior to mixing. A fourfold dilution series of Buf(i+4)13 Q9K and the other antibiotic was dispensed into a 96-well plate across the rows, followed by addition of an equal volume (50 pL) of A. baumannii suspension (final concentration 1 * 10® CFU / mL). After overnight incubation, FICI values were calculated. The FIC index was calculated using the following equation, where CA and CB are the concentrations of compounds A and B, respectively, and MICA and MICB are the MICs of compounds A and B alone, respectively.

[0222] FIC index = CA / MICA + CB / MICB

[0223] The combination is synergistic if FICI is below 0.5, antagonistic if FICI is above 4, and indifferent if FICI is between 0.5 and 1.

[0224] RNAseq transriptome profiling: A. baumannii bacterial culture from the exponential phase was adjusted to 1 x 108CFU / mL and treated with the MIC concentration of Buf(i+4)13 for 0 min and 30 min. After treatment for the specified time, the A. baumannii culture was immediately pelleted at 5,000 x g for 5 min at 4°C, resuspended in 1 mb IX DNA / RNA Shield (Zymo Research, Irvine, CA), and stored at -80°C. RNA was extracted from 800 pL of preserved ATCC 19606^4. baumannii using the Quick-RNA MiniPrep Plus Kit (Zymo Research, Irvine, CA) and lysed with IXDNA / RNA Shield at full speed for 15 min on a Vortex Genie 2 fitted with a microcentrifuge adapter (Scientific Industries, Bohemia, NY). The RNA was eluted in 50 uL nuclease-free water and 40 pL was purified with the RNA Clean and Concentrator-5 kit (Zymo Research, Irvine, CA). RNA quality was assessed using the Agilent RNA 6000 Pico kit (Agilent Technologies, Waldbronn, Bermany) with samples having an average RNA integrity value of 9.2. cDNA libraries were generated with the Zymo-Seq RiboFree Total RNA Library Kit, which included a 3.5 h ribosomal RNA depletion step and 16-cycles of PCR amplification. Final libraries were diluted to 4 nM, pooled equimolarly, and sequenced on an Illumna MiSeq using the v3 150-cycle kit.

[0225] Resistance acquisition assay: Acquired resistance was studied in A. baumannii (ATCC 19606) by sequential passage in sub-MIC levels of antimicrobial peptides over 30 days. Cultures were prepared in 96-well plates containing peptides at 0.25x, 0.5x, lx, 2x, and 4x MIC concentrations in modified MHB. After overnight incubation at 37 °C, 5 pL from wells with visible growth (typically at 0.5x MIC) was transferred into fresh wells containing the same peptide concentration series. Peptide concentrations were increased when MIC shifts were observed. This serial passage process was repeated daily for 30 passages.

[0226] Proteomic differential expression and pathway enrichment analysis:Differential protein expression at each time point relative to the vehicle-treated, non-resistant control (NR) strain was assessed using the limma package (v3.60.6) on normalized protein abundance values. Proteins exhibiting a fold change greater than ±1.5 and a Benjamini-Hochberg adjusted p value <0.05 were considered significantly differentially expressed. Pathway over-representation analysis (ORA) of Gene Ontology (GO) Biological Process and Molecular Function terms, annotated via UniProt, was performed using the topGO package (v2.56.0). GO terms were considered significantly enriched at an adjusted p value <0.05. For Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, ORA was conducted using the enrichKEGG function from the ClusterProfiler package (v4.12.6). KEGG annotations were derived from the baumannii ACICU strain (KEGG code: abc), as no KEGG annotations were available for the ATCC 19606 strain used in the experimental assays. Of the 2,870 proteins analyzed,KEGG terms were available for 777 proteins. KEGG pathways were considered significantly enriched at an adjusted p value of <0.2. In addition to ORA, gene set enrichment analysis (GSEA) was performed using the gseKEGG function from ClusterProfiler, which incorporates both gene identity and fold change information to detect pathway-level enrichment. GSEA was also based on KEGG annotations from the ACICU strain and used the same statistical threshold (p <0.2). All analyses were conducted in R (v4.4.0), and visualizations were generated using ggplot2 (v3.5.1).EXAMPLE 3. ANTIFUNGAL STAPLED BUFORIN II PEPTIDES

[0227] Antibacterial peptides, such as Buforin II (FIG. 1) can have antifungal activity with unique and unpredictable selectivity. Stapled and mutated Buforin II peptides with enhanced antifungal properties were developed, with a focus on achieving maximal potency and selectivity while avoiding mammalian cell cytotoxicity. Buforin II stapled peptides, including staple scans, and Buf(i+4)12 and Buf(i+4)13 lysine mutation scans (FIG. 2), were tested for antifungal activity against C. albicans, C. auris and C. neoformans (FIGS. 27-29). The stapled peptides showed marked improvement in potency compared to Buforin WT (FIGS.27-29), with Buf(i+4)12 exhibiting notably potent antifungal activity, inhibiting C. albicans, C. auris, and C. neoformans at 0.4, 0.8, and 0.4 pM, respectively, while maintaining moderate mammalian cell toxicity (FIG. 27). Lysine mutants V15K (SEQ ID NO:33) and L19K (SEQ ID NO:36) of Buf(i+4)12 showed strikingly little to no toxicity in RBCs, HUVECs and RPTECs, yet exhibited potent antifungal activity, revealing an excellent therapeutic window for these constructs (FIG. 28). Likewise, a series of Buf(i+4)13 lysine mutant constructs showed markedly improved anti-fungal potency as compared to the parent Buf(i+4)13 compound, and with little to no hemolysis or cytotoxicity (FIG. 29). Of the various stapled and mutated Buforin II peptides, Buf(i+4)12 T1K (SEQ ID NO:20) showed the most potent Candida and Cryptococci killing at 0.1 pM, even more potent than the amphotericin B positive control anti-fungal drug, although significant mammalian cell cytotoxicity was observed (FIG. 28). Given the anti-fungal potency but non-selective cytotoxicity of Buf(i+4)12T1K, V15K and / or L19K substitutions were further incorporated, including without T1K, as shown in FIG. 30. The indicated peptides not only retained potent antifungal properties but also showed elimination of hemolysis and of HUVEC and RPTEC cytotoxicity. Potent activity against the C. auris and C. neoformans species of Candida was also evident for these stapled and doubly or triply lysine-mutated Buforin II peptides. It was further found that these doubly and triply lysine-mutated Buf(i+4)12 peptides were remarkably selective antifungal agents, with much weaker to no activity observed in the gram-negative bacterial strains A. baumannii, E. coll, and P. aeruginosa (FIG.31).

