Patent Application
20240002450
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 29, 2021, is named 00530-0373WO1_SL.txt and is 41,134 bytes in size.
This disclosure relates to structurally-stabilized Ebolavirus GP2 antiviral peptides and methods for using such peptides in the prevention and treatment of an Ebola virus infection.
Ebolaviruses (EBOV) are membrane-enveloped, negative-stranded RNA viruses of the filoviridae family. Within this genus, there are four species that are known to infect humans: Zaire ebolavirus, Bundibugyo ebolavirus, Sudan ebolavirus, and Tai Forest ebolavirus.
EBOV requires fusion of the host and virus membranes to allow for delivery of viral genetic material into the host cell. Membrane fusion in Ebola occurs in host endosomal compartments rather than on the cell surface. Upon engagement of the viral surface glycoprotein (GP1,2) with the cell, the EBOV particle is endocytosed, and its GP is enzymatically cleaved, removing the majority of the GP1 subunit and exposing the transmembrane-anchored subunit GP2. GP2 contains an N-terminal and a C-terminal helical heptad repeat (NHR and CHR, respectively). At its N-terminus, GP2 contains a fusion loop, which, upon a conformational transition, can embed into the host endosomal membrane, leading to a transient intermediate known as the “prehairpin” intermediate in which NHR and CHR are exposed and link the viral and host membranes. Upon pH-mediated maturation of the endosome, GP2 collapses into a highly stable six-helix bundle that brings the host and viral membranes into proximity, providing the driving force for membrane fusion, pore formation, and subsequent infection. The six-helix bundle contains a long, central NHR core with three shorter CHR segments packed alongside in an anti-parallel configuration, together forming a trimeric coiled-coil. An additional intramolecular disulfide bond stabilizes a helix-turn-helix motif between the NHR and CHR and is important for overall bundle stability.
EBOV infections result in severe and often fatal disease (Ebola virus Disease, EVD) in humans. Since its discovery in 1976, the virus has caused several epidemics including in Western Africa (2013-2016) and more recently in the Democratic Republic of Congo (2017-2019). Transmission occurs readily upon direct contact of mucus membranes or non-intact skin with infected body fluids or tissues. EVD is characterized by systemic dissemination of the virus, immune suppression, immune overactivation (cytokine storm), coagulation abnormalities, and tissue damage leading to organ failure and death. In EVD survivors, persistent infection in immune-privileged sites (e.g., central nervous system, eyes, male reproductive tract) occurred. Sexual transmission, male-to-female, has been reported. A number of randomized controlled trials testing preventative vaccines have been completed; however, no approved drugs are currently available for the treatment of Ebola infection. A randomized controlled trial is also underway to test several experimental therapies in addition to supportive care. These experimental therapies or other therapeutic agents are needed to treat the acute disease or RNA persistence following recovery that are easy to administer in an outbreak setting.
Accordingly, there is a need for new inhibitors of Ebola virus for both prevention and treatment.
This disclosure provides structurally stabilized (e.g., stapled) alpha-helical peptides mimicking the CHR, or a portion thereof, of the EBOV GP2. These stabilized (e.g., stapled) EBOV peptides can act as direct inhibitors of EBOV, such as by blocking the virus fusion event. In certain aspects, the structurally stabilized (e.g., stapled) EBOV peptides bind to the EBOV GP2 fusion complex. Without being bound by any theory, the stabilized EBOV peptides provided herein inhibit the formation of the GP2 six-helix bundle, thereby inhibiting the EBOV fusion process. This disclosure also features methods for using such stabilized peptides alone or in combination with other therapeutic agents in the prevention of EBOV infection and in the treatment or prevention of EVD.
Provided herein is a peptide comprising an amino acid sequence: (a) HNWTKX1ITNX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:15); (b) HDWTKX1ITDX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:14); (c) X1DWTX2NITDKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:7); (d) X1DWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:8); (e) X1NWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:9); (f) HX1WTKX2ITDKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:10); (g) HX1WTKX2ITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:11); (h) HNWTX1NITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:12); (i) HDWTX1NITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:13); (j) HDWTKNITX1KIDX2IIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:16); (k) HDWTKNITDKIX1QIIX2DFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:17); (1) HDWTKNITDKIDX1IIHX2FVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:18); (m) X1DWTKNIX2DKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:19); (n) HX1WTKNITX2KIDQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:20); (o) HX1WTKNITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:21); (p) HDWTKX1ITDKIDX2IIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:22); (q) HDWTKNITX1KIDQIIX2DFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:23); or (r) HDWTX1NIX2DKIX3QIIHDFVDK, wherein each of X1, X2, and X3 is independently a stitching amino acid (SEQ ID NO:24); and wherein the peptide binds a 5 helix bundle or fusion bundle intermediate of EBOV GP2. In some instances, the peptide further prevents or blocks fusion of an Ebola virus membrane and a host membrane. In some instances, the peptide is 21 to 50 amino acids in length. The amino acid sequence of any one of SEQ ID NOs:7-23 can be trimmed down at the N and/or C-terminus by 1, 2, 3, 4, 5, 6, or 7 amino acids (e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids can be removed from the N- and/or C-terminus of the amino acid sequence of any one of SEQ ID NOs:7-23) while still permitting the resulting peptide to bind a 5 helix bundle or fusion bundle intermediate of EBOV GP2 and prevent or block fusion of an Ebola virus membrane and a host membrane.
Also provided herein is a structurally stabilized peptide comprising an amino acid sequence: (a) HNWTKX1ITNX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:15); (b) HDWTKX1ITDX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:14); (c) X1DWTX2NITDKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:7); (d) X1DWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:8); (e) X1NWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:9); (f) HX1WTKX2ITDKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:10); (g) HX1WTKX2ITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:11); (h) HNWTX1NITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:12); (i) HDWTX1NITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:13); (j) HDWTKNITX1KIDX2IIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:16); (k) HDWTKNITDKIX1QIIX2DFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:17); (1) HDWTKNITDKIDX1IIHX2FVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:18); (m) X1DWTKNIX2DKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:19); (n) HX1WTKNITX2KIDQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:20); (o) HX1WTKNITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:21); (p) HDWTKX1ITDKIDX2IIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:22); (q) HDWTKNITX1KIDQIIX2DFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:23); or (r) HDWTX1NIX2DKIX3QIIHDFVDK, wherein each of X1, X2, and X3 is independently a stitching amino acid (SEQ ID NO:24); wherein the structurally stabilized peptide is stapled or stitched; and wherein the peptide binds a 5 helix bundle or fusion bundle intermediate of EBOV GP2. In some instances, the peptide further prevents or blocks fusion of an Ebola virus membrane and a host membrane. In some instances, the structurally stabilized peptide is 21-50 amino acids in length. The amino acid sequence of any one of SEQ ID NOs:7-23 can be trimmed down at the N and/or C-terminus by 1, 2, 3, 4, 5, 6, or 7 amino acids (e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids can be removed from the N- and/or C-terminus of the amino acid sequence of any one of SEQ ID NOs:7-23) while still permitting the resulting structurally stabilized peptide to bind a 5 helix bundle or fusion bundle intermediate of EBOV GP2 and prevent or block fusion of an Ebola virus membrane and a host membrane.
In some instances, the structurally stabilized peptide comprises the amino acid sequence: (a) HNWTKX1ITNX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:15), and wherein the sidechains of X1 and X2 are cross-linked to each other; (b) HDWTKX1ITDX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:14), and wherein the sidechains of X1 and X2 are cross-linked to each other; (c) X1DWTX2NITDKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:7), and wherein the sidechains of X1 and X2 are cross-linked to each other; (d) X1DWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:8), and wherein the sidechains of X1 and X2 are cross-linked to each other; (e) X1NWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:9), and wherein the sidechains of X1 and X2 are cross-linked to each other; (f) HX1WTKX2ITDKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:10), and wherein the sidechains of X1 and X2 are cross-linked to each other; (g) HX1WTKX2ITDKINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:11), and wherein the sidechains of X1 and X2 are cross-linked to each other; (h) HNWTX1NITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:12), and wherein the sidechains of X1 and X2 are cross-linked to each other; (i) HDWTX1NITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:13), and wherein the sidechains of X1 and X2 are cross-linked to each other; (j) HDWTKNITX1KIDX2IIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:16), and wherein the sidechains of X1 and X2 are cross-linked to each other; (k) HDWTKNITDKIX1QIIX2DFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:17), and wherein the sidechains of X1 and X2 are cross-linked to each other; (1) HDWTKNITDKIDX1IIHX2FVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:18), and wherein the sidechains of X1 and X2 are cross-linked to each other; (m) X1DWTKNIX2DKIDQIIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:19), and wherein the sidechains of X1 and X2 are cross-linked to each other; (n) HX1WTKNITX2KIDQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:20), and wherein the sidechains of X1 and X2 are cross-linked to each other; (o) HX1WTKNITX2KINQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:21), and wherein the sidechains of X1 and X2 are cross-linked to each other; (p) HDWTKX1ITDKIDX2IIHDFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:22), and wherein the sidechains of X1 and X2 are cross-linked to each other; (q) HDWTKNITX1KIDQIIX2DFVDK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:23), and wherein the sidechains of X1 and X2 are cross-linked to each other; or (r) HDWTX1NIX2DKIX3QIIHDFVDK, wherein each of X1, X2, and X3 is independently a stitching amino acid (SEQ ID NO:24), and wherein the sidechains of X1 and X2 are cross-linked to each other and the sidechains of X2 and X3 are cross-linked to each other. In some instances, the structurally stabilized peptide is 21-50 amino acids in length. The amino acid sequence of any one of SEQ ID NOs:7-23 can be trimmed down at the N and/or C-terminus by 1, 2, 3, 4, 5, 6, or 7 amino acids (e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids can be removed from the N- and/or C-terminus of the amino acid sequence of any one of SEQ ID NOs:7-23) while still permitting the resulting structurally stabilized peptide to bind a 5 helix bundle or fusion bundle intermediate of EBOV GP2 and prevent or block fusion of an Ebola virus membrane and a host membrane.
Also provided herein is a structurally stabilized peptide comprising the Formula:
or a pharmaceutically acceptable salt thereof, wherein: each R1 and R2 is H or a C1 to C10 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; z is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and (a) each [Xaa]w is HDWTK (SEQ ID NO:50), each [Xaa]x is ITD, and each [Xaa]y is INQIIHDFVNK (SEQ ID NO:51); (b) each [Xaa]w is HDWT (SEQ ID NO:49), each [Xaa]x is NIT, and each [Xaa]y is KINQIIHDFVNK (SEQ ID NO:48); (c) each [Xaa]w is absent, each [Xaa]x is DWT, and each [Xaa]y is NITDKIDQIIHDFVDK (SEQ ID NO:43); (d) each [Xaa]w is absent, each [Xaa]x is DWT, and each [Xaa]y is NITDKINQIIHDFVNK (SEQ ID NO:44); (e) each [Xaa]w is absent, each [Xaa]x is NWT, and each [Xaa]y is NITDKINQIIHDFVNK (SEQ ID NO:44); (f) each [Xaa]w is H, each [Xaa]x is WTK, and each [Xaa]y is ITDKIDQIIHDFVDK (SEQ ID NO:45); (g) each [Xaa]w is H, each [Xaa]x is WTK, and each [Xaa]y is ITDKINQIIHDFVNK (SEQ ID NO:46); (h) each [Xaa]w is HNWT (SEQ ID NO:47), each [Xaa]x is NIT, and each [Xaa]y is KINQIIHDFVNK (SEQ ID NO:48); (i) each [Xaa]w is HNWTK (SEQ ID NO:52), each [Xaa]x is ITN, and each [Xaa]y is INQIIHDFVNK (SEQ ID NO:51); (j) each [Xaa]w is HDWTKNIT (SEQ ID NO:53), each [Xaa]x is KID, and each [Xaa]y is IIHDFVDK (SEQ ID NO:54); (k) each [Xaa]w is HDWTKNITDKI (SEQ ID NO:55), each [Xaa]x is QII, and each [Xaa]y is DFVDK (SEQ ID NO:56); (1) each [Xaa]w is HDWTKNITDKID (SEQ ID NO:57), each [Xaa]x is IIH, and each [Xaa]y is FVDK (SEQ ID NO:58); (m) each [Xaa]w is absent, each [Xaa]x is DWTKNI (SEQ ID NO:59), and each [Xaa]y is DKIDQIIHDFVDK (SEQ ID NO:60); (n) each [Xaa]w is H, each [Xaa]x is WTKNIT (SEQ ID NO:61), and each [Xaa]y is KIDQIIHDFVNK (SEQ ID NO:62); (o) each [Xaa]w is H, each [Xaa]x is WTKNIT (SEQ ID NO:61), and each [Xaa]y is KINQIIHDFVNK (SEQ ID NO:48); (p) each [Xaa]w is HDWTK (SEQ ID NO:63), each [Xaa]x is ITDKID (SEQ ID NO:64), and each [Xaa]y is IIHDFVDK (SEQ ID NO:65); or (q) each [Xaa]w is HDWTKNIT (SEQ ID NO:53), each [Xaa]x is KIDQII (SEQ ID NO:66), and each [Xaa]y is DFVDK (SEQ ID NO:56); wherein the peptide binds a 5 helix bundle or fusion bundle intermediate of EBOV GP2. In some instances, the peptide further prevents or blocks fusion of an Ebola virus membrane and a host membrane. In some instances, wherein R1 is an alkyl. In some instances, R1 is a methyl group. In some instances, R2 is an alkyl. In some instances, R2 is a methyl group. In some instances, R3 is an alkenyl. In some instances, R3 is 4-octenyl. In some instances, R1 is a methyl group, R3 is 4-octenyl, and R2 is a methyl group. In some instances, the structurally stabilized peptide or pharmaceutically acceptance salt thereof is 21 to 50 amino acids in length. The peptide of Formula (I) can be trimmed down at the N and/or C-terminus by 1, 2, 3, 4, 5, 6, or 7 amino acids (e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids can be removed from the N- and/or C-terminus of the amino acid sequence of any one of SEQ ID NOs:7-23) while still permitting the resulting peptide to bind a 5 helix bundle or fusion bundle intermediate of EBOV GP2 and prevent or block fusion of an Ebola virus membrane and a host membrane. In some instances, the pharmaceutically acceptable salt is an acetate, a sulfate, or a chloride.
Also provided herein is a structurally stabilized peptide comprising the Formula:
or a pharmaceutically acceptable salt thereof, wherein: [Xaa]w is HDWT (SEQ ID NO:49); [Xaa]x is NI; [Xaa]y is DKI; and [Xaa]z is QIIHDFVDK (SEQ ID NO:67); each R1 and R4 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted; each R2 and R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; and wherein the structurally stabilized peptide binds to a 5 helix bundle of EBOV GP2 or fusion bundle intermediate of EBOV GP2. In some instances, the structurally stabilized peptide further prevents or blocks fusion of an Ebola virus membrane and a host membrane. In some instances, R1 is an alkyl. In some instances, R1 is a methyl group. In some instances, R4 is an alkyl. In some instances, R4 is a methyl group. In some instances, R2 is an alkenyl. In some instances, R2 is 4-octenyl. In some instances, R3 is an alkenyl. In some instances, R3 is 4-octenyl. In some instances, R1 is a methyl group, R2 is 4-octenyl, R3 is 4-octenyl, and R4 is a methyl group. In some instances, the structurally stabilized peptide or pharmaceutically acceptable salt thereof is 21 to 50 amino acids in length. The peptide of Formula (II) can be trimmed down at the N and/or C-terminus by 1, 2, 3, 4, 5, 6, or 7 amino acids (e.g., 1, 2, 3, 4, 5, 6, or 7 amino acids can be removed from the N- and/or C-terminus of the amino acid sequence of any one of SEQ ID NOs:7-23) while still permitting the resulting peptide to bind a 5 helix bundle or fusion bundle intermediate of EBOV GP2 and prevent or block fusion of an Ebola virus membrane and a host membrane. In some instances, the pharmaceutically acceptable salt is an acetate, a sulfate, or a chloride.
