Hydrocarbon stapled stabilized alpha-helices of the HIV-1 GP41 membrane proximal external region

Patent metadata

Field	Value
Publication number	US-9822165-B2
Application number	US-201013378929-A
Country	US
Kind code	B2
Filing date	Jun 18, 2010
Priority date	Jun 18, 2009
Publication date	Nov 21, 2017
Grant date	Nov 21, 2017

Abstract

The invention provides structurally constrained viral peptides for use as therapeutic and vaccination agents, and for the production of antibodies for use in a number of applications including as therapeutic agents. The invention further provides methods and kits for use of the structurally constrained peptides and antibodies of the instant invention. The invention is based, at least in part, on the result provided herein demonstrating that viral hydrocarbon stapled helical peptides display excellent proteolytic, acid, and thermal stability, restore the native helical structure of the peptide, are highly effective in interfering with the viral fusogenic process, and possess superior pharmacokinetic properties compared to the corresponding unmodified peptides.

First claim

What is claimed is:

1. A cross-linked polypeptide comprising a stabilized HIV gp41 membrane proximal external region (MPER) domain, wherein said stabilized HIV gp41 MPER domain is stabilized with two hydrocarbon staples, wherein one of said hydrocarbon staples is positioned so as to link amino acid residues i and i+3 relative to amino acid positions of a first helix of the polypeptide, and wherein one of said hydrocarbon staples is positioned so as to link amino acid residues i and i+4 relative to amino acid positions of a second helix of the polypeptide.

2. The cross-linked polypeptide of claim 1 , wherein the stabilized HIV gp41 MPER domain comprises an amino acid mutation or a non-natural amino acid incorporation relative to amino acids 662 to 683 of SEQ ID NO: 1.

3. The cross-linked polypeptide of claim 1 , wherein the stabilized HIV gp41 MPER domain comprises at least two amino acids on a single face of a helix, at least two interacting face amino acids or a conservative substitution of an interacting face amino acid from SEQ ID NO: 1, wherein the interacting face amino acids are selected from positions corresponding to amino acids Trp-672, Phe-673, Asn-674, Ile-675, Thr-676, Leu-679, and W-680 when numbered in accordance with SEQ ID NO: 1.

4. The cross-linked polypeptide of claim 1 , wherein a stacked column of amino acids is defined by positions corresponding to positions on a sequence provided by SEQ ID NO: 1, wherein the stacked column of amino acids comprises a group of amino acids selected from the groups consisting of Glu-662, Lys-665, Trp-666, Leu-669, and Trp-672; Leu-663, Trp-666, Ala-667, and Trp-670; Asp-664, Ala-667, Ser-668, and Asn-671; Lys-665, Ser-668, Leu-669, and Trp-672; Trp-666, Leu-669, and Trp-670; Ala-667, Trp-670, and Asn-671; Ser-668, Asn-671, and Trp-672; Ile-675, Trp-678, and Tyr-681; Asn-676, Leu-679, and Ile-682; Thr-677, Trp-680, and Lys-683; Ile-675, Trp-678, Trp-679, and Ile-682; Thr-676, Leu-679, Trp-680, and Lys-683; Asn-677, Trp-680, and Tyr-681; Trp-678, Tyr-681, and Ile-682; and Leu-679, Ile-682, and Lys-683 when numbered in accordance with SEQ ID NO: 1.

5. The cross-linked polypeptide of claim 3 , wherein said cross-linked polypeptide comprises at least 3 interacting face amino acids or a conservative substitution of an interacting face amino acid from SEQ ID NO: 1, wherein the interacting face amino acids are selected from positions corresponding to amino acids Trp-672, Phe-673, Asn-674, Ile-675, Thr-676, Leu-679, and W-680 when numbered in accordance with SEQ ID NO: 1.

6. The cross-linked polypeptide of claim 1 , wherein the peptide further comprises at least 3 contiguous amino acids of an HR-2 peptide, or at least two amino acids on a single face of a helix of an HR-2 peptide, or at least two interacting face amino acids of an HR-2 peptide or a conservative substitution of an interacting face amino acid from SEQ ID NO: 1.

7. The cross-linked polypeptide of claim 6 , wherein the interacting face amino acids of the HR-2 peptide are selected from the group consisting of amino acids corresponding to positions Thr-627, Trp-628, Trp-631, Asp-632, Arg-633, Ile-635, Tyr-638, Ile-642, Leu-645, Ile-646, Ser-649, Gln-650, Gln-652, Gln-653, Glu-654, Lys-655, Asn-656, Glu-657, Glu-659, Leu-660, Glu-662, and Leu-663 on SEQ ID NO: 1.

8. The cross-linked polypeptide of claim 6 , wherein the interacting face amino acids of the HR-2 peptide are selected from the group consisting of amino acids corresponding to positions Trp-628, Trp-631, Ile-635, Tyr-638, Ile-642, Leu-645, Ser-649, Gln-652, Asn-656, Glu-659, and Leu-663 on SEQ ID NO: 1.