[0228] To further evaluate the membrane impact and selectivity of Buf(i+4)12 V15K,L19K, scanning electron microscopy (SEM) was conducted on treated fungal (C. albicans)', bacterial (A. baumannii), and mammalian (HUVEC) cells, comparing results to Buf(i+4)12 T1K, Buf(i+4)12 T1K,V15K, and Amphotericin B (FIG.32). FIG. 32 shows that Buf(i+4)12 V15K,L19K caused notable fungal membrane damage and structural disruptions, in contrast to untreated cells, which displayed intact membranes.Importantly, this peptide induced no membrane disruptions in A. baumannii or HUVEC cells, highlighting its selective fungicidal action. In contrast, the T1K substitution induced lysis across fungal, bacterial, and mammalian cells (FIG.32). Additionally, a propidium iodide (PI) assay conducted at 1-hour and 3-hour intervals indicated that Buf(i+4)12 V15K,L19K caused a dose-dependent increase in fungal membrane permeability (FIG. 33), similar to that observed with Amphotericin B, a positive control for this assay. Buf WT, however, showed no increase in fluorescence at 1 hour but did achieve membrane permeability by 3 hours (FIG. 33).

[0229] To investigate the potential for peptide binding to specific lipid components of the fungal membrane, MIC assays of Buf(i+4)12 V15K,L19K in the presence of ergosterol, cholesterol, phosphatidylcholine, phosphatidylethanolamine, chitin, and 0-glucan were performed (FIG. 34), comparing results to those of Buf WT and Amphotericin B. The MIC assays revealed that Buf(i+4)12 V15K,L19K specifically binds to 0-glucan, as indicated by a 16-fold change in MIC (FIG.34). As expected, Amphotericin B bound to ergosterol and to a lesser degree to cholesterol, while Buforin WT showed no effect of these added components on its overall relatively weaker MIC(FIG. 34). Strikingly selective and dose-dependent binding of Amphotericin B to ergosterol and Buf(i+4)12 V15K,L19K to P-glucan was further observed, based on the competitive influence of these added lipid components on the corresponding MICs (FIG.35). This unexpected finding indicates that stapled antimicrobial peptides, such as Buf(i+4)12 V15K,L19K, selectively bind P-glucan, providing a mechanistic explanation for the observed selectivity and potency of fungal targeting and killing.[00230J Hemolytic and cytotoxicity profiles of stapled Buf(i+4)12 analogues: To evaluate the mammalian cytotoxicity of the stapled Buf(i+4)12 analogues, hemolysis of human red blood cells (FIG. 36A) and cytotoxicity in HUVEC (FIG.36B) and RPTEC (FIG. 36C) cells were measured. All four variants showed negligible hemolytic activity up to 100 pM, indicating minimal disruption of erythrocyte membranes. In HUVECs, the Buf(i+4)12 T1K, V15K and Buf(i+4)12 T1K, L19K analogues exhibited dose-dependent cytotoxicity, whereas triple-substituted Buf(i+4)12 T1K, V15K, L19K was markedly less toxic and Buf(i+4)12 V15K, L19K showed no cytotoxicity. In RPTECs, all peptides were well tolerated, with little to no cytotoxicity observed even at the highest concentrations tested.

[0231] DNA-dependent sequestration of stapled Buf II peptides reveals strong DNA-binding by Buf(i+4)12 VI 5K, L19K: To assess the functional impact of DNA binding, MICs were determined in the presence of exogenously added bacterial genomic DNA (FIGS. 37A-C). For amphotericin B (Amp B), the presence of DNA had minimal effect on antibacterial activity, consistent with weak or negligible DNA interaction (FIG. 37A).In contrast, Buf WT showed a clear rightward shift in MIC in the presence of DNA, indicating partial sequestration by nucleic acid (FIG.37B). The most pronounced effect was observed for Buf(i+4)12 V15K, L19K, where the addition of DNA caused a substantial loss of activity, reflected by a marked increase in MIC (FIG. 37C) and consistent with strong peptide-DNA binding. These data indicate that peptides exhibiting the largest DNA-dependent MIC shifts display the strongest functional DNA sequestration, with Buf(i+4)12 V15K, L19K showing the highest apparent DNA-binding capacity among the tested peptides.

[0232] Buf(i+4)12 V15K, L19K efficiently eradicates 48-h mature C. albicans and C. neoformans biofilms: To evaluate activity against mature fungal biofilms, 48 h-established biofilms of C. albicans and C. neoformans were treated with Buf WT, Buf(i+4)12 V15K, L19K, and Amp B (FIG. 38A, FIG. 38B). Buf WT showed limited biofilm eradication, with substantial residual biomass even at the highest concentrations tested. In contrast, Buf(i+4)12 V15K, L19K displayed markedly enhanced biofilmdisrupting activity, producing a strong, concentration-dependent reduction in biofilm mass in both C. albicans and C. neoformans . Amp B served as a positive control and efficiently reduced mature biofilms, although higher concentrations were required to achieve comparable effects. These data indicate that stapling and dual Lys substitutions substantially enhance the ability of Buf II peptides to eradicate mature fungal biofilms.