Also provided herein is a structurally stabilized peptide comprising an amino acid sequence set forth in SEQ ID NO:2 with 2 to 12 amino acid substitutions and 0 to 5 amino acid deletions from the N- and/or C-terminus of the amino acid sequence set forth in SEQ ID NO:2, wherein at least two amino acids separated by 2, 3, or 6 amino acids are substituted with non-natural amino acids with olefinic side chains, and at least one aspartic acid in SEQ ID NO:2 is substituted by asparagine, wherein the peptide binds to a 5 helix bundle or fusion bundle intermediate of EBOV GP2. In some instances, the structural stabilization comprises a hydrocarbon staple. In some instances, the peptide further prevents or blocks fusion of an Ebola virus membrane 10 and a host membrane. In some instances, the 2 to 12 amino acid substitutions are at one or more positions selected from the group consisting of position 2, 5, 6, 8, 9, 10 12, 17, and 20 (numbering with respect to SEQ ID NO:2). In some instances, the amino acids at one of these sets of positions (relative to SEQ ID NO:2) are replaced by non-natural amino acids with olefinic side chains: (i) 2 and 6; (ii) 2 and 9; (iii) 6 and 10; (iv) 1 and 8; (v) 5, 8, and 12; or (vi) 12 and 16. In some instances, the amino acids at one or more of positions 2, 9, 12, 17 or 20 are replaced by asparagine. In some instances, the amino acids at one or more of positions 2, 9, 12, or 20 are replaced by asparagine.
Also provided herein is an internally cross-linked peptide comprising a cross-linked form of the aforementioned peptide. In some instances, the internally cross-linked peptide has one or more of the following properties: (i) alpha-helical; (ii) retains alpha-helical propensity at pH 4.6; (iii) interferes with assembly of the six-helix-bundle post-fusion complex; (iv) is cell permeable in eukaryotic cells (e.g., is taken up by endosomes); (v) is positively charged; (vi) localizes with late endosomes; and/or (vii) displays antiviral activity against EBOV.
Also provided herein is a structurally stabilized peptide of a polypeptide, the structurally stabilized peptide comprising the Formula:
or a pharmaceutically acceptable salt thereof, wherein: the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2 with 2 to 12 amino acid substitutions and 0 to 5 amino acid deletions from the N- and/or C-terminus of the amino acid sequence set forth in SEQ ID NO:2, wherein at least two amino acids separated by 2, 3, or 6 amino acids are replaced by non-natural amino acids with olefinic side chains; each R1 and R2 is H or a C1 to C10 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; z is 1, 2, or 3; and w+y is 12, 13, 14, 15, 16, 17, 18, 19, or 20; and the structurally stabilized peptide binds to a 5 helix bundle or fusion bundle intermediate of EBOV GP2 and has one or more of the following properties: (i) alpha-helical; (ii) retains alpha-helical propensity at pH 4.6; (iii) interferes with assembly of the six-helix-bundle post-fusion complex; (iv) is cell permeable in eukaryotic cells (e.g., is taken up by endosomes); (v) is positively charged; (vi) localizes with late endosomes; (vii) displays antiviral activity against EBOV; and/or (viii) prevents or blocks fusion of an Ebola virus membrane and a host membrane. In some instances, [Xaa]x is DWTKNI (SEQ ID NO: 59), WTKNIT (SEQ ID NO: 61), WTK, ITD, ITN, or QII, with 0 to 3 amino acid substitutions. In some instances, the pharmaceutically acceptable salt is an acetate, a sulfate, or a chloride.
Also provided herein is a structurally stabilized peptide of a polypeptide, the structurally stabilized peptide comprising the Formula:
or a pharmaceutically acceptable salt thereof, wherein: the polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2 with 3 to 12 amino acid substitutions and 0 to 5 amino acid deletions from the N- and/or C-terminus of the amino acid sequence set forth in SEQ ID NO:2, wherein at least three amino acids are replaced by non-natural amino acids with olefinic side chains; each R1 and R4 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted; each R2 and R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted; x and y are 2, 3, or 6; w+z is 8, 9, 10, 11, 12, 13, 14, or 15; and wherein the structurally stabilized peptide binds to a 5 helix bundle or fusion bundle intermediate of EBOV GP2 and has one or more of the following properties: (i) alpha-helical; (ii) retains alpha-helical propensity at pH 4.6; (iii) interferes with assembly of the six-helix-bundle post-fusion complex; (iv) is cell permeable in eukaryotic cells (e.g., is taken up by endosomes); (v) is positively charged; (vi) localizes with late endosomes; (vii) displays antiviral activity against EBOV; and/or (viii) prevents or blocks fusion of an Ebola virus membrane and a host membrane. In some instances, [Xaa]w is HDWT (SEQ ID NO:49) with 0 to 1 amino acid substitution; [Xaa]x is NI; [Xaa]y is DKI with 0 to 1 amino acid substitution; and [Xaa]z is QIIHDFVDK (SEQ ID NO:67) with 0 to 3 amino acid substitutions. In some instances, the pharmaceutically acceptable salt is an acetate, a sulfate, or a chloride.
Also provided herein is a structurally stabilized (e.g., stapled or stitched) peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs:2-6 except with 2 to 13 amino acid substitutions and 0 to 5 deletions from the N- and/or C-terminus of the amino acid sequence set forth in any one of SEQ ID NOs:2-6, wherein at least 2 of the 2 to 13 amino acid substitutions are separated by 2, 3, or 6 amino acids and are substituted with non-natural amino acids with olefinic side chains, wherein the at least 2 non-natural amino acids with olefinic side chains are cross-linked to each other, wherein the structurally stabilized peptide does not comprise the amino acids corresponding to positions 610-612 of SEQ ID NO:1, and wherein the peptide binds to a 5 helix bundle or fusion bundle intermediate of EBOV GP2. In some instances, the structurally stabilized peptide is 21 to 50 amino acids in length. In some instances, the structurally stabilized peptide is 21 amino acids in length. In some instances, the structurally stabilized peptide prevents or blocks fusion of an Ebola virus membrane and a host membrane. In some instances, the 2 to 13 amino acid substitutions are at one or more positions selected from the group consisting of position 2, 5, 6, 8, 9, 10 12, 17, and 20 of any one of SEQ ID NOs:2-6. In some instances, the at least 2 non-natural amino acids with olefinic side chains are at positions (relative to any one of SEQ ID NOs:2-6): (i) 2 and 6; (ii) 2 and 9; (iii) 6 and 10; (iv) 1 and 8; (v) (vi) 5, 8, and 12; or (vii) 12 and 16. In some instances, the structurally stabilized peptide has one or more of the following properties: (i) is alpha-helical; (ii) retains alpha-helical propensity at pH 4.6; (iii) interferes with assembly of the six-helix-bundle post-fusion complex; (iv) is cell permeable in eukaryotic cells; (v) is positively charged; (vi) localizes with late endosomes; and/or (vii) displays antiviral activity against EBOV.
In some instances, the aforementioned peptide or structurally stabilized peptide does not comprise one or more (e.g., 1, 2, 3, or 4) of the amino acids corresponding to positions 615, 616, 630, and 631 of the amino acid sequence set forth in SEQ ID NO:1. For example, in some instances, the aforementioned peptide or structurally stabilized peptide does not comprise the amino acids corresponding to positions 615, 616, 630, and 631 of the amino acid sequence set forth in SEQ ID NO:1. As another example, in some instances, the aforementioned peptide or structurally stabilized peptide does not comprise the amino acids corresponding to positions 615 and 616 of the amino acid sequence set forth in SEQ ID NO:1. As another example, in some instances, the aforementioned peptide or structurally stabilized peptide does not comprise the amino acids corresponding to positions 630 and 631 of the amino acid sequence set forth in SEQ ID NO:1. In some instances, the aforementioned peptide or structurally stabilized peptide does not contain the amino acid sequence IEP (corresponding to positions 610-612 of SEQ ID NO:1). In some instances, the aforementioned peptide or structurally stabilized peptide does not contain the amino acid sequence IGI at the N-terminus. In some instances, the aforementioned peptide or structurally stabilized peptide does not contain the amino acid sequence TLPD (corresponding to positions 634-637 of SEQ ID NO:1). In some instances, the aforementioned peptide or structurally stabilized peptide does not contain the amino acid sequence TLPDQG (corresponding to positions 634-639 of SEQ ID NO:1).
In some instances, the aforementioned peptide or structurally stabilized peptide comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the amino acids corresponding to amino acids W615, T616, N618, I619, K622, I623, Q625, I626, I627, D629, F630, and V631 (numbered according to SEQ ID NO:1), or conservative amino acid substitutions thereof.
In some instances, the aforementioned peptide or structurally stabilized peptide comprises a conservative amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) amino acids corresponding to amino acids K617, T620, D621, D624, and H628 (numbered according to SEQ ID NO:1). In some instances, the aforementioned peptide or structurally stabilized peptide comprises a non-conservative amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) amino acids corresponding to amino acids K617, T620, D621, D624, and H628 (numbered according to SEQ ID NO:1).
Also provided herein is a pharmaceutical composition comprising an aforementioned peptide and a pharmaceutically acceptable carrier.
Also provided herein is a pharmaceutical composition comprising an aforementioned structurally stabilized peptide and a pharmaceutically acceptable carrier.
Also provided herein is a method of treating an Ebolavirus infection in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of an aforementioned structurally stabilized peptide. In some instances, the Ebolavirus infection is an infection with a Zaire ebolavirus, a Tai Forest ebolavirus, a Sudan ebolavirus or a Bundibugyo ebolavirus. In some instances, the subject is a human. In some instances the subject is a non-human primate. In some instances, the subject is a fruit bat.
Also provided herein is a method of preventing an Ebolavirus infection in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of an aforementioned structurally stabilized peptide. In some instances, the Ebolavirus infection is an infection with a Zaire ebolavirus, a Tai Forest ebolavirus, a Sudan ebolavirus or a Bundibugyo ebolavirus. In some instances, the subject is a human. In some instances the subject is a non-human primate. In some instances, the subject is a fruit bat.
Also provided herein is a method of treating an Ebolavirus disease in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of an aforementioned structurally stabilized peptide. In some instances, the Ebolavirus disease is caused by an infection with a Zaire ebolavirus, a Tai Forest ebolavirus, a Sudan ebolavirus or a Bundibugyo ebolavirus. In some instances, the subject is a human. In some instances the subject is a non-human primate. In some instances, the subject is a fruit bat.
Also provided herein is a method of preventing an Ebolavirus disease in a subject in need thereof, the method comprising administering to the subject a therapeutically-effective amount of an aforementioned structurally stabilized peptide. In some instances, the Ebolavirus disease is caused by an infection with a Zaire ebolavirus, a Tai Forest ebolavirus, a Sudan ebolavirus or a Bundibugyo ebolavirus. In some instances, the subject is a human. In some instances the subject is anon-human primate. In some instances, the subject is a fruit bat.
Also provided herein is a method of preventing transmission of an Ebolavirus infection from a first subject to a second subject, the method comprising administering to the first subject a therapeutically-effective amount of an aforementioned structurally stabilized peptide, wherein the first subject is a human or a non-human primate or a fruit bat. In some instances, the second subject is a human.
Also provided herein is a method of making a structurally stabilized peptide, the method comprising: (a) providing a peptide having the sequence set forth in any one of SEQ ID NOs:7-42, or an aforementioned peptide, and (b) cross-linking the peptide to make a structurally stabilized peptide; and optionally, further comprising formulating the structurally stabilized peptide into a pharmaceutical composition.
Fusion of the host and EBOV membranes is required for delivery of EBOV genetic material into the host cell. Membrane fusion in Ebola occurs in host endosomal compartments rather than on the cell surface. Upon engagement of the viral surface glycoprotein (GP1,2) with the cell, the EBOV particle is endocytosed, and its GP is enzymatically cleaved, removing the majority of the GPT subunit and exposing the transmembrane-anchored subunit GP2. GP2 contains an N-terminal and a C-terminal helical heptad repeat (NHR and CHR, respectively). At its N-terminus, GP2 contains a fusion loop, which, upon a conformational transition, can embed into the host endosomal membrane, leading to a transient intermediate known as the “prehairpin” intermediate in which NHR and CHR are exposed and link the viral and host membranes. Upon pH-mediated maturation of the endosome, GP2 collapses into a highly stable six-helix bundle that brings the host and viral membranes into proximity, providing the driving force for membrane fusion, pore formation, and subsequent infection. The six-helix bundle contains a long, central NHR core with three shorter CHR segments packed alongside in an anti-parallel configuration, together forming a trimeric coiled-coil. An additional intramolecular disulfide bond stabilizes a helix-turn-helix motif between the NHR and CHR and is important for overall bundle stability.
This disclosure provides structurally stabilized (e.g., stapled) alpha-helical peptides mimicking the CHR, or a portion thereof, of the EBOV GP2. These stabilized (e.g., stapled) EBOV peptides can act as direct inhibitors of EBOV, such as by blocking the virus fusion event. In certain aspects, the structurally stabilized (e.g., stapled) EBOV peptides bind to the EBOV GP2 fusion complex. Without being bound by any theory, the stabilized EBOV peptides provided herein inhibit the formation of the GP2 six-helix bundle, thereby inhibiting the EBOV fusion process. This disclosure also features methods for using such stabilized peptides alone or in combination with other therapeutic agents in the prevention of EBOV infection and in the treatment or prevention of EVD.
The amino acid sequence of an exemplary Ebola virus GP2 protein of the Zaire species is provided below (GenBank Accession No. AK184258):
GP2 contains an N-terminal helical heptad repeat and a C-terminal helical heptad repeat (NHR and CHR, respectively), separated by a turn/linker. In some instances, the EBOV GP2 NHR comprises amino acid residues 557-597 of the amino acid sequence set forth in SEQ ID NO:1. In some instances, the EBOV GP2 NHR comprises amino acid residues 554-595 of the amino acid sequence set forth in SEQ ID NO:1. In some instances, the turn/linker separating the EBOV GP2 NHR and EBOV GP2 CHR comprises amino acids 598-612 of the amino acid sequence set forth in SEQ ID NO:1. In some instances, the turn/linker separating the EBOV GP2 NHR and EBOV GP2 CHR comprises amino acids 596-614 of the amino acid sequence set forth in SEQ ID NO:1. In some instances, the EBOV CHR comprises amino acids 613-633 of the amino acid sequence set forth in SEQ ID NO:1. In some instances, the EBOV GP2 CHR comprises amino acids 615-631 of the amino acid sequence set forth in SEQ ID NO:1. In some instances, an EBOV GP2 CHR peptide comprises the amino acid sequence HDWTKNITDKIDQIIHDFVDK (SEQ ID NO:2). In some instances, an EBOV GP2 CHR peptide consists of the amino acid sequence HDWTKNITDKIDQIIHDFVDK (SEQ ID NO:2). In some instances, an EBOV GP2 CHR peptide comprises the amino acid sequence HDWTKNIT DKIDQIIHDF (SEQ ID NO:73). In some instances, an EBOV GP2 CHR peptide consists of the amino acid sequence HDWTKNIT DKIDQIIHDF (SEQ ID NO:73). At its N-terminus, GP2 contains a fusion loop, which, upon a conformational transition, can embed into the host endosomal membrane, leading to a transient intermediate known as the “prehairpin” intermediate in which NHR and CHR are exposed and link the viral and host membranes. Upon pH-mediated maturation of the endosome, GP2 collapses into a highly stable six-helix bundle that brings the host and viral membranes into proximity, providing the driving force for membrane fusion, pore formation, and subsequent infection. The six-helix bundle contains a long, central NHR core with three shorter CHR segments packed alongside in an anti-parallel configuration, together forming a trimeric coiled-coil. An additional intramolecular disulfide bond stabilizes a helix-turn-helix motif between the NHR and CHR and is important for overall bundle stability.
The GP2 proteins among the different Ebolavirus species have high homology. See
The amino acid sequence of an exemplary HR1 (NHR) and HR2 (CHR) separated by a GG linker for Tai Forest ebolavirus species is provided below:
The amino acid sequence of an exemplary HR1 (NHR) and HR2 (CHR) separated by a GG linker for Bundibugyo ebolavirus species is provided below:
The amino acid sequence of an exemplary HR1 (NHR) and HR2 (CHR) separated by a GG linker for Sudan ebolavirus species is provided below:
In some instances, an EBOV GP2 CHR peptide comprises the amino acid sequence of SEQ ID NO:73, except that it contains a Q at position 1 of SEQ ID NO:73. In some instances, an EBOV GP2 CHR peptide comprises the amino acid sequence of SEQ ID NO:73, except that it contains an N at position 11 of SEQ ID NO:73.