9. The cross-linked polypeptide of claim 6 , wherein said cross-linked polypeptide comprises at least two amino acids on a single face of a helix or at least two interacting face amino acids of an amino acid sequence selected from the group consisting of amino acids 37-57 of SEQ ID NO: 17-23; amino acids 19-36 of SEQ ID NO: 50-57 and 64-70; SEQ ID NO: 24, SEQ ID NO: 41-43, and SEQ ID NO: 75-89 operably linked either directly or indirectly to the carboxy-terminus to a peptide that comprises at least 3 contiguous amino acids, or at least two amino acids on a single face of a helix, or at least two interacting face amino acids of an amino acid sequence selected from the group consisting of SEQ ID NO: 10-23, SEQ ID NO: 26-40, SEQ ID NO: 45-48, SEQ ID NO: 58-63, SEQ ID NO: 71-74, and amino acids 1-25 of SEQ ID NO: 49-57, amino acids 1-25 of SEQ ID NO: 64-70; and the sequence of SEQ ID NO: 76-128 and 135-140.

10. The cross-linked peptide of claim 1 in a pharmaceutically acceptable carrier.

11. A kit comprising at least one cross-linked polypeptide of claim 1 and instructions for use.

12. The cross-linked polypeptide of claim 1 , wherein one of the hydrocarbon staples comprises a pairing selected from the group consisting of an R3-S6 pairing, an R6-S3 pairing, an R3-S5 pairing, and an R5-S3 pairing.

13. The cross-linked polypeptide of claim 1 , wherein the polypeptide comprises SEQ ID NO: 137 comprising a hydrocarbon staple between amino acid positions 17 and 20 and a hydrocarbon staple between amino acid positions 3 and 7.

14. The cross-linked peptide of claim 1 , wherein the polypeptide comprises at least two interacting face amino acids of an amino acid sequence selected from the group consisting of amino acids 37-57 of SEQ ID NO: 17-23; amino acids 19-36 of SEQ ID NO: 50-57 and 64-70; SEQ ID NO: 24-25, SEQ ID NO: 41-43, and SEQ ID NO: 75-128 and 135-140.

15. The cross-linked polypeptide of claim 1 , wherein the polypeptide comprises: amino acids 37-57 of SEQ ID NO: 17-23 or SEQ ID NO: 43.

16. The cross-linked polypeptide of claim 1 , wherein the polypeptide comprises:
SEQ ID NO: 41 comprising a hydrocarbon staple between amino acid positions 17 and 20;
SEQ ID NO: 42 comprising a hydrocarbon staple between amino acid positions 20 and 24;
SEQ ID NO: 75 comprising a hydrocarbon staple between amino acid positions 6 and 10;
SEQ ID NO: 76 comprising a hydrocarbon staple between amino acid positions 5 and 9;
SEQ ID NO: 77 comprising a hydrocarbon staple between amino acid positions 4 and 8;
SEQ ID NO: 78 comprising a hydrocarbon staple between amino acid positions 3 and 7;
SEQ ID NO: 79 comprising a hydrocarbon staple between amino acid positions 2 and 6;
SEQ ID NO: 80 comprising a hydrocarbon staple between amino acid positions 1 and 5;
SEQ ID NO: 81 comprising a hydrocarbon staple between amino acid positions 13 and 17;
SEQ ID NO: 82 comprising a hydrocarbon staple between amino acid positions 9 and 13;
SEQ ID NO: 83 comprising a hydrocarbon staple between amino acid positions 19 and 22;
SEQ ID NO: 84 comprising a hydrocarbon staple between amino acid positions 18 and 21;
SEQ ID NO: 85 comprising a hydrocarbon staple between amino acid positions 17 and 20;
SEQ ID NO: 86 comprising a hydrocarbon staple between amino acid positions 16 and 19;
SEQ ID NO: 87 comprising a hydrocarbon staple between amino acid positions 15 and 18;
SEQ ID NO: 88 comprising a hydrocarbon staple between amino acid positions 14 and 17;
SEQ ID NO: 89 comprising a hydrocarbon staple between amino acid positions 13 and 16;
SEQ ID NO: 98 comprising a hydrocarbon staple between amino acid positions 8 and 11;
SEQ ID NO: 99 comprising a hydrocarbon staple between amino acid positions 8 and 11;
SEQ ID NO: 100 comprising a hydrocarbon staple between amino acid positions 8 and 11;
SEQ ID NO: 135 comprising a hydrocarbon staple between amino acid positions 17 and 21;
SEQ ID NO: 136 comprising a hydrocarbon staple between amino acid positions 17 and 20;
SEQ ID NO:

Assignees

• Walensky Loren D
• Bird Gregory
• Dana Farber Cancer Inst Inc

Inventors

• Walensky Loren D
• Bird Gregory

Classifications

• A61P31/18

  for HIV · CPC title

• A61P37/04

  Immunostimulants · CPC title

• A61P31/12

  Antivirals · CPC title

• C07K2317/34

  Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues · CPC title

• C12N2740/16034

  Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein · CPC title