[0233] Protease resistance of Buf(i+4)12 V15K, L19K: The impact of proteinase K treatment on the antifungal activity of Buf WT and Buf(i+4)12 VI 5K and L19K was evaluated (FIG.39). Buf WT was highly sensitive to proteinase K, showing complete inactivation at the lowest tested enzyme concentration of 0.002 mg / ml. In contrast, the Buf(i+4)12 V15K, L19K displayed resistance to enzymatic degradation / inactivation, requiring concentrations of 0.1 mg / ml or higher of proteinase K to achieve inactivation.

[0234] Methods used in Example 3

[0235] Antifungal activity testing: Candida albicans, Candida auris. and Cryptococcus neoformans were sourced from ATCC, USA. The strains were stored as glycerol stocks at -80°C and -20°C. For routine use, fungi were plated on YPD agar plates. The assays were done using 96-well polypropylene plates following standard literature procedures. Briefly, overnight cultures were grown at 37°C in modified YMB media until a turbidity of 0.08-0.13 at 625 nm (1 x 108CFU / mL) was obtained. Peptide stock solutions were prepared in modified YMB media and then further diluted to achieve a range of concentrations. The antifungal assay was performed by adding diluted cultures (1 x 106CFU / ml) to the peptide solution in each well of the plates. The plates, which also contain suitable growth and sterility controls, were then incubated at 37°C for 24 hours to determine MIC. The antifungal activity of the peptides was compared to the positive control, amphotericin B. Optical density measurements at 600 nm fordetermination of MIC were performed using a spectrophotometer plate reader. MIC is reported as the concentration at which no visible growth was observed. Three biological replicate experiments were performed, each with three technical replicates, to determine the average MIC values. Amphotericin B served as the antifungal control.

[0236] Cytotoxicity assay was performed as described in Example 2.

[0237] Hemolysis was assayed as described in Example 2.

[0238] Inner Membrane Permeability Assay: Membrane permeability in C. albicans was assessed using propidium iodide (PI) as a fluorescent indicator in a black 96-well polystyrene plate. Mid-logarithmic phase C. albicans cultures in modified YMB were washed in PBS and diluted to 1 x 106CFU / mL. PI was added to the fungal suspension at a final concentration of 5 pM, following treatment with peptides at various concentrations for 1 and 3 hours. Fluorescence was measured on a Tecan microplate reader with excitation / emission wavelengths set at 535 nm / 615 nm.

[0239] Scanning Electron Microscopy (SEM) Imaging: Overnight cultures of C. albicans (grown in YMB media) and A. baumannii (grown in MHB media) were incubated at 37°C in fresh media until reaching an OD600 of 0.3. Peptide solutions prepared in modified media (at 2x MIC) were mixed with an equal volume of fungal or bacterial culture in Eppendorf tubes and incubated at 37°C for 3 hours. Controls were prepared using drug-free media. Following incubation, samples were centrifuged at 6000 rpm for 5 minutes, supernatants were removed, and pellets were washed twice with PBS. Cells were fixed in 2.5% glutaraldehyde (in PBS) at 4°C overnight, washed twice with PBS and twice with water, and dehydrated using a graded ethanol series (25%, 50%, 75%, 90%, and 100%). Dried samples were mounted on carbon-coated stubs and sputter-coated before imaging with a field emission scanning electron microscope (Hitachi S-4700). For imaging HUVEC cells, cells were adhered to poly-lysine coated coverslips, treated with peptides or amphotericin B for 90 minutes, then fixed and processed as described above.

[0240] Minimum Inhibitory Concentration (MIC) Assay in the Presence of Fungal Membrane Components: MIC assays in the presence of fungal membrane componentswere conducted following the standard MIC protocol. Briefly, overnight fungal cultures were grown in modified YMB media at 37°C to a turbidity of 0.08-0.13 at 625 nm (1 x 108CFU / mL). Peptide stock solutions (4x MIC concentration) were prepared and diluted to yield a range of concentrations. Fungal membrane components, including ergosterol, cholesterol, phosphatidylcholine, phosphatidylethanolamine, chitin, and P-glucan, were added at a final concentration of 100 mg / mL and incubated with the peptides for 1 hour prior to the assay. Diluted fungal cultures (1 x 106CFU / mL) were then added to each well. Plates, containing appropriate growth and sterility controls, were incubated at 37°C for 24 hours, and optical density at 600 nm was measured. MIC values, defined as the concentration with no visible growth, were determined in three separate experiments, each performed in triplicate, and averaged across experiments.

[0241] Fungal biofilm eradication assay: To form mature biofilms, 100 mL of C. albicans and C. neoformans cultures (grown overnight) were adjusted to 1x10sCFU / mL in RPMI 1640 and loaded into polystyrene 96-well plates, followed by incubation at 37 °C for 48 h. The wells were gently washed twice with sterile distilled water to remove planktonic cells, and fresh RPMI 1640 containing serially diluted peptides was added for an additional 24-h treatment. After treatment, the wells were washed, stained with 0.1% crystal violet for 15 min, and excess dye removed by washing. Stained biofilms were solubilized in 30% glacial acetic acid and absorbance at 560 nm was measured using a SpectraMax M5 microplate reader. Biofilm eradication was calculated by comparing absorbance of peptide-treated versus vehicle-treated wells.