The EBOV GP2 CHR contains multiple hydrophobic residues at its binding interface with the EBOV GP2 NHR (see
An overall positive charge in solution may modulate or enhance the cell permeability of stapled peptides (e.g., the ability to be taken up by endosomes). The EBOV GP2 CHR peptide having the amino acid sequence set forth in SEQ ID NO:2 has a charge of −2 at physiological pH. To engineer EBOV GP2 CHR peptides having a positive charge, one or more aspartic acid residues in the EBOV GP2 CHR peptide can be replaced with a positively charged or neutral amino acid, such as, e.g., arginine, histidine, or lysine. In some instances, one, two, three, four, or all five of the aspartic acid residues in an EBOV GP2 CHR peptide (e.g., in a peptide comprising or consisting of the amino acid sequence of SEQ ID NO:2) are replaced with a positively charged amino acid. In some instances, one, two, three, four, or all five of the aspartic acid residues in an EBOV GP2 CHR peptide (e.g., in a peptide comprising or consisting of the amino acid sequence of SEQ ID NO:2) are replaced with aspartic acid's isosteric counterpart, asparagine. In some instances, one or more (e.g., 1, 2, 3, 4, 5, 6, 7) of the NHR-interacting residues in an EBOV GP2 CHR peptide are replaced with an arginine, histidine, or lysine. In some instances, one, two, three, four, five, six, seven, eight, nine, 10, 11, 12, 13, or 14 of the non-NHR-interacting residues in an EBOV GP2 CHR peptide are replaced with an arginine, histidine, or a lysine. In some instances, the overall charge of an EBOV GP2 CHR peptide is −3 to +3. In some instances, the overall charge of an EBOV GP2 CHR peptide is −2 to +2. In some instances, the overall charge of an EBOV GP2 CHR peptide is −1 to +3. In some instances, the overall charge of an EBOV GP2 CHR peptide is 0 to +3. In some instances, the overall charge of an EBOV GP2 CHR peptide is 0 to +2.
In certain instances, also provided herein are peptides comprising a modified amino acid sequence of an EBOV GP2 CHR peptide described herein. In some cases, the peptides are modified to introduce moieties (e.g., non-natural amino acids with olefinic side chains) that permit structural stabilization to the peptide (e.g., to maintain alpha-helicity of the peptide). The structural stabilization may be by, e.g., “stapling” or “stitching” the peptide. In some cases, the staple or stitch is a hydrocarbon staple or stitch. The modification(s) to introduce structural stabilization (e.g., internal cross-linking, e.g., stapling, stitching) into the EBOV GP2 CHR peptides described herein may be positioned on the face of the EBOV GP2 CHR peptide that does not interact with EBOV GP2 NHR (i.e., the face of the EBOV GP2 CHR peptide that does not comprise one or more NHR-interacting residues). Alternatively, the modification(s) to introduce stabilization (e.g., internal cross-linking, e.g., stapling or stitching) into the EBOV GP2 CHR peptides described herein may be positioned on the face of the EBOV GP2 CHR peptide that does interact with EBOV GP2 NHR (i.e., the face of the EBOV GP2 CHR peptide that comprises one or more NHR-interacting residues). In some cases, an EBOV GP2 CHR peptide is stabilized by introducing a staple or stitch (e.g., a hydrocarbon staple or stitch) at the interface of the NHR-interacting and NHR-non-interacting faces of the EBOV GP2 CHR (i.e., on a face of the EBOV GP2 CHR peptide comprising one or more NHR-interacting residues and one or more non-NHR-interacting residues). In some instances, the modifications to introduce structural stabilization (e.g., internal cross-linking, e.g., stapling or stitching) into the EBOV GP2 CHR peptides described herein—e.g., substitution of two or more amino acids with non-natural amino acids with olefinic side chains—are positioned at the amino acid positions in the EBOV GP2 peptide corresponding to residues:
In certain instances, the EBOV GP2 CHR peptides of this disclosure can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in any one of SEQ ID NOs:2-6 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids are conservatively or non-conservatively substituted). For example, in certain instances, the EBOV GP2 CHR peptide of this disclosure comprises a modified amino acid sequence of the sequence set forth in SEQ ID NO:2, wherein the modified amino acid sequence comprises SEQ ID NO:2 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in the SEQ ID NO:2 sequence (e.g., the modified amino acid sequence comprises SEQ ID NO:2, except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of SEQ ID NO:2 are conservatively or non-conservatively substituted). In another example, in certain instances, the EBOV GP2 CHR peptide of this disclosure comprises a modified amino acid sequence of the sequence set forth in SEQ ID NO:3, wherein the modified amino acid sequence comprises SEQ ID NO:3 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in the SEQ ID NO:3 sequence (e.g., the modified amino acid sequence comprises SEQ ID NO:3, except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of SEQ ID NO:3 are conservatively or non-conservatively substituted). In another example, in certain instances, the EBOV GP2 CHR peptide of this disclosure comprises a modified amino acid sequence of the sequence set forth in SEQ ID NO:4, wherein the modified amino acid sequence comprises SEQ ID NO:4 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in the SEQ ID NO:4 sequence (e.g., the modified amino acid sequence comprises SEQ ID NO:4, except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of SEQ ID NO:4 are conservatively or non-conservatively substituted). In yet another example, in certain instances, the EBOV GP2 CHR peptide of this disclosure comprises a modified amino acid sequence of the sequence set forth in SEQ ID NO:5, wherein the modified amino acid sequence comprises SEQ ID NO:5 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in the SEQ ID NO:5 sequence (e.g., the modified amino acid sequence comprises SEQ ID NO:5, except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of SEQ ID NO:5 are conservatively or non-conservatively substituted). In yet another example, in certain instances, the EBOV GP2 CHR peptide of this disclosure comprises a modified amino acid sequence of the sequence set forth in SEQ ID NO:6, wherein the modified amino acid sequence comprises SEQ ID NO:6 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in the SEQ ID NO:6 sequence (e.g., the modified amino acid sequence comprises SEQ ID NO:6, except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of SEQ ID NO:6 are conservatively or non-conservatively substituted). A “conservative amino acid substitution” means that the substitution replaces one amino acid with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), and acidic side chains and their amides (e.g., aspartic acid, glutamic acid, asparagine, glutamine). In some instances, one to three amino acids of any one of SEQ ID NOs:2-6 are substituted. The amino acid substitutions in any one of SEQ ID NOs:2-6 can be of amino acids that NHR-interacting residues or non-NHR-interacting residues. Much greater variability is permitted in the EBOV GP2 CHR amino acids that do not engage in direct interaction with EBOV GP2 NHR (i.e., non-NHR-interacting residues). In fact, just about every one of the non-NHR-interacting residues can be substituted (e.g., conservative or non-conservative amino acid substitutions or substitution with alanine). In certain instances, one, two, or three NHR-interacting residues are substituted with another amino acid. In some instances, the substitution(s) is/are a conservative amino acid substitution. In other instances, the substitution(s) is/are a non-conservative amino acid substitution. In some instances, where there are more than one amino acid substitutions, the substitutions are both conservative and non-conservative amino acid substitutions. In some instances, where there are more than one amino acid substitutions, each of the substitutions are conservative amino acid substitutions. In some cases, where one to three amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs:2-6 are substituted, the substitutions are all of non-NHR-interacting residues. In some cases, where one to three amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs:2-6 are substituted, the substitutions are all of NHR-interacting residues. In some cases, where one to three amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs:2-6 are substituted, the substitutions are of both non-NHR-interacting residues and NHR-interacting residues. In certain instances, the substituted amino acid(s) are selected from the group consisting of L-Ala, D-Ala, Aib, Sar, Ser, a substituted alanine, or a substituted glycine derivative.
In some instances, the EBOV GP2 CHR peptides of this disclosure comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) of the amino acids corresponding to amino acids W615, T616, N618, I619, K622, I623, Q625, I626, I627, D629, F630, and V631 (numbered according to SEQ ID NO:1), or conservative amino acid substitutions thereof.
In some instances, the EBOV GP2 CHR peptides of this disclosure comprise a conservative amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) amino acids corresponding to amino acids K617, T620, D621, D624, and H628 (numbered according to SEQ ID NO:1). In some instances, the EBOV GP2 CHR peptides of this disclosure comprise a non-conservative amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) amino acids corresponding to amino acids K617, T620, D621, D624, and H628 (numbered according to SEQ ID NO:1).
In some instances, the EBOV GP2 CHR peptides of this disclosure do not comprise one or more (e.g., 1, 2, 3, or 4) of the amino acids corresponding to positions 615, 616, 630, and 631 of the amino acid sequence set forth in SEQ ID NO:1. For example, in some instances, the EBOV GP2 CHR peptides of this disclosure do not comprise the amino acids corresponding to positions 615, 616, 630, and 631 of the amino acid sequence set forth in SEQ ID NO:1. As another example, in some instances, the EBOV GP2 CHR peptides of this disclosure do not comprise the amino acids corresponding to positions 615 and 616 of the amino acid sequence set forth in SEQ ID NO:1. As another example, in some instances, the EBOV GP2 CHR peptides of this disclosure do not comprise the amino acids corresponding to positions 630 and 631 of the amino acid sequence set forth in SEQ ID NO:1. In certain instances, the EBOV GP2 CHR peptides of this disclosure can have 1, 2, 3, 4, or 5, amino acids removed/deleted from the C-terminus of the sequence set forth in any one of SEQ ID NOs:2-6. In certain instances, the EBOV GP2 CHR peptides of this disclosure can have 1, 2, 3, 4, or 5, amino acid removed/deleted from the N-terminus of the sequence set forth in any one of SEQ ID NOs:2-6. In certain instances, these removed amino acids can be replaced with 1-6 (e.g., 1, 2, 3, 4, 5, or 6) amino acids selected from the group consisting of L-Ala, D-Ala, Aib, Sar, Ser, a substituted alanine, or a substituted glycine derivative.
The disclosure also encompasses EBOV GP2 CHR peptides that are at least 14% (e.g., at least 14 to 50%, at least 14 to 45%, at least 14 to 40%, at least 14 to 35%, at least 14 to 30%, at least 14 to 25%, at least 14 to 20%, at least 20% to 50%, at least 20% to 45%, at least 20% to 40%, at least 20% to 35%, at least 20% to 30%, at least 20% to 25%, at least 15%, at least 20%, at least 27%, at least 34%, at least 40% at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) identical to any one of SEQ ID NOs:2-6. In some instances, the at least 14% identity is at the binding interface as shown in
In certain instances, the EBOV GP2 CHR peptide is a variant having an amino acid sequence set forth in Table 1 below. In certain instances, the EBOV GP2 CHR peptide is a variant having an amino acid sequence set forth in Table 1 below, except that it lacks the three C-terminal amino acids in the sequences in Table 1 below.
The EBOV GP2 CHR peptides described herein can be optimized for therapeutic use. For example, if any of the above-described EBOV GP2 CHR peptides cause membrane disruption (cell lysis), the peptides can be optimized by lowering the overall peptide hydrophobicity. This can for example be achieved by substituting especially hydrophobic residues with an amino acid with lower hydrophobicity (e.g., alanine). Membrane disruption can also be lowered by reducing the overall positive charge of the peptide. This can be accomplished by substituting basic residues with uncharged or acidic residues. In certain instances, both the overall peptide hydrophobicity and the overall positive charge of the peptide are lowered. In some instances, the overall charge of an EBOV GP2 CHR peptide is −3 to +3. In some instances, the overall charge of an EBOV GP2 CHR peptide is −2 to +2. In some instances, the overall charge of an EBOV GP2 CHR peptide is −1 to +3. In some instances, the overall charge of an EBOV GP2 CHR peptide is 0 to +3. In some instances, the overall charge of an EBOV GP2 CHR peptide is 0 to +2.
In certain instances, each of the EBOV GP2 CHR peptides described above binds to a 5 helix bundle of EBOV GP2 or fusion bundle intermediate of EBOV GP2. In certain instances, each of the EBOV GP2 CHR peptides described above binds to a 5 helix bundle of EBOV GP2 or fusion bundle intermediate of EBOV GP2 and prevents or blocks fusion of an Ebola virus membrane and a host membrane.
Methods of determining whether a peptide (e.g., a structurally stabilized peptide described herein) binds to a 5 helix bundle or a fusion bundle intermediate of EBOV GP2 are known in the art, such as, high resolution clear native electrophoresis (hrCNE). See, e.g., the working Examples section below and Harrison et al., Protein Science, 2011, 20:1587-1596, which is incorporated by reference herein in its entirety. For instance, a tagged (e.g., hexa-histidine (SEQ ID NO:75) tagged) EBOV GP2 ectodomain construct that lacks one of the CHR helices in the post-fusion complex (e.g., a construct having the amino acid sequence MGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDC AIEPHDWTKNITDKIDQIIHDFGSSGGLRQLANETTQALQLFLRATTELRTFSIL NRKAIDFLLQRWGGTCHILGPDCAIEPHDWTKNITDKIDQIIHDFGSSGGLRQL ANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGHHHHHH (SEQ ID NO:74)) is expressed and incubated with a labeled (e.g., FITC-labeled) control peptide (e.g., a peptide comprising the amino acid sequence of SEQ ID NO:2) or a labeled (e.g., FITC-labeled) test peptide (e.g., a structurally-stabilized peptide described herein) at 37° C. for, e.g., 30 minutes. After incubation, the mixture is analyzed by native PAGE electrophoresis and the native PAGE gel is imaged in a fluorescence scanner to detect the migration of the labeled species and immunoblotted to reveal the location of the tagged EBOV GP2 ectodomain construct. Co-migration of the labeled test peptide and the tagged EBOV GP2 ectodomain construct indicates binding of the test peptide to a 5 helix bundle or a fusion bundle intermediate of EBOV GP2. In some instances, the control peptide is an unstapled version of the test peptide. For example, the control peptide may have the amino acid sequence of the test peptide except that the control peptide contains the corresponding wild type amino acids at the positions of the staple(s) or stitch(es) in the test peptide.
Methods of determining whether a peptide (e.g., a structurally stabilized peptide described herein) prevents or blocks fusion of an Ebola virus membrane and a host membrane are known in the art, such as, e.g., cytotoxicity and immunofluorescence. In some instances, a peptide prevents or blocks fusion of an Ebola virus membrane and a host membrane if less than 1%, less than 5%, less than 10%, less than 15% less than 20%, less than 30%, less than 40%, or less than 50% of cells are infected with Ebola virus at a multiplicity of infection of 0.1, 0.5, 1, or 10 in the presence the peptide. In some instances, a peptide prevents or blocks fusion of an Ebola virus membrane and a host membrane if less than 1%, less than 5%, less than 10%, less than 15% less than 20%, less than 30%, less than 40%, or less than 50% of cells exhibit fusion of the Ebola virus membrane and the host membrane after infection with Ebola virus at a multiplicity of infection of 0.1, 0.5, 1, or 10 in the presence the peptide.
This disclosure also features stabilized versions (e.g., internally cross-linked, e.g., stapled or stitched) of the above-described EBOV GP2 CHR peptides.
A peptide helix is an important mediator of key protein-protein interactions that regulate many important biological processes such as apoptosis; however, when such a helix is taken out of its context within a protein and prepared in isolation, it usually adopts a random coil conformation, leading to a drastic reduction in biological activity and thus diminished therapeutic potential. The present disclosure provides structurally stabilized EBOV GP2 CHR peptides. The present disclosure includes structurally stabilized EBOV GP2 CHR peptides (such as structurally stabilized versions of the EBOV GP2 CHR peptides described above) comprising at least two modified amino acids joined by an internal (intramolecular) cross-link (a staple or stitch). Stabilized peptides as described herein include stapled peptides and stitched peptides as well as peptides containing multiple stitches, multiple staples or a mix of staples and stitches, or other chemical strategies for structural reinforcement (see. e.g., Balaram P. Cur. Opin. Struct. Biol. 1992; 2:845; Kemp D S, et al., J. Am. Chem. Soc. 1996; 118:4240; Omer B P, et al., J Am. Chem. Soc. 2001; 123:5382; Chin J W, et al., Int. Ed. 2001; 40:3806; Chapman R N, et al., J Am. Chem. Soc. 2004; 126:12252; Home W S, et al., Chem., Int. Ed. 2008; 47:2853; Madden et al., Chem Commun (Camb). 2009 Oct. 7; (37): 5588-5590; Lau et al., Chem. Soc. Rev., 2015, 44:91-102; and Gunnoo et al., Org. Biomol. Chem., 2016, 14:8002-8013; all of which are incorporated by reference herein in its entirety).