[0242] Protease resistance study: Protease resistance of the peptides was assessed by C. albicans growth inhibition assay. The two-fold serially diluted concentration of proteinase K (from 0.4 to 0.002 mg / mL) in PBS buffer were incubated with peptides at MIC final concentration for 1 h at 37°C in a 96-well plate. After treatment of peptides with the enzyme, fungal cultures adjusted to 1 x 106CFU / mL were added, and the plates incubated overnight. The plate was read at OD 600 nm to determine fungal growth inhibition.EXAMPLE 4. ANTICANCER STAPLED BUFORIN II PEPTIDES

[0243] An i, i+4 staple scanning library of Buforin II (FIG. 2) was screened at a 10 pM dose against a panel of diverse cancer cell subtypes, including 0CI-AML3, K562, MV4;11, U937, SJSA-1, and A549 cells, and a series of non-transformed mammalian cells (HUVEC, RPTEC, RBC) to evaluate the effect of treatment on cellular lysis at 90 minutes by LDH release assay for all cells except RBCs, which were evaluated by absorbance assay (FIG. 40). Whereas some of the stapled positions yielded peptides with little to no lytic effect on cancer or non-transformed mammalian cells (e.g., Buf(i+4) 0, 1, 2, 4, 5, 6, 8, 10, 13, 15, 17 (SEQ ID NOs:2-4, 6-8, 10, 12, 15, 17, 19, respectively)), other positions resulted in lytic peptides (e.g., Buf(i+4) 3, 7, 9, 11, 12, 14, 16 (SEQ ID NOs:5, 9, 11, 13, 14, 15, 18, respectively)) (FIG. 40). Certain peptides were selectively lytic to cancer cells but not to non-transformed mammalian cells (e.g., Buf(i+4) 3, 16 (SEQ ID NOs:5, 18, respectively)) (FIG. 40). In contrast, other peptides were non-specifically lytic to all of the cell types tested (e.g., Buf(i+4) 7, 9, 11, 12 (SEQ ID NOs:9, 11, 13, 14, respectively)) (FIG. 40).

[0244] Comparing the percent lytic activity and percent viability at 90 minutes and 24 hours of 0CI-AML3 cancer cells upon treatment with wild-type Buforin II (SEQ ID NO:1), Buf(i+4)12 (SEQ ID NO: 14, broadly lytic), Buf(i+4)13 (SEQ ID NO: 15, non-lytic), and the Q9K mutant of Buf(i+4)13 (SEQ ID NO:50, non-lytic), it was observed that the lytic Buf(i+4)12 (SEQ ID NO: 14) peptide caused prompt LDH release and impairment of cancer cell viability, reflective of a membrane-disruptive effect (FIGS. 41A-41D). Whereas the non-lytic wild-type Buforin II peptide had no lytic or cytotoxic effect at 90 minutes or 24 hours, the non-lytic Buf(i+4)13 (SEQ ID NO: 15) and Buf(i+4)13 Q9K (SEQ ID NO:50) peptides showed dose-responsive LDH release and cytotoxicity only at the 24 hour time point, reflective of an anti-cancer effect unrelated to a direct membrane lysis (FIGS. 41A-41D). Expanding the study to a selection of diverse hematologic cancer cells, differential dose-responsive susceptibility as measured by LDH release and cell viability assay at 24 hours after Buf(i+4)13 (SEQ ID NO: 15) treatment was observed, with SUDHL8 showing the most sensitivity, EOL-1 the least sensitivity, and OCI-AML3 moderate sensitivity (FIGS. 42A-42C). Overall, these data indicate thatsubstitution of native residues with the all -hydrocarbon staple at distinct positions and lysine mutagenesis at distinct positions differentially effected the selectivity of the stapled Buforin II peptides for cells, generating a spectrum of activities from non-specific lytic activity, selective lytic activity, to no lytic activity, with differential outcomes on the type of cytotoxicity, ranging from early non-specific cytotoxicity to delayed dose-responsive cytotoxicity that does not derive from a direct membrane lytic effect.[00245J Buf(i+4)13 (SEQ ID NO: 15), which had no direct membrane-lytic effect, was selected for Profiling Relative Inhibition Simultaneously in Mixtures (PRISM) analysis (Broad Institute). This technology allows for high-throughput multiplexed screening of over 900 robustly characterized (genomics, proteomics, metabolomics, drug susceptibilities) cancer cell lines. Buf(i+4)13 (SEQ ID NO: 15) treatment demonstrated a range of dose-responsive activity upon cancer cell exposure for 5 days, as assessed by cell viability assay (FIG. 43A). The lineage-based sensitivity profiles of cancer cells to Buf(i+4)13 revealed that ovarian, skin and lung cancers were among the most susceptible, and cancers of the bowel, colon and lymphoid systems were relatively less susceptible (FIGS. 43B-43C). The ovarian and skin cancer subsets demonstrated dose-responsive reduction in cell viability (FIGS. 43D-43E). Intriguingly, when comparing the overall cancer cell susceptibility profile of Buf(i+4)13 (SEQ ID NO: 15) to a broad spectrum of drugs, profiles that correlated and anti -correlated with Buf(i+4)13 (SEQ ID NO: 15) were discerned (FIG. 44A). The most highly correlated drugs included a spectrum of agents with DNA-targeting mechanisms of action (FIG. 44B), reinforcing that Buf(i+4)13 manifests broad anti-cancer properties with a specific, non-membrane lytic, mechanism of action.