In certain instances, one or more of the EBOV GP2 CHR peptides described herein can be structurally stabilized by peptide stapling (see, e.g., Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are incorporated by reference herein in its entirety). A peptide is “structurally stabilized” in that it maintains its native secondary structure. For example, stapling allows a peptide, predisposed to have an α-helical secondary structure, to maintain its native α-helical conformation. This secondary structure increases resistance of the peptide to proteolytic cleavage and heat, and also may increase target binding affinity, hydrophobicity, and cell permeability (e.g., the ability to be taken up by endosomes). Accordingly, the stapled (cross-linked) peptides described herein have improved biological activity relative to a corresponding non-stapled (un-cross-linked) peptide.
“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 et al., J Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). As used herein, the term “peptide stapling” includes the joining of two (e.g., at least one pair of) double bond-containing side-chains, triple bond-containing side-chains, or double bond-containing and triple bond-containing side chain, which may be present in a peptide chain, using any number of reaction conditions and/or catalysts to facilitate such a reaction, to provide a singly “stapled” peptide. The term “multiply stapled” peptides refers to those peptides containing more than one individual staple, and may contain two, three, or more independent staples of various spacing. Additionally, the term “peptide stitching,” as used herein, refers to multiple and tandem “stapling” events in a single peptide chain to provide a “stitched” (e.g., tandem or multiply stapled) peptide, in which two staples, for example, are linked to a common residue. Peptide stitching is disclosed, e.g., in WO 2008/121767 and WO 2010/068684, which are both hereby incorporated by reference in their entirety. In some instances, staples, as used herein, can retain the unsaturated bond or can be reduced.
In certain instances, one or more of the EBOV GP2 CHR peptides described herein can be structurally stabilized. In some instances, the EBOV GP2 CHR peptides of this disclosure are structurally stabilized by a hydrocarbon staple or stitch, a lactam staple or stitch; a UV-cycloaddition staple or stitch; an oxime staple or stitch; a thioether staple or stitch; a double-click staple or stitch; a bis-lactam staple or stitch; a bis-arylation staple or stitch; or a combination of any two or more thereof. In some instances, the EBOV GP2 CHR peptides of this disclosure are structurally stabilized by a hydrocarbon staple. In some instances, the EBOV GP2 CHR peptides of this disclosure are structurally stabilized by a hydrocarbon stitch.
In some instances, the structurally stabilized (e.g., stapled or stitched) peptide is a cross-linked version of a peptide comprising or consisting of any one of the amino acids sequences of SEQ ID NOs:2-6, as listed in Table 1. In some instances, the stapled peptide is a hydrocarbon stapled version of a peptide comprising or consisting of any one of the amino acids sequences of SEQ ID NOs:2-6, as listed in Table 1.
In some instances, the stapled peptide is a peptide comprising or consisting of any one of the amino acids sequences of SEQ ID NOs:2-6, except that at least two (e.g., 2, 3, 4) amino acids of SEQ ID NOs:2-6 are replaced with a non-natural amino acid capable of forming a staple or stitch (e.g., non-natural amino acids with olefinic side chains, e.g., (S)-2-(4′-pentenyl)Alanine, (R)-2-(7′-octenyl)Alanine). In some instances, the stapled peptide is a peptide comprising or consisting of any one of the amino acids sequences of SEQ ID NOs:2-6 or comprising 1 to 13 (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acid substitutions, deletions and/or insertions therein. In certain instances, the stapled peptide includes at least two (e.g., 2, 3, 4, 5, 6) amino acid substitutions, wherein the substituted amino acids are separated by two, three, or six amino acids, and wherein the substituted amino acids are non-natural amino acids with olefinic side chains (e.g., (S)-2-(4′-pentenyl)Alanine or (R)-2-(7′-octenyl)Alanine). There are many known non-natural amino acids that may be used as stapling amino acids or stitching amino acids, any of which may be included in the peptides of the present invention. Some examples of non-natural amino acids that may be used as stapling amino acids or stitching amino acids are: (R)-2-(7′-octenyl)Alanine, (S)-2-(7′-octenyl)Alanine, (S)-2-(4′-pentenyl)Alanine, (R)-2-(4′-pentenyl)Alanine, 2,2-bis(4-pentenyl)glycine, 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine, 1-amino-cyclopropanecarboxylic acid, 1-amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta- and/para-substituted phenylalanines (e.g., substituted with —C(═O)C6H5; —CF3; —CN; -halo; —NO2; CH3), disubstituted phenylalanines, substituted tyrosines (e.g., further substituted with —C═O)C6H5; —CF3; —CN; -halo; —NO2; CH3), and statine. Additionally, amino acids can be derivatized to include amino acid residues that are hydroxylated, phosphorylated, sulfonated, acylated, or glycosylated. In some instances, the amino acids forming the staple or stitch (also referred to as the “stapling amino acids” or the “stitching amino acids”, respectively) are (S)-2-(4′-pentenyl)Alanine at each of positions i and i+4 of the staple. In some instances, the amino acids forming the staple or stitch are (R)-2-(4′-pentenyl)Alanine at each of positions i and i+4 of the staple. In some instances, the amino acids forming the staple or stitch are (R)-2-(7′-octenyl)Alanine and (S)-2-(4′-pentenyl)Alanine at positions i and i+7, respectively, of the staple. In some instances, the amino acids forming the staple or stitch are (S)-2-(4′-pentenyl)Alanine, 2,2-bis(4-pentenyl)glycine, and (S)-α-(7′-octenyl)alanine at positions i, i+4, and i+11, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (R)-α-(4′-pentenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (R)-α-(7′-octenyl)alanine at positions i, i+4, and i+11, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (R)-2-(4′-pentenyl)Alanine, 2,2-bis(4-pentenyl)glycine, and (R)-2-(4′-pentenyl)alanine at positions i, i+3, and i+7, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (S)-2-(4′-pentenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (S)-2-(4′-pentenyl)alanine at positions i, i+3, and i+7, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (R)-α-(propenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (R)-2-(4′-pentenyl)alanine at positions i, i+3, and i+7, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (R)-2-(4′-pentenyl)Alanine, 2,2-bis(4-pentenyl)glycine, and (S)-2-(4′-pentenyl)Alanine at positions i, i+4, and i+8, respectively, of the stitch.
Hydrocarbon stapled peptides include one or more tethers (linkages) between two non-natural amino acids, which tether significantly enhances the α-helical secondary structure of the peptide. Generally, the tether extends across the length of one or two helical turns (i.e., about 3.4 or about 7 amino acids). Accordingly, amino acids positioned at i and i+3; i and i+4; or i and i+7 are ideal candidates for chemical modification and cross-linking. Thus, for example, where a peptide has the sequence . . . X1, X2, X3, X4, X5, X6, X7, X8, X9 . . . , cross-links between X1 and X4, or between X1 and X5, or between X1 and X8 are useful hydrocarbon stapled forms of that peptide, as are cross-links between X2 and X5, or between X2 and X6, or between X2 and X9, etc. (i.e., forming an “[i, i+3] staple”, an “[i, i+4] staple”, or an “[i, i+7] staple”, respectively). The use of multiple cross-links (e.g., 2, 3, 4, or more) is also contemplated. The use of multiple cross-links is very effective at stabilizing and optimizing the peptide, especially with increasing peptide length. Thus, the disclosure encompasses the incorporation of more than one cross-link within the peptide sequence to either further stabilize the sequence or facilitate the structural stabilization, proteolytic resistance, acid stability, thermal stability, cellular permeability, and/or biological activity enhancement of longer peptide stretches. Additional description regarding making and use of hydrocarbon stapled peptides can be found, e.g., in U.S. Patent Publication Nos. 2012/0172285, 2010/0286057, and 2005/0250680, the contents of all of which are incorporated by reference herein in their entireties.
In certain instances when a staple is at the i and i+3 residues, (R)-2-(2-propenyl)alanine and (S)-2-(4′-pentenyl)Alanine; or (R)-2-(4-pentenyl)alanine and (S)-2-(4′-pentenyl)Alanine are substituted for the amino acids at those positions. In certain instances when a staple is at the i and i+4 residues, (S)-2-(4′-pentenyl)Alanine is substituted for the amino acids at i and i+4. In certain instances when a staple is at the i and i+7 residues, (S)-2-(4′-pentenyl)Alanine and (R)-2-(7′-octenyl)Alanine are substituted for the amino acids at i and i+7. In some instances, when the peptide is stitched, the amino acids of the peptide to be involved in the “stitch” are substituted with: (i) 2,2-bis(4-pentenyl)glycine, (S)-2-(4′-pentenyl)Alanine, and (S)-2-(7′-octenyl)Alanine; or (ii) 2,2-bis(4-pentenyl)glycine, (R)-2-(4′-pentenyl)Alanine, and (R)-2-(7′-octenyl)Alanine; or (iii) 2,2-bis(4-pentenyl)glycine, (R)-2-(7′-octenyl)Alanine, and (R)-2-(7′-octenyl)Alanine; or (iv) 2,2-bis(4-pentenyl)glycine, (S)-2-(7′-octenyl)Alanine, and (S)-2-(7′-octenyl)Alanine; (v) 2,2-bis(4-pentenyl)glycine, (S)-2-(4′-pentenyl)Alanine, (R)-2-(4′-pentenyl)Alanine; or (vi) 2,2-bis(4-pentenyl)glycine, (S)-2-(4′-pentenyl)Alanine, (S)-2-(4′-pentenyl)Alanine; or (vii) 2,2-bis(4-pentenyl)glycine, (R)-2-(propenyl)Alanine, (R)-2-(4′-pentenyl)Alanine; and other such combinations (see, e.g., Bird et al., ACS Chemical Biology, 2020, 15:1340-1348; Bird et al., PNAS, 2010, 107(32):14093-14098; Hilinski et al., JACS, 2014, 136:12314-12322; Bird et al., Nature Structure & Molecular Biology, 2014, 21(12):1058-1067; Kim et al., Organic Letters, 2010, 12(13):3046-3049; Schafmeister et al., J. Am Chem. Soc., 2000, 122:5891-5892; Shim et al., 2013, Chem Biol Drug Des, 2013, 82:635-642; each of which is incorporated by reference herein in its entirety).
In a peptide to be stapled, amino acids that interfere with (e.g., inhibit or reduce the efficiency of) the stapling reaction should be substituted with amino acids that do not interfere with (e.g., do not inhibit or do not substantially reduce the efficiency of) the stapling reaction. For example, methionine (Met, M) may interfere with the stapling reaction; thus, in certain instances, the methionine(s) in a peptide to be stapled is replaced with, e.g., norleucine(s).
In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 1 and 5 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 2 and 6 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 5 and 9 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 6 and 10 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 9 and 13 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 12 and 16 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 13 and 17 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 1 and 8 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 2 and 9 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 6 and 13 of SEQ ID NO:2. In some instances, the staple is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 9 and 16 of SEQ ID NO:2. In some instances, the stitch is located at the amino acid positions in a EBOV GP2 CHR peptide corresponding to positions 5, 8, and 12 of SEQ ID NO:2.
In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:4, the stitch is located at amino acid positions 1 and 5 of SEQ ID NO:4 (i.e., positions 1 and 5 of SEQ ID NO:2). In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:5, the stitch is located at amino acid positions 1 and 5 of SEQ ID NO:5 (i.e., positions 1 and 5 of SEQ ID NO:2). In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:4, the stitch is located at amino acid positions 2 and 6 of SEQ ID NO:4 (i.e., positions 2 and 6 of SEQ ID NO:2). In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:4, the stitch is located at amino acid positions 5 and 9 of SEQ ID NO:4 (i.e., positions 5 and 9 of SEQ ID NO:2). In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:5, the stitch is located at amino acid positions 5 and 9 of SEQ ID NO:5 (i.e., positions 5 and 9 of SEQ ID NO:2). In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:4, the stitch is located at amino acid positions 6 and 10 of SEQ ID NO:4 (i.e., positions 6 and 10 of SEQ ID NO:2). In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:6, the stitch is located at amino acid positions 6 and 10 of SEQ ID NO:6 (i.e., positions 6 and 10 of SEQ ID NO:2). In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:3, the stitch is located at amino acid positions 2 and 9 of SEQ ID NO:3 (i.e., positions 2 and 9 of SEQ ID NO:2). In some instances in which the EBOV GP2 CHR peptide comprises or consists of the amino acid sequence of SEQ ID NO:4, the stitch is located at amino acid positions 2 and 9 of SEQ ID NO:4 (i.e., positions 2 and 9 of SEQ ID NO:2).
In some instances, the stabilized EBOV GP2 CHR peptide comprises a stapled or stitched form of a peptide described in Table 2 (i.e., the stapled or stitched peptide is the product of one or more ring-closing metathesis reaction(s) on a peptide of Table 2).
X
1DWTX2NITDKIDQIIHDFVDK, wherein each of X1 and X2
X
1DWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2
X
1
NWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2
X
1DWTKNIX2DKIDQIIHDFVDK, wherein each of X1 and X2
X
1DWTX2NITDKIDQIIHDFVDK, wherein each of X1 and X2
X
1DWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2
X
1
NWTX2NITDKINQIIHDFVNK, wherein each of X1 and X2
X
1DWTKNIX2DKIDQIIHDFVDK, wherein X1 is (R)-2-(7′-
This disclosure also encompasses peptides or stabilized peptides that are identical to those listed in Table 2 but which have 1, 2, 3, 4, or 5 of the aspartic residues therein replaced by asparagine. In some instances, D2, D12, D20, D2 and D12, D2 and D20, D12 and D20, and D2, D12, and D20 (the numbering corresponding to SEQ ID NO:2) are substituted by asparagine.
In some instances, the disclosure features structurally stabilized (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs:7-9 and 25-27 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 1 and 5 of the amino acid sequence of any one of SEQ ID NOs:7-9 and 25-27 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs:10, 11, 28, and 29 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 2 and 6 of the amino acid sequence of any one of SEQ ID NOs:10, 11, 28, and 29 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs:12, 13, 30, and 31 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 5 and 9 of the amino acid sequence of any one of SEQ ID NOs:12, 13, 30, and 31 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs:14, 15, 32, and 33 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 6 and 10 of the amino acid sequence of any one of SEQ ID NOs:14, 15, 32, and 33 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of SEQ ID NO:16 or 34 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 9 and 13 of the amino acid sequence of SEQ ID NO:16 or 34 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of SEQ ID NO:17 or 35 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 12 and 16 of the amino acid sequence of SEQ ID NO:17 or 35 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of SEQ ID NO:18 or 36 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 13 and 17 of the amino acid sequence of SEQ ID NO:18 or 36 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of SEQ ID NO:19 or 37 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 1 and 8 of the amino acid sequence of SEQ ID NO:19 or 37 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of any one of SEQ ID NOs:20, 21, 38, and 39 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 2 or 9 of the amino acid sequence of any one of SEQ ID NOs:20, 21, 38, and 39 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of SEQ ID NO:22 or 40 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 6 and 13 of the amino acid sequence of SEQ ID NO:22 or 40 are cross-linked (stapled) to each other. In some instances, the disclosure features internally cross-linked (e.g., stapled or stitched) peptides comprising or consisting of the amino acid sequence of SEQ ID NO:23 or 41 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 9 and 16 of the amino acid sequence of SEQ ID NO:23 or 41 are cross-linked (stapled) to each other. In some instances, the disclosure feature an internally cross-linked (e.g., stitched) peptide comprising the amino acid sequence of SEQ ID NO:24 or 42 (or a modified version thereof), wherein the sidechains of the stapling amino acid at positions 5 and 8 of SEQ ID NO:24 or 42 are cross-linked (“stitched”) to each other and the side chains of positions 8 and 12 of SEQ ID NO:24 or 42 are cross-linked to each other, thereby forming a stitch between positions 5, 8, and 12 of SEQ ID NO:24 or 42.
In one aspect, the structurally stabilized EBOV GP2 CHR peptide comprises Formula (I),
or a pharmaceutically acceptable salt thereof, wherein:
In certain instances, the sequences set forth above in Table 3 can have at least one (e.g., 1, 2, 3, 4, 5, 6) amino acid substitution or deletion. The EBOV GP2 CHR peptides can include any amino acid sequence described herein.
The tether of Formula (I) can include an alkyl, alkenyl, or alkynyl moiety (e.g., C5, C8, C11, or C12 alkyl, a C5, C8, or C11 alkenyl, or C5, C8, C11, or C12 alkynyl). The tethered amino acid can be alpha disubstituted (e.g., C1-C3 or methyl).
In some instances of Formula (I), x is 2, 3, or 6. In some instances of Formula (I), each y is independently an integer between 0 and 15, or 3 and 15. In some instances of Formula (I), R1 and R2 are each independently H or C1-C6 alkyl. In some instances of Formula (I), R1 and R2 are each independently C1-C3 alkyl. In some instances or Formula (I), at least one of R1 and R2 are methyl. For example, R1 and R2 can both be methyl. In some instances of Formula (I), R3 is alkyl (e.g., C8 alkyl) and x is 3. In some instances of Formula (I), R3 is C11 alkyl and x is 6. In some instances of Formula (I), R3 is alkenyl (e.g., C8 alkenyl) and x is 3. In some instances of Formula (I), x is 6 and R3 is C11 alkenyl. In some instances, R3 is a straight chain alkyl, alkenyl, or alkynyl. In some instances, R3 is —CH2—CH2—CH2—CH═CH—CH2—CH2—CH2—.
In one aspect, a structurally stabilized EBOV GP2 CHR peptide comprises Formula (I), or a pharmaceutically acceptable salt thereof, wherein:
In another aspect of Formula (I), the two alpha, alpha disubstituted stereocenters are both in the R configuration or S configuration (e.g., i, i+4 cross-link), or one stereocenter is R and the other is S (e.g., i, i+7 cross-link). Thus, where Formula (I) is depicted as:
In some instances of Formula (I), R3 is [R4—K—R4]n; and R4 is a straight chain alkyl, alkenyl, or alkynyl.
In one aspect, a structurally stabilized EBOV GP2 CHR peptide comprises Formula (II):
or a pharmaceutically acceptable salt thereof, wherein:
In some instances of Formula (II), R1 and R4 are each independently H or C1-C6 alkyl. In some instances of Formula (II), R1 and R4 are each independently C1-C3 alkyl. In some instances of Formula (II), at least one of R1 and R4 are methyl. For example, R1 and R4 can both be methyl. In some instances of Formula (II), R2 and R3 are each independently alkyl (e.g., C12 alkyl). In some instances of Formula (II), R2 and R3 are each independently a C12 alkyl. In some instances of Formula (II), R2 and R3 are each independently a straight chain alkyl, alkenyl, or alkynyl (e.g., a straight chain C12 alkyl, alkenyl, or alkynyl. In some instances of Formula (II), R2 is —CH2—CH2—CH2—CH2—CH2—CH2—CH═CH—CH2—CH2—CH2—CH2—. In some instances of Formula (II), R3 is —CH2—CH2—CH2—CH2—CH═CH—CH2—CH2—CH2—CH2—CH2—CH2—.
In some instances, the structurally stabilized EBOV GP2 CHR peptide comprises Formula (II), or a pharmaceutically acceptable salt thereof, wherein:
In another aspect of Formula (II), of the three alpha, alpha disubstituted stereocenters: (i) two stereocenters are in the R configuration and one stereocenter is in the S configuration; or (ii) two stereocenters are in the S configuration and one stereocenter is in the R configuration. Thus, where Formula (II) is depicted as:
In some instances of Formula (II), R3 is [R4—K—R4]n; and R4 is a straight chain alkyl, alkenyl, or alkynyl.
As used herein, the term “Ci-j,” where i and j are integers, employed in combination with a chemical group, designates a range of the number of carbon atoms in the chemical group with i-j defining the range. For example, C1-6 alkyl refers to an alkyl group having 1, 2, 3, 4, 5, or 6 carbon atoms.
As used herein, the term “alkyl,” employed alone or in combination with other terms, refers to a saturated hydrocarbon group that may be straight-chain or branched. In some instances, the alkyl group contains 1 to 7, 1 to 6, 1 to 4, or 1 to 3 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 2-methyl-1-butyl, 3-pentyl, n-hexyl, 1,2,2-trimethylpropyl, n-heptyl, and the like. In some instances, the alkyl group is methyl, ethyl, or propyl. The term “alkylene” refers to a linking alkyl group.
As used herein, “alkenyl,” employed alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon double bonds. In some instances, the alkenyl moiety contains 2 to 6 or 2 to 4 carbon atoms. Example alkenyl groups include, but are not limited to, ethenyl, n-propenyl, isopropenyl, n-butenyl, sec-butenyl, and the like.
As used herein, “alkynyl,” employed alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some instances, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.
As used herein, “alkynyl,” employed alone or in combination with other terms, refers to an alkyl group having one or more carbon-carbon triple bonds. Example alkynyl groups include, but are not limited to, ethynyl, propyn-1-yl, propyn-2-yl, and the like. In some instances, the alkynyl moiety contains 2 to 6 or 2 to 4 carbon atoms.
As used herein, the term “cycloalkylalkyl,” employed alone or in combination with other terms, refers to a group of formula cycloalkyl-alkyl-. In some instances, the alkyl portion has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom(s). In some instances, the alkyl portion is methylene. In some instances, the cycloalkyl portion has 3 to 10 ring members or 3 to 7 ring members. In some instances, the cycloalkyl group is monocyclic or bicyclic. In some instances, the cycloalkyl portion is monocyclic. In some instances, the cycloalkyl portion is a C3-7 monocyclic cycloalkyl group.
As used herein, the term “heteroarylalkyl,” employed alone or in combination with other terms, refers to a group of formula heteroaryl-alkyl-. In some instances, the alkyl portion has 1 to 4, 1 to 3, 1 to 2, or 1 carbon atom(s). In some instances, the alkyl portion is methylene. In some instances, the heteroaryl portion is a monocyclic or bicyclic group having 1, 2, 3, or 4 heteroatoms independently selected from nitrogen, sulfur and oxygen. In some instances, the heteroaryl portion has 5 to 10 carbon atoms.
As used herein, the term “substituted” means that a hydrogen atom is replaced by a non-hydrogen group. It is to be understood that substitution at a given atom is limited by valency.
As used herein, “halo” or “halogen”, employed alone or in combination with other terms, includes fluoro, chloro, bromo, and iodo. In some instances, halo is F or Cl.
In some instances, the disclosure features structurally stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NOs:2-6 (or a modified version thereof), wherein: the side chains of two amino acids separated by two, three, or six amino acids are replaced by an internal staple, the side chains of three amino acids are replaced by an internal stitch, the side chains of four amino acids are replaced by two internal staples, or the side chains of five amino acids are replaced by the combination of an internal staple and an internal stitch. In some instances, the disclosure features structurally stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NOs:2-6 (or a modified version thereof), wherein the side chains of two amino acids separated by two, three, or six amino acids are replaced by an internal staple. In some instances, the disclosure features structurally stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NOs:2-6 (or a modified version thereof), wherein the side chains of two amino acids separated by three amino acids are replaced by an internal staple. In some instances, the disclosure features structurally stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NOs:2-6 (or a modified version thereof), wherein the side chains of two amino acids separated by six amino acids are replaced by an internal staple. In some instances, the disclosure features structurally stabilized (e.g., stapled or stitched) peptides comprising the amino acid sequence of any one of SEQ ID NOs:2-6 (or a modified version thereof), wherein the side chains of three amino acids are replaced by an internal stitch. In certain instances, the amino acids corresponding to one or more of positions Trp3, Thr4, Ile7, Ile11, Ile14, Ile15, and Phe18 of SEQ ID NO:2 are not replaced with a staple or stitch. The stapled or stitched peptide can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In a specific instance, the stapled or stitched peptide is 5-21 amino acids (i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) in length. In a specific instance, the stapled or stitched peptide is 10-21 amino acids (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) amino acids in length. In a specific instance, the stapled or stitched peptide is 15-21 amino acids (i.e., 15, 16, 17, 18, 19, 20, 21) amino acids in length. In a specific instance, the stapled or stitched peptide is 21 amino acids in length. In a specific instance, the stapled or stitched peptide is 5-30 amino acids (i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) in length. In a specific instance, the stapled or stitched peptide is 10-30 amino acids (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) amino acids in length. In a specific instance, the stapled or stitched peptide is 15-30 amino acids (i.e., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) amino acids in length. In a specific instance, the stapled or stitched peptide is 13 amino acids in length. Exemplary EBOV GP2 CHR stapled or stitched peptides are shown in Table 2 and described in Formula (I) and Table 3. In one instance, the EBOV GP2 CHR stapled or stitched peptide comprises or consists of a stapled or stitched version of the amino acid sequence of any one of SEQ ID NOs: 7-42 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of any one of SEQ ID NOs:7-42, respectively). In one instance, the EBOV GP2 CHR stapled or stitched peptide comprises or consists of a stapled or stitched version of the amino acid sequence of SEQ ID NO: 15 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of SEQ ID NO:15). In one instance, the EBOV GP2 CHR stapled or stitched peptide comprises or consists of a stapled or stitched version of the amino acid sequence of SEQ ID NO: 14 (e.g., the product of a ring-closing metathesis reaction performed on a peptide comprising the amino acid sequence of SEQ ID NO:14).
EBOV GP2 CHR stapled and stitched peptides are shown in Tables 2 to Tables 4. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:7. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:25. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:8. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:26. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:9. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:27. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:10. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:28. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:11. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:29. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:12. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:30. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:13. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:31. In one instance, the EBOV GP2 CHR stapled peptide comprises the amino acid sequence of SEQ ID NO:14. In one instance, the EBOV GP2 CHR stapled peptide consists of the amino acid sequence of SEQ ID NO:14. In one instance, the EBOV GP2 CHR stapled peptide comprises the amino acid sequence of SEQ ID NO:32. In one instance, the EBOV GP2 CHR stapled peptide consists of the amino acid sequence of SEQ ID NO:32. In one instance, the EBOV GP2 CHR stapled peptide comprises the amino acid sequence of SEQ ID NO:15. In one instance, the EBOV GP2 CHR stapled peptide consists of the amino acid sequence of SEQ ID NO:15. In one instance, the EBOV GP2 CHR stapled peptide comprises the amino acid sequence of SEQ ID NO:33. In one instance, the EBOV GP2 CHR stapled peptide consists of the amino acid sequence of SEQ ID NO:33. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:16. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:34. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:17. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:35. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:18. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:36. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:19. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:37. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:20. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:38. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:21. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:39. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:22. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:40. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:23. In one instance, the EBOV GP2 CHR stapled peptide comprises or consists of the amino acid sequence of SEQ ID NO:41. In one instance, the EBOV GP2 CHR stitched peptide comprises or consists of the amino acid sequence of SEQ ID NO:24. In one instance, the EBOV GP2 CHR stitched peptide comprises or consists of the amino acid sequence of SEQ ID NO:42.
In certain instances, the stapled peptide comprises or consists of a variant of the amino acid sequence set forth in any one of SEQ ID NOs:2-6, wherein two amino acids each separated by 3 amino acids (i.e., positions i and i+4) are modified to structurally stabilize the peptide (e.g., by substituting them with non-natural amino acids to permit hydrocarbon stitching, i.e., stapling amino acids). In certain instances, the two amino acids each separated by three amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 1 and 5 of SEQ ID NO:2. In certain instances, the two amino acids each separated by three amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 2 and 6 of SEQ ID NO:2. In certain instances, the two amino acids each separated by three amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 5 and 9 of SEQ ID NO:2. In certain instances, the two amino acids each separated by three amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 6 and 10 of SEQ ID NO:2. In certain instances, the two amino acids each separated by three amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 9 and 13 of SEQ ID NO:2. In certain instances, the two amino acids each separated by three amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 12 and 16 of SEQ ID NO:2. In certain instances, the two amino acids each separated by three amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 13 and 17 of SEQ ID NO:2.
In certain instances, the stapled peptide comprises or consists of a variant of the amino acid sequence set forth in any one of SEQ ID NOs:2-6, wherein two amino acids each separated by 6 amino acids (i.e., positions i and i+7) are modified to structurally stabilize the peptide (e.g., by substituting them with non-natural amino acids to permit hydrocarbon stapling, i.e., with stapling amino acids). In certain instances, the two amino acids each separated by six amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 1 and 8 of SEQ ID NO:2. In certain instances, the two amino acids each separated by six amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 2 and 9 of SEQ ID NO:2. In certain instances, the two amino acids each separated by six amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 6 and 13 of SEQ ID NO:2. In certain instances, the two amino acids each separated by six amino acids are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 9 and 16 of SEQ ID NO:2.
In certain instances, the stitched peptide comprises or consists of a variant of the amino acid sequence set forth in any one of SEQ ID NOs:2-6, wherein three amino, at positions i, i+3, and i+7, are modified to structurally stabilize the peptide (e.g., by substituting them with non-natural amino acids to permit hydrocarbon stitching, i.e., with stitching amino acids). In certain instances, the three amino acids at positions i, i+3, and i+7 are at the amino acid positions in the EBOV GP2 CHR peptide corresponding to positions 5, 8, and 12 of SEQ ID NO:2.
While hydrocarbon tethers are common, other tethers can also be employed in the structurally stabilized EBOV GP2 CHR peptides described herein. For example, the tether can include one or more of an ether, thioether, ester, amine, or amide, or triazole moiety. In some cases, a naturally occurring amino acid side chain can be incorporated into the tether. For example, a tether can be coupled with a functional group such as the hydroxyl in serine, the thiol in cysteine, the primary amine in lysine, the acid in aspartate or glutamate, or the amide in asparagine or glutamine. Accordingly, it is possible to create a tether using naturally occurring amino acids rather than using a tether that is made by coupling two non-naturally occurring amino acids. It is also possible to use a single non-naturally occurring amino acid together with a naturally occurring amino acid. Triazole-containing (e.g., 1, 4 triazole or 1, 5 triazole) crosslinks can be used (see, e.g., Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137; WO 2010/060112). In addition, other methods of performing different types of stapling are well known in the art and can be employed with the EBOV GP2 CHR peptides described herein (see, e.g., Lactam stapling: Shepherd et al., J Am. Chem. Soc., 127:2974-2983 (2005); UV-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); Photoswitchable 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 et al., J. Am. Chem. Soc., 135:5946-5949 (2013)).
It is further envisioned that the length of the tether can be varied. For instance, a shorter length of tether can be used where it is desirable to provide a relatively high degree of constraint on the secondary alpha-helical structure, whereas, in some instances, it is desirable to provide less constraint on the secondary alpha-helical structure, and thus a longer tether may be desired.
Additionally, while tethers spanning from amino acids i to i+3, i to i+4, and i to i+7 are common in order to provide a tether that is primarily on a single face of the alpha helix, the tethers can be synthesized to span any combinations of numbers of amino acids and also used in combination to install multiple tethers.
In some instances, the hydrocarbon tethers (i.e., cross links) described herein can be further manipulated. In one instance, a double bond of a hydrocarbon alkenyl tether, (e.g., as synthesized using a ruthenium-catalyzed ring closing metathesis (RCM)) can be oxidized (e.g., via epoxidation, aminohydroxylation or dihydroxylation) to provide one of compounds below.
Either the epoxide moiety or one of the free hydroxyl moieties can be further functionalized. For example, the epoxide can be treated with a nucleophile, which provides additional functionality that can be used, for example, to attach a therapeutic agent. Such derivatization can alternatively be achieved by synthetic manipulation of the amino or carboxy-terminus of the peptide or via the amino acid side chain. Other agents can be attached to the functionalized tether, e.g., an agent that facilitates entry of the peptide into cells.
In some instances, alpha disubstituted amino acids are used in the peptide to improve the stability of the alpha helical secondary structure. However, alpha disubstituted amino acids are not required, and instances using mono-alpha substituents (e.g., in the tethered amino acids) are also envisioned.
The structurally stabilized (e.g., stapled or stitched) peptides can include a drug, a toxin, a derivative of polyethylene glycol; a second peptide; a carbohydrate, etc. Where a polymer or other agent is linked to the structurally stabilized (e.g., stapled or stitched) peptide, it can be desirable for the composition to be substantially homogeneous.
The addition of polyethelene glycol (PEG) molecules can improve the pharmacokinetic and pharmacodynamic properties of the peptide. For example, PEGylation can reduce renal clearance and can result in a more stable plasma concentration. PEG is a water soluble polymer and can be represented as linked to the peptide as formula:
XO—(CH2CH2O)n—CH2CH2—Y where n is 2 to 10,000 and X is H or a terminal modification, e.g., a C1-4 alkyl; and Y is an amide, carbamate or urea linkage to an amine group (including but not limited to, the epsilon amine of lysine or the N-terminus) of the peptide. Y may also be a maleimide linkage to a thiol group (including but not limited to, the thiol group of cysteine). Other methods for linking PEG to a peptide, directly or indirectly, are known to those of ordinary skill in the art. The PEG can be linear or branched. Various forms of PEG including various functionalized derivatives are commercially available.
PEG having degradable linkages in the backbone can be used. For example, PEG can be prepared with ester linkages that are subject to hydrolysis. Conjugates having degradable PEG linkages are described in WO 99/34833; WO 99/14259, and U.S. Pat. No. 6,348,558.