[0246] Methods used in Example 4

[0247] Cell Culture and Conditions: RPTECs sourced from Sigma (SA7K Clone) were maintained in MEM (Invitrogen) supplemented with RPTEC complete supplement (Sigma). OCLAML3 (DSMZ, Germany), EOL1 (Sigma), K562, SJSA1, SW1463, MV4-11, U937, A549 and HUVEC (ATCC, USA) were maintained in RPMI supplemented with 10% FBS and 1% each of streptomycin, penicillin, and L-glutamine. Cells were verified mycoplasma free by the My coAlert mycoplasma detection kit (Lonza Biologies).

[0248] Cell Lysis, Cell Viability and Hemolysis Assay: Mycoplasma free cells were plated at 2xl04cells / mL in 96-well format. Non-adherent cancer cells were immediately used for screening, whereas adherent cells are further incubated for 24 hours at 37 °C without agitation. For the Lactate Dehydrogenase (LDH) assay, cells were treated with a screening dose (e.g., 10 pM) of stapled Buforin peptides for 90 minutes or 24 hours, followed by centrifugation of the plates, transfer of 50 pL media from each well to a new plate, incubation with 50 pL of LDH reagent for 10 minutes, and recording absorbance at 490 nm on a microplate reader. Vehicle and 1% Triton X-100 served as negative and positive controls, respectively. For Cell Titer Gio viability assay, cells were plated and treated as above for the LDH assay for 90 minutes or 24 hours, the plate and its contents then equilibrated at room temperature for approximately 30 minutes, followed by addition of equal volume i.e.,50 pL of CellTiter-glo reagent (Promega), with mixing for 2 minutes on an orbital shaker to induce cellular lysis. Luminescence was then recorded after 30 minute incubation. For quantitation of RBC lysis, whole blood cells (Innovative Research) were centrifuged at lOOOxg for 5 minutes to the remove buffy coat and isolate RBCs. RBCs (pellet) are washed thrice with PBS and resuspended in PBS to a final concentration of 1% v / v. The cell suspension was then added to a Buforin II peptide solution prepared in PBS to achieve a final concentration of 10 pM in 96-well plates, which were then incubated for 1 hour at 37 °C without agitation. The plates were centrifuged at 3500xg for 10 minutes, supernatant (100 pL) transferred to new plates, and absorbance measured at 540 nm. Vehicle and 1% Triton X-100 were used for controls, as above. Percentage hemolysis was calculated as (absorbance of drug treated-treated minus vehicle-treated) x 100 / (absorbance of Triton X-100-treated minus vehicle-treated).

[0249] PRISM Analysis: For PRISM analyses, Buf(i+4)13 was submitted for PRISM analysis as a stock solution of 5 mM in PBS for 8-point serial 3 -fold dilutions from 20 pM into barcoded and pooled cancer cells (>900 genomically characterized cell lines across 45 cancer lineages). Cells were treated for 5 days in triplicate with each plate containing both positive and negative benchmark controls. To determine differential sensitivity profiles, the entire dataset was parsed by cancer type and mean sensitivity of cell lines for each cancer type was computed at each dose (log2 fold change in viabilityfor peptide treatment vs PBS). Two PRISM cell line collections were used in the assay, PR500 (including only adherent cell lines), and PR300+ (including adherent and suspension cell lines). Cell-lines were maintained in RPMI-1640 media without phenol red and supplemented with fetal bovine serum (FBS) at 10% for adherent cell lines and at 20% for suspension cell lines. All standard PRISM assay 384-well plates were prepared by seeding adherent pools at 1,250 cells per well and suspension pools at 2,000 cells per well. PRISM provided a detailed report of the quality of the assay, viability of 900 celllines, feature set correlation analysis, and the validation compound reports.

Claims

1. WHAT IS CLAIMED IS:

1. A stapled peptide comprising the structure of formula (I):Formula (I)or a pharmaceutically acceptable salt thereof, wherein:each Xaa is independently an amino acid;each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclyl alkyl, any of which is substituted or unsubstituted, optionally wherein each Ri and R2 is a methyl;each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted, optionally wherein R3 is Cs alkylene, Cs alkenylene, or C& alkynylene, optionally wherein R3 is (CH2)3-CH=CH-(CH2)3; andwherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 3 to 6 substitutions relative to the sequence TRSSRAGLQWPVGRVHRLLRK (SEQ ID NO: 1),wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 1 , 3, 5, 6, 7, 8, 9, 11, 12, 15, 18, 19, or 20 of the sequence of SEQ ID NO: 1, andwherein two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at (i) positions 12 and 16 of the sequence of SEQ ID NO: 1, or (ii) positions 13 and 17 of the sequence of SEQ ID NO: 1;wherein [Xaa]xis (i) positions 13-15 of the sequence of SEQ ID NO:1, optionally comprising one or two amino acid substitutions, or (ii) positions 14-16 of the sequence of SEQ ID NO: 1, optionally comprising one or two amino acid substitutions; andwherein the stapled peptide, or the pharmaceutically acceptable salt thereof, exhibits an antimicrobial effect against at least one microbe, optionally wherein the at least one microbe is E. coli, or wherein the stapled peptide, or the pharmaceutically acceptable salt thereof exhibits an anticancer effect.

2. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 1, wherein:Ri is methyl,R3is (CH2)3-CH=CH-(CH2)3, andR2is methyl.

3. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 1 or 2, wherein:(a) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:63), [Xaa]xconsists of the sequence GRV, and[Xaa]yconsists of the sequence RLLRK (SEQ ID NO:85);(b) [Xaa]wconsists of the sequence TRSSRAGLQWP (SEQ ID NO:64), [Xaa]xconsists of the sequence GRK, and[Xaa]yconsists of the sequence RLLRK (SEQ ID NO:86);(c) [Xaa]wconsists of the sequence TRSSRAGLQWP (SEQ ID NO:65), [Xaa]xconsists of the sequence GRV, and[Xaa]yconsists of the sequence RLKRK (SEQ ID NO:87);(d) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:66), [Xaa]xconsists of the sequence GRK, and[Xaa]yconsists of the sequence RLLRK (SEQ ID NO:88);(e) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:67),[Xaa]xconsists of the sequence GRV, and[Xaa]yconsists of the sequence RLKRK (SEQ ID NO:89);(f) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:68), [Xaa]xconsists of the sequence GRK, and[Xaa]yconsists of the sequence RLKRK (SEQ ID NO:90);(g) [Xaa]wconsists of the sequence TRSSRAGLQWP (SEQ ID NO:69), [Xaa]xconsists of the sequence GRK, and[Xaa]yconsists of the sequence RLKRK (SEQ ID NO:91);(h) [Xaa]wconsists of the sequence KRSSRAGLQWPV (SEQ ID NQ:70), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:92);(i) [Xaa]wconsists of the sequence TRKSRAGLQWPV (SEQ ID NO:71), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 93);(j) [Xaa]wconsists of the sequence TRSSKAGLQWPV (SEQ ID NO:72), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:94);(k) [Xaa]wconsists of the sequence TRSSRKGLQWPV (SEQ ID NO:73), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:95);(l) [Xaa]wconsists of the sequence TRSSRAKLQWPV (SEQ ID NO:74), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 96);(m) [Xaa]wconsists of the sequence TRSSRAGKQWPV (SEQ ID NO:75), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:97);(n) [Xaa]wconsists of the sequence TRSSRAGLKWPV (SEQ ID NO.76), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:98);(o) [Xaa]wconsists of the sequence TRSSRAGLQWKV (SEQ ID NO:77), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:99);(p) [Xaa]wconsists of the sequence TRSSRAGLQWPK (SEQ ID NO:78), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 100);(q) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:79), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence KLRK (SEQ ID NO: 101);(r) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:80), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LKRK (SEQ ID NO: 102);(s) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:81), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLKK (SEQ ID NO: 103);(t) [Xaa]wconsists of the sequence KRSSRAGLKWPV (SEQ ID NO:82), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 104);(u) [Xaa]wconsists of the sequence TRKSRAGLKWPV (SEQ ID NO:83), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 105); or (v) [Xaa]wconsists of the sequence TRSSRKGLKWPV (SEQ ID NO:84), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 106).

4. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 1 or 2, wherein:(a) two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 12 and 16 of the sequence of SEQ ID NO: 1, wherein [Xaa]xcorresponds to positions 13-15 of the sequence of SEQ ID NO: 1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 1, 15, or 19 of the sequence of SEQ ID NO : 1; or(b) two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 13 and 17 of the sequence of SEQ ID NO:1, wherein [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 3, 5, 6, 7, 8, 11, 12, 18, 19, or 20 of the sequence of SEQ ID NO: 1.

5. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 1 or 2, wherein:(a) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:66), [Xaa]xconsists of the sequence GRK, and[Xaa]yconsists of the sequence RLLRK (SEQ ID NO:88);(b) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:67), [Xaa]xconsists of the sequence GRV, and[Xaa]yconsists of the sequence RLKRK (SEQ ID NO: 89);(c) [Xaa]wconsists of the sequence KRSSRAGLQWP (SEQ ID NO:68), [Xaa]xconsists of the sequence GRK, and[Xaa]yconsists of the sequence RLKRK (SEQ ID NO:90);(d) [Xaa]wconsists of the sequence TRSSRAGLQWP (SEQ ID NO:69), [Xaa]xconsists of the sequence GRK, and[Xaa]yconsists of the sequence RLKRK (SEQ ID NO:91);(e) [Xaa]wconsists of the sequence TRKSRAGLQWPV (SEQ ID NO:71),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:93);(f) [Xaa]wconsists of the sequence TRSSKAGLQWPV (SEQ ID NO:72),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:94);(g) [Xaa]wconsists of the sequence TRSSRKGLQWPV (SEQ ID NO:73),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:95);(h) [Xaa]wconsists of the sequence TRSSRAKLQWPV (SEQ ID NO:74),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:96);(i) [Xaa]wconsists of the sequence TRSSRAGKQWPV (SEQ ID NO:75),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:97);(j) [Xaa]wconsists of the sequence TRSSRAGLQWKV (SEQ ID NO:77), [Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:99);(k) [Xaa]w consists of the sequence TRSSRAGLQWPK (SEQ ID NO:78),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 100);(l) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:79),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence KLRK (SEQ ID NO: 101);(m) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:80),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LKRK (SEQ ID NO: 102); or (n) [Xaa]wconsists of the sequence TRSSRAGLQWPV (SEQ ID NO:81),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLKK (SEQ ID NO: 103).

6. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 1 or 2, wherein two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 13 and 17 of the sequence of SEQ ID NO:1, wherein [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1, and wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 is with a lysine at position 1, 3, 6, or 9 of the sequence of SEQ ID NO: 1.

7. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 1 or 2, wherein:(a) [Xaa]wconsists of the sequence KRSSRAGLQWPV (SEQ ID NO:70),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:92);(b) [Xaa]wconsists of the sequence TRKSRAGLQWPV (SEQ ID NO:71),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:93);(c) [Xaa]wconsists of the sequence TRSSRKGLQWPV (SEQ ID NO:73),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:95);(d) [Xaa]w consists of the sequence TRSSRAGLKWPV (SEQ ID NO:76),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:98);(e) [Xaa]wconsists of the sequence KRSSRAGLKWPV (SEQ ID NO:82),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 104);(f) [Xaa]wconsists of the sequence TRKSRAGLKWPV (SEQ ID NO:83),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 105); or (g) [Xaa]wconsists of the sequence TRSSRKGLKWPV (SEQ ID NO:84),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO: 106).

8. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 1 or 2, wherein two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are at positions 13 and 17 of the sequence of SEQ ID NO:1, wherein [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1, and wherein one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO:1 is with a lysine at position 9 of the sequence of SEQ ID NO: 1.

9. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 1 or 2, wherein:[Xaa]wconsists of the sequence TRSSRAGLKWPV (SEQ ID NO:76),[Xaa]xconsists of the sequence RVH, and[Xaa]yconsists of the sequence LLRK (SEQ ID NO:98).

10. A stapled peptide comprising the sequence of any one of SEQ ID NOs:20, 33, 36, 38-42, 44, 46-50, 52, 53, and 57-62, except for 0, 1, 2 or 3 amino acid substitutions relative to the sequence of any one of SEQ ID NOs:20, 33, 36, 38-42, 44, 46-50, 52, 53, and 57-62, respectively, or a pharmaceutically acceptable salt thereof,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:20, the amino acid substitutions are not at any of positions 1, 12, and 16 of SEQ ID NO:20,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:33, the amino acid substitutions are not at any of positions 12, 15, and 16 of SEQ ID NO:33,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:36, the amino acid substitutions are not at any of positions 12, 16, and 19 of SEQ ID NO:36,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:38, the amino acid substitutions are not at any of positions 1, 12, 15, and 16 of SEQ ID NO:38,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:39, the amino acid substitutions are not at any of positions 1, 12, 16, and 19 of SEQ ID NO:39,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:40, the amino acid substitutions are not at any of positions 1, 12, 15, 16, and 19 of SEQ ID NO:40,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:41, the amino acid substitutions are not at any of positions 12, 15, 16, and 19 of SEQ ID N0:41,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:42, the amino acid substitutions are not at any of positions 1, 13, and 17 of SEQ ID NO:42,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:44, the amino acid substitutions are not at any of positions 3, 13, and 17 of SEQ ID NO:44,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:46, the amino acid substitutions are not at any of positions 5, 13, and 17 of SEQ ID NO:46,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:47, the amino acid substitutions are not at any of positions 6, 13, and 17 of SEQ ID NO:47,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:48, the amino acid substitutions are not at any of positions 7, 13, and 17 of SEQ ID NO:48,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:49, the amino acid substitutions are not at any of positions 8, 13, and 17 of SEQ ID NO:49,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ IDNO:50, the amino acid substitutions are not at any of positions 9, 13, and 17 of SEQ ID NO:50,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:52, the amino acid substitutions are not at any of positions 11, 13, and 17 of SEQ ID NO:52,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:53, the amino acid substitutions are not at any of positions 12, 13, and 17 of SEQ ID NO:53,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:57, the amino acid substitutions are not at any of positions 13, 17, and 18 of SEQ ID NO:57,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:58, the amino acid substitutions are not at any of positions 13, 17, and 19 of SEQ ID NO:58,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:59, the amino acid substitutions are not at any of positions 13, 17, and 20 of SEQ ID NO:59,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:60, the amino acid substitutions are not at any of positions 1, 9, 13, and 17 of SEQ ID NO: 60,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:61, the amino acid substitutions are not at any of positions 3, 9, 13, and 17 of SEQ ID NO:61,wherein if stapled peptide or the pharmaceutically acceptable salt thereof comprises the 1, 2, or 3 amino acid substitutions relative to the sequence of SEQ ID NO:62, the amino acid substitutions are not at any of positions 6, 9, 13, and 17 of SEQ ID NO: 62,wherein positions 12 and 16 of any one of SEQ ID NOs: 20, 33, 36, 38-41 are cross-linked to each other,wherein positions 13 and 17 of any one of SEQ ID NOs: 42, 44, 46-50, 52, 53, and 57-62 are cross-linked to each other, andwherein the stapled peptide, or the pharmaceutically acceptable salt thereof, exhibits an antimicrobial effect against at least one microbe, optionally wherein the at least one microbe is E. coli, or wherein the stapled peptide, or the pharmaceutically acceptable salt thereof exhibits an anti cancer effect.

11. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 10, comprising the sequence of any one of SEQ ID NOs:38-41, 44, 46-49, 52, 53, and 57-59, except for the 0, 1, 2 or 3 amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 38-41, 44, 46-49, 52, 53, and 57-59, respectively.

12. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 10, comprising the sequence of any one of SEQ ID NOs:42, 44, 47, 50, and 60-62, except for the 0, 1, 2 or 3 amino acid substitutions relative to the sequence of any one of SEQ ID NOs: 42, 44, 47, 50, and 60-62, respectively.

13. The stapled peptide, or the pharmaceutically acceptable salt thereof, of claim 10, comprising the sequence of SEQ ID NO:50, except for the 0, 1, 2 or 3 amino acid substitutions relative to the sequence of SEQ ID NO:50.

14. A pharmaceutical composition comprising the stapled peptide, or the pharmaceutically acceptable salt thereof, of any one of claims 1 to 13; and a pharmaceutically acceptable carrier.

15. A method of treating a fungal infection in a subject in need thereof, the method comprising administering to the subject the stapled peptide, or the pharmaceutically acceptable salt thereof, of any one of claims 1, 2, 4, 5, and 11, optionally wherein the subject is a human, optionally wherein the fungal infection is a C. albicans, a C. auris, or a C. neoformans fungal infection.

16. A method of treating a bacterial infection in a subject in need thereof, the method comprising administering to the subject the stapled peptide, or the pharmaceutically acceptable salt thereof, of any one of claims 1, 2, 6, 7, and 12, optionally wherein the subject is a human, optionally wherein the bacterial infection is a gram-negative bacterial infection, optionally wherein the bacterial infection is an 4. baumannii, E. coli, P. aeruginosa, S. aureus, or B. cereus bacterial infection.