In certain instances, macromolecular polymer (e.g., PEG) is attached to a structurally stabilized (e.g., stapled or stitched) peptide described herein through an intermediate linker. In certain instances, the linker is made up of from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those in the art. In other instances, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other instances, a linker is made up of a majority of amino acids that are sterically unhindered, such as glycine and alanine. Non-peptide linkers are also possible. For example, alkyl linkers such as —NH(CH2)nC(O)—, wherein n=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. U.S. Pat. No. 5,446,090 describes a bifunctional PEG linker and its use in forming conjugates having a peptide at each of the PEG linker termini.
The structurally stabilized (e.g., stapled or stitched) peptides can also be modified, e.g., to further facilitate cellular uptake or increase in vivo stability, in some instances. For example, acylating or PEGylating a structurally stabilized peptide facilitates cellular uptake, increases bioavailability, increases blood circulation, alters pharmacokinetics, decreases immunogenicity and/or decreases the needed frequency of administration.
In some instances, the structurally stabilized (e.g., stapled or stitched) peptides disclosed herein have an enhanced ability to penetrate cell membranes (e.g., relative to non-stabilized peptides). See, e.g., International Publication No. WO 2017/147283, which is incorporated by reference herein in its entirety.
In some instances, the pharmaceutically acceptable salt is an acetate, a sulfate, or a chloride. Lists of other suitable salts are found in Remington's Pharmaceutical Sciences, 17th Ed., (Mack Publishing Company, Easton, 1985), p. 1418, Berge et al., J. Pharm. Sci., 1977, 66(1), 1-19 and in Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, (Wiley, 2002).
In certain instances, each of the stabilized EBOV GP2 CHR peptides described above bind to a 5 helix bundle of EBOV GP2 or fusion bundle intermediate of EBOV GP2. In some instances, each of the stabilized EBOV GP2 CHR peptides described above bind to a 5 helix bundle of EBOV GP2 and prevents or blocks fusion of an Ebola virus membrane and a host membrane.
Properties of the stabilized (e.g., stapled or stitched) peptides of the invention can be assayed, for example, using the methods described below and in the Examples.
Assays to Determine α-Helicity: Compounds are dissolved in an aqueous solution (e.g., 5 mM potassium phosphate solution at pH 7, or distilled H2O, to concentrations of 25-50 μM). Circular dichroism (CD) spectra are obtained on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard measurement parameters (e.g., temperature, 20° C.; wavelength, 190-260 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; path length, 0.1 cm). The α-helical content of each peptide is calculated by dividing the mean residue ellipticity by the reported value for a model helical decapeptide (Yang et al., Methods Enzymol. 130:208 (1986)).
Assays to Determine Melting Temperature (Tm): Cross-linked or the unmodified template peptides are dissolved in distilled H2O or other buffer or solvent (e.g., at a final concentration of 50 μM) and Tm is determined by measuring the change in ellipticity over a temperature range (e.g., 4 to 95° C.) on a spectropolarimeter (e.g., Jasco J-710, Aviv) using standard parameters (e.g., wavelength 222 nm; step resolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1° C./min; path length, 0.1 cm).
In vitro Protease Resistance Assays: The amide bond of the peptide backbone is susceptible to hydrolysis by proteases, thereby rendering peptidic compounds vulnerable to rapid degradation in vivo. Peptide helix formation, however, typically buries and/or twists and/or shields the amide backbone and therefore may prevent or substantially retard proteolytic cleavage. The stabilized peptides of the present invention may be subjected to in vitro enzymatic proteolysis (e.g., trypsin, chymotrypsin, pepsin) to assess for any change in degradation rate compared to a corresponding unstabilized or alternatively stapled or stitched peptide. For example, the stabilized peptide and a corresponding unstabilized peptide are incubated with trypsin agarose and the reactions quenched at various time points by centrifugation and subsequent HPLC injection to quantitate the residual substrate by ultraviolet absorption at 280 nm. Briefly, the stabilized peptide and its precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S/E ˜125) for 0, 10, 20, 90, and 180 minutes. Reactions are quenched by tabletop centrifugation at high speed; remaining substrate in the isolated supernatant is quantified by HPLC-based peak detection at 280 nm. The proteolytic reaction displays first order kinetics and the rate constant, k, is determined from a plot of In[S] versus time.
Stabilized peptides and/or a corresponding unstabilized peptide can be each incubated with fresh mouse, rat and/or human serum (e.g., 1-2 mL) at 37° C. for, e.g., 0, 1, 2, 4, 8, and 24 hours. Samples of differing stabilized peptide concentration may be prepared by serial dilution with serum. To determine the level of intact compound, the following procedure may be used: The samples are extracted, for example, by transferring 100 μL of sera to 2 ml centrifuge tubes followed by the addition of 10 μL of 50% formic acid and 500 μL acetonitrile and centrifugation at 14,000 RPM for 10 min at 4+/−2° C. The supernatants are then transferred to fresh 2 ml tubes and evaporated on Turbovap under N2<10 psi, 37° C. The samples are reconstituted in 100 μL of 50:50 acetonitrile:water and submitted to LC-MS/MS analysis. Equivalent or similar procedures for testing ex vivo stability are known and may be used to determine stability of stabilized peptides in serum.
In vivo Protease Resistance Assays: A key benefit of peptide stapling or stitching is the translation of in vitro protease resistance into markedly improved pharmacokinetics in vivo.
In vitro Binding Assays: To assess the binding and affinity of stabilized peptides and their precursors to acceptor proteins, a fluorescence polarization assay (FPA) can be used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g., FITC-labeled peptides bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g., FITC-labeled peptides that are free in solution).
In some instances, structurally stabilized (e.g., stapled or stitched) peptides can be made by modifying (e.g., by amino acid substitution) a peptide of any one of SEQ ID NOs:2-6 or a modified version thereof. In some instances, an internal staple replaces the side chains of 2 amino acids, i.e., each staple is between two amino acids separated by, for example, 2, 3, or 6 amino acids. In some instances, an internal stitch replaces the side chains of 3 amino acids, i.e., the stitch is a pair of crosslinks between three amino acids separated by, for example, 2, 3, or 6 amino acids. In some instances, the internal stitch replaces the side chain of a first amino acid and a second and a third amino acid thereby cross-linking the first amino acid (which lies between the second and third amino acids) to the second and third amino acid via an internal cross-link, wherein the first and second amino acid are separated by two, three, or six amino acids, the first and the third amino acids are separated by three or six amino acids, and the second and third amino acids are distinct amino acids.
The structurally stabilized (e.g., stapled or stitched) peptide comprises at least two modified amino acids (relative to an EBOV GP2 CHR peptide) joined by an internal intramolecular cross-link (or “staple”), wherein the at least two amino acids are separated by 2, 3, or 6 amino acids. Structurally stabilized peptides herein include stapled peptides, including peptides having two staples and/or stitched peptides. The at least two modified amino acids can be non-natural alpha-amino acids (including, but not limited to α,α-disubstituted and N-alkylated amino acids). There are many known non-natural amino acids that may be used as stapling amino acids or stitching amino acids, any of which may be included in the peptides of the present invention. Some examples of non-natural amino acids that may be used as stapling amino acids or stitching amino acids are: (R)-2-(7′-octenyl)Alanine, (S)-2-(7′-octenyl)Alanine, (S)-2-(4′-pentenyl)Alanine, (R)-2-(4′-pentenyl)Alanine, bis-S5/R5, 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-leucine, 1-amino-cyclopropanecarboxylic acid, 1-amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-1-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta- and/para-substituted phenylalanines (e.g., substituted with —C(═O)C6H5; —CF3; —CN; -halo; —NO2; CH3), disubstituted phenylalanines, substituted tyrosines (e.g., further substituted with -Q=O)C6H5; —CF3; —CN; -halo; —NO2; CH3), and statine. In some instances, the amino acids forming the staple or stitch (also referred to as the “stapling amino acids” or the “stitching amino acids” are (S)-2-(4′-pentenyl)Alanine at each of positions i and i+4. In some instances, the amino acids forming the staple or stitch are (R)-2-(4′-pentenyl)Alanine at each of positions i and i+4. In some instances, the amino acids forming the staple or stitch are (R)-2-(7′-octenyl)Alanine and (S)-2-(4′-pentenyl)Alanine at positions i and i+7, respectively. In some instances, the amino acids forming the staple or stitch are (S)-2-(4′-pentenyl)Alanine, 2,2-bis(4-pentenyl)glycine, and (S)-α-(7′-octenyl)alanine at positions i, i+4, and i+11, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (R)-α-(4′-pentenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (R)-α-(7′-octenyl)alanine at positions i, i+4, and i+11, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (R)-2-(4′-pentenyl)Alanine, 2,2-bis(4-pentenyl)glycine, and (R)-2-(4′-pentenyl)alanine at positions i, i+3, and i+7, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (S)-2-(4′-pentenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (S)-2-(4′-pentenyl)alanine at positions i, i+3, and i+7, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (R)-α-(propenyl)alanine, 2,2-bis(4-pentenyl)glycine, and (R)-2-(4′-pentenyl)alanine at positions i, i+3, and i+7, respectively, of the stitch. In some instances, the amino acids forming the staple or stitch are (R)-2-(4′-pentenyl)Alanine, 2,2-bis(4-pentenyl)glycine, and (S)-2-(4′-pentenyl)Alanine at positions i, i+4, and i+8, respectively, of the stitch. The skilled artisan will appreciate that other combinations for staples and stitches are permitted, such as, e.g., i, i+4 and i, i+4; i, i+4 and i, i+7; i, i+7 and i, i+4; and i, i+7 and i, i+7. See, e.g., Bird et al., ACS Chemical Biology, 2020, 15:1340-1348; Bird et al., PNAS, 2010, 107(32):14093-14098; Hilinski et al., JACS, 2014, 136:12314-12322; Bird et al., Nature Structure & Molecular Biology, 2014, 21(12):1058-1067; Kim et al., Organic Letters, 2010, 12(13):3046-3049; Schafmeister et al., J. Am Chem. Soc., 2000, 122:5891-5892; Shim et al., 2013, Chem Biol Drug Des, 2013, 82:635-642 (each of which is incorporated by reference herein in its entirety) for exemplary staples and stitches.
In some instances, structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide variants of the disclosure are prepared from a peptide of any one of SEQ ID NOs:2-6 and having e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids are conservatively or non-conservatively substituted) and/or having, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid deletions from the N- and/or C-terminus (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids from the N- and/or C-terminus are deleted). Exemplary EBOV GP2 CHR peptides, including variants, are provided in Table 1 and in the amino acid sequence of SEQ ID NO:2. For example, in certain instances, the structurally stabilized EBOV GP2 CHR peptide variants of this disclosure can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in any one of SEQ ID NOs:2-6 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids are conservatively or non-conservatively substituted). In some instances, one to three amino acids of any one of SEQ ID NOs:2-6 are substituted. The amino acid substitutions in any one of SEQ ID NOs:2-6 can be of non-NHR-interacting amino acids. Much greater variability is permitted in the non-NHR-interacting amino acids. In fact, just about every one of non-NHR-interacting amino acids can be substituted (e.g., conservative or non-conservative amino acid substitutions or substitution with alanine). In certain instances, one, two, or three NHR-interacting amino acids amino acids are substituted with another amino acid. In some instances, the substitution(s) is/are a conservative amino acid substitution. In other instances, the substitution(s) is/are a non-conservative amino acid substitution.
In some instances, where there are more than one amino acid substitutions, the substitutions are both conservative and non-conservative amino acid substitutions. In some instances, where there are more than one amino acid substitutions, each of the substitutions are conservative amino acid substitutions. In some cases, where one to three amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs:2-6 are substituted, the substitutions are all of non-NHR-interacting amino acids. In some cases, where one to three amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs:2-6 are substituted, the substitutions are all of NHR-interacting amino acids. In some cases, where one to three amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs:2-6 are substituted, the substitutions are of both non-NHR-interacting amino acids and NHR-interacting amino acids. In certain instances, the substituted amino acid(s) are selected from the group consisting of L-Ala, D-Ala, Aib, Sar, Ser, a substituted alanine, or a substituted glycine derivative.
In some instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12) amino acids corresponding to amino acids W615, T616, N618, I619, K622, 1623, Q625, I626, I627, D629, F630, and V631 (numbered according to SEQ ID NO:1), or conservative amino acid substitutions thereof.
In some instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides comprise a conservative amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) amino acids corresponding to amino acids K617, T620, D621, D624, and H628 (numbered according to SEQ ID NO: 1). In some instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides comprise a non-conservative amino acid substitution at one or more (e.g., 1, 2, 3, 4, or 5) amino acids corresponding to amino acids K617, T620, D621, D624, and H628 (numbered according to SEQ ID NO:1).
In some instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides do not comprise one or more (e.g., 1, 2, 3, or 4) of the amino acids corresponding to positions 615, 616, 630, and 631 of the amino acid sequence set forth in SEQ ID NO:1. For example, in some instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides do not comprise the amino acids corresponding to positions 615, 616, 630, and 631 of the amino acid sequence set forth in SEQ ID NO:1. As another example, in some instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides do not comprise the amino acids corresponding to positions 615 and 616 of the amino acid sequence set forth in SEQ ID NO:1. As another example, in some instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides do not comprise the amino acids corresponding to positions 630 and 631 of the amino acid sequence set forth in SEQ ID NO:1.
In certain instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides of this disclosure can have 1, 2, 3, 4, or 5, amino acids removed/deleted from the C-terminus of the sequence set forth in any one of SEQ ID NOs:2-6. In certain instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR variant peptides of this disclosure can have 1, 2, 3, 4, or 5, amino acid removed/deleted from the N-terminus of the sequence set forth in any one of SEQ ID NOs:2-6. In certain instances, these removed amino acids can be replaced with 1-6 (e.g., 1, 2, 3, 4, 5, or 6) amino acids selected from the group consisting of L-Ala, D-Ala, Aib, Sar, Ser, a substituted alanine, or a substituted glycine derivative.
The structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide variants described herein can be optimized for therapeutic use. For example, if any of the above-described structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide variants cause membrane disruption (cell lysis), the peptides can be optimized by lowering the overall peptide hydrophobicity. This can for example be achieved by substituting especially hydrophobic residues with an amino acid with lower hydrophobicity (e.g., alanine). Membrane disruption can also be lowered by reducing the overall positive charge of the peptide. This can be accomplished by substituting basic residues with uncharged or acidic residues. In certain instances, both the overall peptide hydrophobicity and the overall positive charge of the peptide are lowered. In some instances, the overall charge of the peptide is −2 to +2. In some instances, the overall charge of the peptide is −3 to +3. In some instances, the overall charge of the peptide is −1 to +3. In some instances, the overall charge of the peptide is 0 to +3. In some instances, the overall charge of the peptide is 0 to +2.
In certain instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide variants described herein are from 5 to 50 (i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50) amino acids in length, from 5 to 35 (i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35) amino acids in length, from 5 to 30 (i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) amino acids in length, from 5 to 25 (i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25) amino acids in length, from 5 to 21 (i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) amino acids in length, from 10 to 21 (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) amino acids in length, from 15 to 21 (i.e., 15, 16, 17, 18, 19, 20, 21) amino acids in length, from 10 to 30 (i.e., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) amino acids in length, or from 15 to 30 (i.e., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30) amino acids in length. In certain instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide variants described herein are 21 amino acids in length.
In certain instances, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide variant comprises or consists of the amino acid sequence set forth in Table 2. In certain instances, the internally cross-linked (e.g., stapled or stitched) EBOV GP2 CHR peptide variant comprises or consists of any one of constructs 1-17 of Table 3 or construct 18 or Table 4.
In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:6. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide comprises the amino acid sequence of SEQ ID NO:6 with: (i) two or more amino acid substitutions with stapling amino acids, and (ii) 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) additional amino acid substitutions, insertions, and/or deletions. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide consists of the amino acid sequence of SEQ ID NO:6 with: (i) two or more amino acid substitutions with stapling amino acids, and (ii) 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) additional amino acid substitutions, insertions, and/or deletions. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:6 with 0 to 3 amino acid deletions from the N-terminus. In certain instances, the stabilized peptide is based on the amino acid sequence of SEQ ID NO:6 with 0 to 3 amino acid deletions from the C-terminus. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of HNWTKX1ITNX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:15). In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide comprises the amino acid sequence of HNWTKX1ITNX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:15), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions relative to SEQ ID NO:15 at position(s) other than X1 and X2. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide consists of the amino acid sequence of HNWTKX1ITNX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:15), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions relative to SEQ ID NO:15 at position(s) other than X1 and X2. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:15 with 0 to 3 amino acid deletions from the N-terminus. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:15 with 0 to 3 amino acid deletions from the C-terminus. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide comprises the amino acid sequence of HNWTKX1ITNX21NQIIHDFVNK, wherein each of X1 and X2 is (S)-2-(4′-pentenyl)Alanine (SEQ ID NO:33), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions relative to SEQ ID NO:33 at position(s) other than X1 and X2. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide consists of the amino acid sequence of HNWTKX1ITNX2INQIIHDFVNK, wherein each of X1 and X2 is (S)-2-(4′-pentenyl)Alanine (SEQ ID NO:33), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions relative to SEQ ID NO:33 at position(s) other than X1 and X2. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:33 with 0 to 3 amino acid deletions from the N-terminus. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:33 with 0 to 3 amino acid deletions from the C-terminus. In certain instances, the 1 to 3 amino acid in SEQ ID NO:6, 15, or 33 that are removed from the N-terminus are replaced with 1 to 6 amino acids from the croup consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methyl glycine, serine, a substituted alanine, and a glycine derivative. In certain instances, the 1 to 3 amino acid in SEQ ID NO:6, 15, or 33 that are removed from the C-terminus are replaced with 1 to 6 amino acids from the croup consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methyl glycine, serine, a substituted alanine, and a glycine derivative. In certain instances, the 1 to 6 amino acid substitutions relative to SEQ ID NO:6, 15, or 33 are of non-NHR-interacting residues. In certain instances, the 1 to 6 amino acid substitutions relative to SEQ ID NO:6, 15, or 33 are of NHR-interacting residues. In certain instances, the 1 to 6 amino acid substitutions relative to SEQ ID NO:6, 15, or 33 are of non-NHR-interacting residues and NHR-interacting residues.
In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide comprises an amino acid sequence of Formula (I), wherein [Xaa]w is HNWTK (SEQ ID NO:52), [Xaa]x is ITN, and [Xaa]y is INQIIHDFVNK (SEQ ID NO:51), R1 is a methyl group, R2 is a methyl group, and R3 is 4-octenyl. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide consists of an amino acid sequence of Formula (I), wherein [Xaa]w is HNWTK (SEQ ID NO:52), [Xaa]x is ITN, and [Xaa]y is INQIIHDFVNK (SEQ ID NO:51), R1 is a methyl group, R2 is a methyl group, and R3 is 4-octenyl.
In a specific instance, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide comprises a stapled form of the amino acid sequence of SEQ ID NO:15 (e.g., the product of a ring-closing metathesis reaction on SEQ ID NO:15). In a specific instance, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide consists of a stapled form of the amino acid sequence of SEQ ID NO:15 (e.g., the product of a ring-closing metathesis reaction on SEQ ID NO:15).
In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:4 with 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:14 with 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:4. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide comprises the amino acid sequence of SEQ ID NO:4 with: (i) two or more amino acid substitutions with stapling amino acids, and (ii) 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) additional amino acid substitutions, insertions, and/or deletions. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide consists of the amino acid sequence of SEQ ID NO:4 with: (i) two or more amino acid substitutions with stapling amino acids, and (ii) 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) additional amino acid substitutions, insertions, and/or deletions. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:4 with 0 to 3 amino acid deletions from the N-terminus. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:4 with 0 to 3 amino acid deletions from the C-terminus. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of HDWTKX1ITDX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:14). In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide comprises the amino acid sequence of HDWTKX1ITDX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:14), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions relative to SEQ ID NO:14 at position(s) other than X1 and X2. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide consists of the amino acid sequence of HDWTKX1ITDX2INQIIHDFVNK, wherein each of X1 and X2 is independently a stapling amino acid (SEQ ID NO:14), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions relative to SEQ ID NO:14 at position(s) other than X1 and X2. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:14 with 0 to 3 amino acid deletions from the N-terminus. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:14 with 0 to 3 amino acid deletions from the C-terminus. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide comprises the amino acid sequence of HDWTKX1ITDX2INQIIHDFVNK, wherein each of X1 and X2 is (S)-2-(4′-pentenyl)Alanine (SEQ ID NO:32), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions relative to SEQ ID NO:32 at position(s) other than X1 and X2. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide consists of the amino acid sequence of HDWTKX1ITDX2INQIIHDFVNK, wherein each of X1 and X2 is (S)-2-(4′-pentenyl)Alanine (SEQ ID NO:32), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and/or deletions relative to SEQ ID NO:32 at position(s) other than X1 and X2. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:32 with 0 to 3 amino acid deletions from the N-terminus. In certain instances, the structurally stabilized (e.g., stapled or stitched) peptide is based on the amino acid sequence of SEQ ID NO:32 with 0 to 3 amino acid deletions from the C-terminus. In certain instances, the 1 to 3 amino acid in SEQ ID NO:4, 14, or 32 that are removed from the N-terminus are replaced with 1 to 6 amino acids from the croup consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methyl glycine, serine, a substituted alanine, and a glycine derivative. In certain instances, the 1 to 3 amino acid in SEQ ID NO: 4, 14, or 32 that are removed from the C-terminus are replaced with 1 to 6 amino acids from the croup consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methyl glycine, serine, a substituted alanine, and a glycine derivative. In certain instances, the 1 to 6 amino acid substitutions relative to SEQ ID NO: 4, 14, or 32 are of non-NHR-interacting residues. In certain instances, the 1 to 6 amino acid substitutions relative to SEQ ID NO: 4, 14, or 32 are of NHR-interacting residues. In certain instances, the 1 to 6 amino acid substitutions relative to SEQ ID NO: 4, 14, or 32 are of non-NHR-interacting residues and NHR-interacting residues.
In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide comprises an amino acid sequence of Formula (I), wherein [Xaa]W is HDWTK (SEQ ID NO:50), [Xaa]x is ITD, and [Xaa]y is INQIIHDFVNK (SEQ ID NO:51), R1 is a methyl group, R2 is a methyl group, and R3 is 4-octenyl. In a specific instance, the structurally stabilized (e.g., stapled or stitched) peptide consists of an amino acid sequence of Formula (I), wherein [Xaa]w is HDWTK (SEQ ID NO:50), [Xaa]x is ITD, and [Xaa]y is INQIIHDFVNK (SEQ ID NO:51), R1 is a methyl group, R2 is a methyl group, and R3 is an alkenyl.
In a specific instance, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide comprises a stapled form of the amino acid sequence of SEQ ID NO:14 (e.g., the product of a ring-closing metathesis reaction on SEQ ID NO:14). In a specific instance, the structurally stabilized (e.g., stapled or stitched) EBOV GP2 CHR peptide consists of a stapled form of the amino acid sequence of SEQ ID NO:14 (e.g., the product of a ring-closing metathesis reaction on SEQ ID NO:14).
In certain instances, each of the EBOV GP2 CHR structurally stabilized peptides described above bind to a 5 helix bundle of EBOV GP2 or fusion bundle intermediate of EBOV GP2. In some instances, the EBOV GP2 CHR structurally stabilized peptides described above prevent or block fusion of the virus and host membranes.
The disclosure features methods of using any of the structurally stabilized (e.g., stapled or stitched) peptides (or pharmaceutical compositions comprising said structurally stabilized peptides) described herein for the prevention and/or treatment of an Ebola virus infection or Ebola virus disease. The terms “treat” or “treating,” as used herein, refers to alleviating, inhibiting, or ameliorating the disease or infection from which the subject is suffering.
The structurally stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can be useful for treating a subject (e.g., human, non-human primate, or fruit bat) having an Ebolavirus infection. The structurally stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can also be useful for treating a subject (e.g., human, non-human primate, or fruit bat) having an Ebolavirus disease. In certain instances, the Ebolavirus infection is a Zaire ebolavirus infection. In certain instances, the Ebolavirus disease is caused by a Zaire ebolavirus infection. In certain instances, the Ebolavirus infection is a Bundibugyo ebolavirus infection. In certain instances, the Ebolavirus disease is caused by a Bundibugyo ebolavirus infection. In certain instances, the Ebolavirus infection is a Sudan ebolavirus infection. In certain instances, the Ebolavirus disease is caused by a Sudan ebolavirus infection. In certain instances, the Ebolavirus infection is a Tai Forest ebolavirus infection. In certain instances, the Ebolavirus disease is caused by a Tai Forest ebolavirus infection.
The structurally stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can be useful for preventing an Ebola virus infection in a subject. The peptides (or compositions comprising the peptides) described herein can also be useful for preventing an Ebola virus disease in a subject. In certain instances, the Ebola virus infection is a Zaire ebolavirus infection. In certain instances, the Ebola virus disease is caused by a Zaire ebolavirus infection. In some instances, the subject is a human. In some instances, the subject is a non-human primate. In some instances, the subject is a fruit bat.
In certain instances, the subject in need thereof is administered a peptide described in Table 2 (i.e., any one of SEQ ID NOs: 7-42). In certain instances, the subject in need thereof is administered a stapled EBOV GP2 CHR peptide comprising or consisting of SEQ ID NO:15 or a modified version thereof. In certain instances, the subject in need thereof is administered a stapled EBOV GP2 CHR peptide comprising or consisting of SEQ ID NO:14 or a modified version thereof. In some instances, the subject is a human. In some instances, the subject is anon-human primate. In some instances, the subject is a fruit bat.
In certain instances, the subject in need thereof is administered any one of constructs 1-17 described in Table 3 or construct 18 described in Table 4. In certain instances, the subject in need thereof is administered construct 9 described in Table 3. In certain instances, the subject in need thereof is administered construct 8 described in Table 3. In some instances, the subject is a human. In some instances, the subject is a non-human primate. In some instances, the subject is a fruit bat.
In certain instances, the subject in need thereof is administered a peptide described in Table 4 (i.e., any one of SEQ ID NOs: 2, 25, 28, 37, 42, 40, 34, 41, 35, and 36). In certain instances, the subject in need thereof is administered a peptide having SEQ ID NO:28. In certain instances, the subject in need thereof is administered a peptide having SEQ ID NO:37. In certain instances, the subject in need thereof is administered a peptide having SEQ ID NO:42. In certain instances, the subject in need thereof is administered a peptide having SEQ ID NO:35. In some instances, the subject is infected with an Ebola virus. In some instances, the subject is at risk of being infected with an Ebola virus. In some instances, the subject is at risk of developing an Ebola virus disease. In some instances, a subject is at risk of being infected with an Ebola virus or at risk of developing an Ebola virus disease if he or she lives in an area (e.g., city, state, country) subject to an active Ebola virus outbreak (e.g., an area where at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or more people have been diagnosed as infected with Ebola virus). In some instances, a subject is at risk of being infected with an Ebola virus or developing an Ebola virus disease if he or she lives in an area near (e.g., a bordering city, state, country) a second area (e.g., city, state, country) subject to an active Ebola virus outbreak (e.g., an area near (e.g., bordering) a second area where at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, at least 40, or more people have been diagnosed as infected with Ebola virus). In some instances, the Ebola virus is Zaire ebolavirus. In some instances, the subject is a human. In some instances, the subject is a non-human primate. In some instances, the subject is a fruit bat.
In general, methods include selecting a subject and administering to the subject an effective amount of one or more of the structurally stabilized (e.g., stapled or stitched) peptides herein, e.g., in or as a pharmaceutical composition, and optionally repeating administration as required for the prevention or treatment of an Ebola virus infection or Ebola virus disease and can be administered orally, intravenously or topically. A subject can be selected for treatment based on, e.g., determining that the subject has an Ebola virus infection. The peptides of this disclosure can be used to determine if a subject's is infected with an Ebola virus.
The structurally stabilized (e.g., stapled or stitched) peptides (or compositions comprising the peptides) described herein can be useful for preventing transmission of an Ebolavirus infection from a first subject to a second subject, the method comprising administering to the first subject a therapeutically-effective amount of a structurally stabilized peptide described herein, wherein the first subject is a human or a non-human primate or a fruit bat. In some instances, the second subject is a human.
Specific dosage and treatment regimens for any particular patient 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 or symptoms, the patient's disposition to the disease, condition or symptoms, and the judgment of the treating physician.
An effective amount can be administered in one or more administrations, applications or dosages. A therapeutically effective amount of a therapeutic compound (i.e., an effective dosage) depends on the therapeutic compounds selected.
The compositions can be administered one 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 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.
One or more of any of the structurally stabilized (e.g., stapled or stitched) peptides described herein can be formulated for use as or in pharmaceutical compositions. The pharmaceutical compositions may be used in the methods of treatment or prevention described herein (see above). In certain instances, the pharmaceutical composition comprises a structurally stabilized (e.g., stapled or stitched) peptide comprising or consisting of an amino acid sequence that is identical to an amino acid sequence set forth in Table 2, except for 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid substitution, insertion, or deletion. These changes to the amino acid sequences can be made on the non-NHR-interacting alpha-helical face of these peptides (i.e., to the amino acids that do not interact with the EBOV GP2 NHR) and/or on the NHR-interacting alpha-helical face (i.e., to the amino acids that interact with the EBOV GP2 NHR). Such compositions can be formulated 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, 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, intra-arterial, subdermally, intraperitoneally, intramuscularly, and/or subcutaneously); and/or for oral administration, transmucosal administration, and/or topical administration (including topical (e.g., nasal) sprays and/or solutions).
In some instances, pharmaceutical compositions can include an effective amount of one or more structurally stabilized (e.g., stapled or stitched) peptides. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more structurally stabilized (e.g., stapled or stitched) peptides 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 infection).
Pharmaceutical compositions of this invention can include one or more structurally stabilized (e.g., stapled or stitched) peptides described herein and any pharmaceutically acceptable carrier and/or vehicle. In some instances, pharmaceuticals can further include one or more additional therapeutic agents in amounts effective for achieving a modulation of disease or disease symptoms.
The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that may be administered to a patient, together with a compound of this invention, 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.
The pharmaceutical compositions of this invention 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, intra-venous, intra-muscular, intra-articular, intra-arterial, intra-synovial, intra-sternal, intra-thecal, intra-lesional and intra-cranial injection or infusion techniques.
In some instances, one or more structurally stabilized (e.g., stapled or stitched) peptides disclosed herein can be conjugated, for example, to a carrier protein. Such conjugated compositions can be monovalent or multivalent. For example, conjugated compositions can include one structurally stabilized (e.g., stapled or stitched) peptide disclosed herein conjugated to a carrier protein. Alternatively, conjugated compositions can include two or more structurally stabilized (e.g., stapled or stitched) peptides disclosed herein conjugated to a carrier.
As used herein, when two entities are “conjugated” to one another they are linked by a direct or indirect covalent or non-covalent interaction. In certain instances, the association is covalent. In other instances, the association is non-covalent. Non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. An indirect covalent interaction is when two entities are covalently connected, optionally through a linker group.
Carrier proteins can include any protein that increases or enhances immunogenicity in a subject. Exemplary carrier proteins are described in the art (see, e.g., Fattom et al., Infect. Immun., 58:2309-2312, 1990; Devi et al., Proc. Natl. Acad. Sci. USA 88:7175-7179, 1991; Li et al., Infect. Immun. 57:3823-3827, 1989; Szu et al., Infect. Immun. 59:4555-4561, 1991; Szu et al., J Exp. Med. 166:1510-1524, 1987; and Szu et al., Infect. Immun. 62:4440-4444, 1994). Polymeric carriers can be a natural or a synthetic material containing one or more primary and/or secondary amino groups, azido groups, or carboxyl groups. Carriers can be water soluble.
Also provided herein are methods of making structurally-stabilized peptides (e.g., a structurally-stabilized peptide described herein). In some instances, the method comprises (a) providing a peptide described herein, wherein the peptide comprises two or more stapling amino acids (e.g., a peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 7-42) or a variant thereof, and (b) performing a ring-closing metathesis reaction. In some instances, the method further comprises repeating step (b) one to four additional times (i.e., performing a total of 3 to 5 ring-closing metathesis reactions. In some instances, the method comprises: (a) providing a peptide having the sequence set forth in any one of SEQ ID NOs: 2-6 or a variant thereof, and (b) cross-linking the peptide. In some instances, the method further comprises formulating the structurally-stabilized peptide as a pharmaceutical composition.