17. The method of claim 16, wherein the method further comprises administering to the subject an antibiotic, optionally wherein the antibiotic is selected from the group consisting of a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof, optionally wherein the P-lactam is selected from ampicillin, ceftazidime, cefotaxime, or a combination thereof, the fluoroquinolone is ciprofloxacin, the aminoglycoside is selected from gentamicin, tobramycin, or a combination thereof, the carbapenem is meropenem, or a combination thereof.

18. A method of treating a multi-drug resistant bacterial infection in a subject in need thereof, the method comprising administering to the subject the stapled peptide, or the pharmaceutically acceptable salt thereof, of any one of claims 1, 2, 6, 7, and 12, optionally wherein the subject is a human, optionally wherein the bacterial infection is a gram-negative bacterial infection, optionally wherein the multi-drug resistant bacterial infection is an A. baumannii, E. coli, or K. pneumonia bacterial infection, optionally wherein the multi-drug resistant bacterial infection is a bacterial infection resistant to treatment with a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof; optionally wherein the P-lactam is selected from ampicillin, ceftazidime, cefotaxime, or a combination thereof, the fluoroquinolone is ciprofloxacin,the aminoglycoside is selected from gentamicin, tobramycin, or a combination thereof, the carbapenem is meropenem, or a combination thereof.

19. A method of synergistically killing bacteria in a subject in need thereof, the comprising administering to the subject the stapled peptide, or the pharmaceutically acceptable salt thereof, of any one of claims 1, 2, 6, 7, and 12, optionally wherein the subject is a human, optionally wherein the bacteria are gram-negative bacteria, optionally wherein the bacteria are multi-drug resistant, optionally wherein the bacteria are A. baumannii, E. coh, or K. pneumonia, optionally wherein the bacteria are resistant to treatment with a P-lactam, a fluoroquinolone, an aminoglycoside, a carbapenem, or a combination thereof; optionally wherein the P-lactam is selected from ampicillin, ceftazidime, cefotaxime, or a combination thereof, the fluoroquinolone is ciprofloxacin, the aminoglycoside is selected from gentamicin, tobramycin, or a combination thereof, the carbapenem is meropenem, or a combination thereof.

20. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject the stapled peptide, or the pharmaceutically acceptable salt thereof, of any one of claims 1, 2, 8, 9, and 13, optionally wherein the subject is a human, and optionally wherein the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

21. A method of treating a cancer in a subject in need thereof, the method comprising administering to the subject a stapled peptide, or a pharmaceutically acceptable salt thereof, wherein the stapled peptide comprises:(a) the structure of formula (I):"Formula (I)wherein:each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, aiylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted, optionally wherein each Ri and R2 is a methyl;each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted, optionally wherein R3 is Cs alkylene, Cs alkenylene, or Cs alkynylene, optionally wherein R3 is (CH2)3-CH=CH-(CH2)3;each Xaa is independently an amino acid;each w and y is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; andeach x is 3 or 6; andwherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 2 to 6 substitutions relative to the sequence of TRSSRAGLQWPVGRVHRLLRK (SEQ ID NO: 1),wherein 2 of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are substitutions with non-natural amino acids, the side chains of which are cross-linked to form R3, optionally wherein the 2 of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 with non-natural amino acids, the side chains of which are cross-linked to form R3 are at positions 13 and 17 of the sequence of SEQ ID NO: 1 and [Xaa]xcorresponds to positions 14-16 of the sequence of SEQ ID NO: 1; or(b) 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1, wherein two of the 2 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are with stapling amino acids that are cross-linked to each other; andoptionally wherein the subject is a human, and optionally wherein the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

22. A method of making a stapled peptide, the method comprising:(a) providing a peptide comprising the sequence of SEQ ID NO: 1 with 3 to 6 substitutions, wherein at least one of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 is with a lysine at position 1, 3, 5, 6, 7, 8, 9, 11, 12, 18, 19, or 20 of the sequence of SEQ ID NO:1, and wherein two of the 3 to 6 substitutions relative to the sequence of SEQ ID NO: 1 are with stapling amino acids at (i) positions 12 and 16 of the sequence of SEQ ID NO:1, or (ii) positions 13 and 17 of the sequence of SEQ ID NO:1; and (b) cross-linking the peptide thereby making the stapled peptide,optionally wherein the stapling amino acids are (S)-2-(4’-pentenyl)alanine, optionally wherein the cross-linking is by a ruthenium catalyzed metathesis reaction, and optionally wherein the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition.

23. A pharmaceutical composition comprising: (a) a means for treating a fungal infection in a subject, and (b) a pharmaceutically acceptable carrier; optionally wherein the subject is a human, plant, animal, insect, aquatic species, bird, amphibian, or reptile, and optionally wherein the fungal infection is a C. albicans, a C. auris, or a C. neoformans fungal infection.

24. A pharmaceutical composition comprising: (a) a means for treating a bacterial infection in a plant or animal, and (b) a pharmaceutically acceptable carrier; optionally wherein the animal is a human, and optionally wherein the bacterial infection is an A. baumannii, E. coli, P. aeruginosa, S. aureus, or B. cereus bacterial infection.

25. A pharmaceutical composition comprising: (a) a means for treating cancer in an animal, and (b) a pharmaceutically acceptable carrier; optionally wherein the animal is a human, and optionally wherein the cancer is an ovarian cancer, a skin cancer, a lung cancer, a leukemia, a lymphoma, or osteosarcoma.

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