In some instances, the peptides produced by the methods of making the structurally-stabilized peptides are the structurally stabilized peptides such as those set forth in SEQ ID NOs: 7-42, as well as variants thereof.
Fmoc-based solid-phase peptide synthesis may be used to synthesize the structurally stabilized peptides described herein (e.g., in accordance with reported methods for generating all-hydrocarbon stapled peptides, e.g., Bird, G. H., Crannell, W. C. & Walensky, L. D. Chemical synthesis of hydrocarbon-stapled peptides for protein interaction research and therapeutic targeting. Curr. Protoc. Chem. Biol. 3, 99-117 (2011)). For example, to achieve the various staple lengths, α-methyl, α-alkenyl amino acids may be installed at i, i+4 positions using two (S)-pentenyl alanine residues (S5) and at i, i+7 positions by inserting (R)-octenyl alanine (R8) at the i position and S5 at the i+7 position, or by inserting (R)-pentenyl alanine (R5) at the i position and (S)-octenyl alanine (S8) at the i+7 position. For the stapling reaction, Grubbs first-generation ruthenium catalyst dissolved in dichloroethane is added to the resin-bound peptides. To ensure maximal conversion, 3-5 rounds of stapling may be performed. The peptides are then cleaved off of the resin using, e.g., trifluoroacetic acid, precipitated using, e.g., a hexane:ether (1:1) mixture, air dried and purified by, e.g., LC-MS. Peptides may be quantified by amino acid analysis. TFA-HCl exchange may be performed on peptides to be used in animal studies.
Methods of synthesizing the structurally stabilized (e.g., stapled or stitched) peptides described herein are known in the art. Nevertheless, the following exemplary method may be used. It will be appreciated that the various steps may 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 compounds described herein are known in the art and include, for example, 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., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic 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.
The peptides of this invention can be made by chemical synthesis methods, which are well known to the ordinarily skilled artisan. See, for example, 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 can be synthesized using the automated Merrifield techniques of solid phase synthesis with the α-NH2 protected by either t-Boc or Fmoc chemistry using side chain protected amino acids on, for example, an Applied Biosystems Peptide Synthesizer Model 430A or 431.
One manner of making of the 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.
Longer peptides could 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. Peptide bonds can be replaced, e.g., to increase physiological stability of the 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 trans-olefin 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 or F or CH3.
The peptides can be further modified by: cholesterolization, acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfurylation. As indicated above, peptides can be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight or branched alkyl groups); fatty acid radicals; and combinations thereof. α, α-Disubstituted non-natural amino acids containing olefinic side chains of varying length can be synthesized by known methods (Williams et al. J. Am. Chem. Soc., 113:9276, 1991; Schafmeister et al., J. Am. Chem Soc., 122:5891, 2000; and Bird et al., Methods Enzymol., 446:369, 2008; Bird et al, Current Protocols in Chemical Biology, 2011). For peptides where an i linked to i+7 staple is used (two turns of the helix stabilized) either: a) one (S)-2-(4′-pentenyl)Alanine amino acid and one (R)-2-(7′-octenyl)Alanine is used; or b) one (S)-2-(7′-octenyl)Alanine amino acid and one (R)-2-(4′-pentenyl)Alanine amino acid is used. (R)-2-(7′-octenyl)Alanine is synthesized using the same route, except that the starting chiral auxiliary confers the R-alkyl-stereoisomer. Also, 8-iodooctene is used in place of 5-iodopentene. Inhibitors are synthesized on a solid support using solid-phase peptide synthesis (SPPS) on MBHA resin (see, e.g., WO 2010/148335).
Fmoc-protected α-amino acids (other than the olefinic amino acids Fmoc-(S)-2-(4′-pentenyl)Alanine-OH, Fmoc-(R)-2-(7′-octenyl)Alanine-OH, Fmoc-(S)-2-(7′-octenyl)Alanine-OH, and Fmoc-(R)-2-(4′-pentenyl)Alanine-OH), 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available from, e.g., Novabiochem (San Diego, CA). Dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (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).
Again, methods suitable for obtaining (e.g., synthesizing), stapling or stitching, and purifying the peptides disclosed herein are also known in the art (see, e.g., Bird et. al., Methods in Enzymol., 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 Ser. No. 12/525,123, filed Mar. 18, 2010; and U.S. Pat. No. 7,723,468, issued May 25, 2010, each of which are hereby incorporated by reference in their entirety).
In some instances, the structurally stabilized (e.g., stapled or stitched) peptides are substantially free of non-stabilized peptide contaminants or are isolated. Methods for purifying peptides include, for example, synthesizing the peptide on a solid-phase support. Following cyclization, 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 instance, 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 instance, the resin is washed with NMP. Shaking and bubbling an inert gas into the solution may be performed.
Also provided herein is a method of producing a structurally stabilized (e.g., stapled or stitched) peptide comprising: (a) stapling or stitching an EBOV GP2 CHR peptide (or variant thereof); and (b) isolating the stapled or stitched peptide. The method may further involve formulating the stapled or stitched peptide into a pharmaceutical composition.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art can develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention.
In designing compounds that could block the formation of the hemifusion complex, a helical wheel analysis was performed of residues 613-630 within the 613-633 amino acid sequence of EBOV GP2 (SEQ ID NO: 1) (
The helical propensity of the designed compounds was evaluated by circular dichroism (CD) spectroscopy in water (
Having demonstrated the pH and thermal stability of SEboV peptides as well as their ability to assemble with GP2, the ability of these peptides to enter cells was examined. To ascertain the endosomal localization of SEboV peptides upon uptake, live cell fluorescence microscopy was performed. Due to the acidic nature of the endosomal compartment, compounds with N,N-dimethylaminocoumarin (DMACA), a pH-insensitive fluorescent dye were tagged. Huh-7 hepatocellular carcinoma cells were exposed to DMACA-labeled WT EboV, SEboV-3, and SEboV-9 peptides and to either an early or late endosomal marker (
Once it was determined that SEboV peptides enter cells by endosomal uptake in a similar manner as EBOV, the ability of the compounds to block viral infection was evaluated. To examine the efficacy of the compounds, Huh-7 hepatocellular carcinoma cells were pretreated with Ac-SEboV peptides and were then exposed to the Makona variant of EBOV. After exposure, the cells were incubated for 48 hours, fixed, and analyzed for drug efficacy and cell toxicity. The WT EboV parent peptide was ineffective at preventing infection; however, compounds SEboV-1, 2, and 9 all displayed antiviral activity (
Similar experiments were performed with peptides Ac1-Ac10, which are N-terminally acetylated versions of peptides comprising the amino acid sequence set forth in SEQ ID NO: 2, 25, 28, 37, 42, 40, 34, 41, 35, and 36, respectively (see Table 5, below). Zaire Ebola virus was pretreated with 25 μM of Ac1, Ac2, Ac3, Ac4, Ac5, Ac5, Ac6, Ac7, Ac8, Ac9, or Ac10 for 30 minutes. Vero cells were then infected with the peptide/virus mixture at a multiplicity of infection of 1. The infected cells were evaluated at six and 24 hours post-infection by immunofluorescence to determine the level of virus infection. At six hours post-infection, many of the peptides, especially Ac3, Ac4, Ac5, and Ac9, inhibited the virus (
X
1DWTX2NITDKIDQIIHDFVDK,
X
1DWTKNIX2DKIDQIIHDFVDK,
SEboV Peptide Synthesis: Peptides were synthesized on a Tetras Peptide Synthesizer (Advanced ChemTech, Louisville, KY) using Fmoc-based solid phase peptide chemistry on Rink amide resin (30 μmol peptide per reaction). In brief, the synthesis protocol consisted of removal of the Fmoc protective group with 25% piperidine in NMP, washing with NMP, and subsequent amino acid coupling using 10 equiv. amino acid (1 mL, 0.3 M), HCTU (0-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate) (0.99 mL, 0.3 M) and DIPEA (N,N-Diisopropylethylamine) (2.0 mL, 0.3 M) in NMP (N-methyl-2-pyrrolidone) for 60 minutes before draining and washing. All peptide sequences included an N-terminal β-alanine spacing residue. Stapled peptides underwent a further ring-closing metathesis reaction on the resin-bound N-terminal Fmoc-protected peptide. After washing with NMP and 1,2-dichloroethane, the resin was exposed to three 6-hour cycles of bis(tricyclohexylphosphine)benzylidine ruthenium (IV) dichloride (Grubbs' I catalyst, 3 mL, 1 mM) at room temperature in 1,2-dichloroethane. Peptides were primarily N-terminal acetylated for use in biochemical and cell-based assays. The acetylation reaction consisted of deprotection of the Fmoc group as outlined above, followed by reaction with neat acetic anhydride (1 mL) and DIPEA (2 mL, 0.3 M in NMP) for 1 hour. Other experiments made use of N-terminal peptides with fluorescent tags. For peptides tagged with fluorescein (FITC), Fmoc-deprotected peptides were exposed to fluorescein isothiocyanate (2.8 mL, 25 mM) and DIPEA (0.2 mL, 0.3 M) in DMF for 12 hours. For peptides tagged with N,N-dimethylaminocoumarin (DMACA), Fmoc-deprotected peptides were exposed to N-hydroxysuccinimidyl-7-dimethylaminocoumarin-4-acetate (2.8 mL, 25 mM) and DIPEA (0.2 mL, 0.3 M) in DMF for 12 hours. Final peptides were cleaved from the resin and fully deprotected by exposure to a solution containing 95% TFA (trifluoroacetic acid), 2.5% water, and 2.5% triisopropyl silane for 2.5-3 hours. The cleaved peptides were precipitated into ice-cold 1:1 methyl-tert-butyl ether (MTBE), re-suspended in water and lyophilized as crude peptides overnight. The lyophilized peptides were purified by C-18 reverse phase high performance liquid chromatography (HPLC) on an Agilent 1200 HPLC system (Santa Clara, CA).
Circular dichroism (CD) spectroscopy: Acetylated compounds were dissolved in water or pH 4.6 acetate buffer to concentrations ranging from 60 to 75 μM. Final compound concentrations were determined by measuring sample absorbance at 205 nm using a NanoDrop2000 spectrophotometer (ThermoScientific, Wilmington, DE). Spectra were obtained on an Aviv Circular Dichroism Spectrometer, Model 420 (Aviv Biomedical, Inc, Lakewood, New Jersey) at 25° C. or at 80° C. for thermal denaturation study. The spectra were collected using a 0.1 cm path length quartz cuvette (Hellma Analytics, Germany) with the following measurement parameters: wavelength, 240-190 nm; step resolution, 0.50 nm; averaging time, 5.0 sec per step. Spectra were processed using Aviv CDS Program software and converted to mean residue molar ellipticity using the cuvette path length (0.1 cm), the measured concentration, and the number of amino acids in the peptide (cross-linking amino acids and β-alanine cap were included as amino acids in this count).
High Resolution Clear Native Electrophoresis (hrCNE): hrCNE was performed as previously described (see, e.g., Bird et al., 2014, JCI, 124(5):2113-2124, Bird et al., 2010, PNAS, 107(32):14093-14098, and Harrison et al., 2011, Protein Sci., 20(9):1587-1596, each of which is incorporated by reference herein in its entirety). Briefly, 2.5 μM of either FITC WT EboV or FITC-SEboV-9 were incubated in the presence or absence of 50 μg/mL of histidine-tagged 5Helix protein (SEQ ID NO:74) in 1× ubiquitylation buffer (Enzo life sciences, BML-KW9885-0001). Binding reactions were incubated at 37° C. for 2 hours, and then loaded onto an 8.0% hrCNE acrylamide resolving gel. Proteins were resolved at 80 mV for 7 hours at 4° C. Fluorescent peptide bands were visualized using a Typhoon FLA 7000 imager (GE Healthcare Life Sciences). After imaging of the FITC channel, proteins were transferred to FL-PVDF (Millipore) membranes. The membranes were blocked and incubated overnight with a His-Tag antibody (Santa Cruz Biotechnology, sc-8036). His Tag-5Helix protein bands were visualized using IRDye mouse (LI-COR) secondary antibodies on a Li-Cor Odyssey 9120 Imaging System (LI-COR).
Fluorescence microscopy: Huh-7 hepatocyte carcinoma cells were plated in wells (1×105 cells/well) of a 12-well glass bottom plate (Mattek P12G-1.5-14-F) in DMEM (Dulbecco's Modified Eagle Medium) medium containing 10% fetal bovine serum, 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, and 250 ng/mL amphotericin B. The following day, cells were transfected with CellLight Early Endosome-RFP (ThermoFisher, C10587) or CellLight Late Endosome-RFP (ThermoFisher, C10589) following the manufacturer's protocol. After 24 hours, to minimize autofluorescence, the medium was changed to transparent DMEM lacking phenol red (Lonza 12-917F), supplemented with 2% fetal bovine serum, 100 U/mL penicillin G, 100 μg/mL streptomycin sulfate, and 250 ng/mL amphotericin B. Cells were then treated with 1 μM SEboV-WT, 2 μM SEboV-3, or 1 μM SEboV-9 peptides labeled with DMACA. Images were taken every 20 minutes for a total of 24 hours using a Nikon TiE inverted fluorescence microscope (Nikon) with a custom incubation chamber to maintain constant 37° C. temperature, elevated humidity, and 5% CO2 levels. Images were taken using a 20× Plan Apochromat 0.75 NA objective (Nikon MRD30205), a red filter set with a 560/40-nm excitation filter, 585-nm dichroic filter, and a 630/75-nm emission filter (Chroma Technologies 49008), a blue filter set with a 436/20-nm excitation filter, 455-nm dichroic, and a 480/40-nm emission filter (Chroma 49001), and an iXon Ultra 888 electron multiplying charged coupled device camera (Andor). Images were analyzed using NIS-Elements v 4.13.00 (Nikon).
Virus: The C05 isolate of the Makona variant of EBOV (Ebola virus/H.sapiens-tc/GIN/2014/Makona-C05; GenBank: KX000398) was propagated in Vero E6 cells (BEI NR-596) and used after one or two passages. All procedures using infectious EBOV/Mak were performed under biosafety level 4 (BSL-4) conditions at the National Institute of Allergy and Infectious Diseases (NIAID) Integrated Research Facility at Fort Detrick (IRF-Frederick).
Cells: Huh-7 (human hepatocellular carcinoma) cells were obtained from Dr. Hideki Ebihara (NIAID), Rocky Mountain Laboratories, Hamilton, MT.
Cell-based efficacy and cytotoxicity assays: The hydrocarbon-stapled α-helical GP2 heptad repeat peptides were tested in Huh-7 cells seeded for 24 hours at a density of 3×104 in 96 well Operetta plates. The peptides were diluted 2-fold starting at a concentration of 50 μM and continuing for 8 dilutions to obtain a dose-response curve. The cells were treated with the peptides for 1 hour prior to infection with EBOV/Mak variant at a multiplicity of infection (MOI) of 0.2. After 48 hours, cells were fixed with 10% neutral-buffered formalin and analyzed using a chemiluminescent assay for efficacy. A mouse primary antibody against EBOV VP40 matrix protein (B-MD04-BD07-AE11, prepared by US Army Medical Research Institute of Infectious Diseases, Frederick, MD under Centers for Disease Control and Prevention contract) was used to detect EBOV. Peroxidase labeled anti-mouse IgG (Cat #074-1802, KPL Inc., Gaithersburg, MD) was used as a secondary antibody. Luminescence was detected with the SuperSignal® ELISA Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) kit and a Tecan Infinite M1000 Pro plate reader (Tecan, Morrisville, NC).
To analyze toxicity, Huh-7 cells were seeded in 96-well black opaque plates and treated with the peptides in the same manner as the efficacy plates. After 48 hours, the CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, Madison, WI) was used to quantify metabolically active cells. Luminescence was read on the Tecan Infinite M1000 Pro plate reader.
Inhibition was determined as percent relative to untreated infected cells, after subtracting background. IC50 and CC50 values were interpolated from the nonlinear regression analysis performed on the data points. GraphPad Software (La Jolla, CA) was used for fitted curves (log [agonist] vs response [variable slope] with constraint to remain above 0). Duplicate efficacy plates and one toxicity plate with triplicate wells per dose were run. The experiment was performed on two separate days. Error bars of dose-response curves represent the standard deviation of 12 replicates for efficacy and 6 replicates for cytotoxicity.
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the priority benefit of U.S. provisional application No. 63/110,169, filed Nov. 5, 2020, which is hereby incorporated by reference herein in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/058023 | 11/4/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63110169 | Nov 2020 | US |
