IP Library › Granted Patent US 7,811,577

Granted Patent B2

US 7,811,577 · App. 11/628,483 · Granted Oct 12, 2010

Covalently stabilized chimeric coiled-coil HIV gp41 N-peptides with improved antiviral activity

View Patent ↗

Loading inventors, assignments & file history…

Monitor This Case

Get email alerts when status or documents change.

Order Certified Copies

Most orders are placed with the USPTO same day — all within 24 business hours.

Order via The Patent Place →

Pre-filled with this patent's details

Quick Facts

Patent No.: US 7,811,577
App. No.: 11/628,483
Granted: Oct 12, 2010
Kind: B2

External Links

USPTO Patent Center↗ Full Patent PDF↗

Abstract

Methods of covalently-stabilizing alpha-helical, chimeric peptides constrained within a homotrimeric or heterotrimeric coiled-coil structure are disclosed. The coiled-coil structures made by the methods disclosed within this specification mimic all or a portion of the internal, trimeric coiled-coil motif contained within the fusogenic conformation of an enveloped virus membrane-fusion protein, particularly the internal coiled-coil domain of the HIV gp41 ectodomain. The HIV-derived, chimeric peptides disclosed comprise a non-HIV, soluble, trimeric form of a coiled-coil fused in helical phase to all or a portion of the N-helix of HIV gp41 and are covalently-stabilized in a homotrimeric or heterotrimeric coiled-coil structure through the formation of disulfide or chemoselective bonds between said peptides. The covalently-stabilized, HIV-derived, homotrimeric or heterotrimeric coiled-coil structures made by the methods disclosed herein represent a close mimetic of a HIV gp41 fusion intermediate and are potent inhibitors of HIV infectivity. These HIV-derived chimeric peptides may provide for therapeutic treatment against HIV infection by inhibiting the virus-host cell membrane fusion process.

Claims (134)

1. A soluble chimeric peptide which comprises:

(a) a scaffold portion comprising a soluble, α-helical region capable of forming a trimeric coiled-coil;

(b) a N-peptide portion comprising all or a portion of the HIV gp41 NH 2 -terminal heptad repeat domain; and,

wherein said scaffold portion in (a) is fused in helical phase to said N-peptide portion in (b), forming an α-helical domain, and said cysteine portion in (c) is located outside of said α-helical domain at either the NH 2 - or COOH-terminus.

2. The chimeric peptide of claim 1 , wherein said N-peptide portion is fused to the COOH-terminus of the scaffold portion of said peptide.

3. The chimeric peptide of claim 2 , wherein said N-peptide portion comprises a sufficient portion of the HIV gp41NH 2 -terminal heptad repeat domain to include amino acid residues which form the N-helix coiled-coil hydrophobic pocket.

4. The chimeric peptide of claim 3 , wherein said N-peptide portion comprises N17 (LLQLTVWGIKQLQARIL (SEQ ID NO:44)).

5. The chimeric peptide of claim 2 , wherein the scaffold portion of said peptide comprises all or a portion of a coiled-coil motif selected from the group consisting of:

(a) the Suzuki-IZ coiled-coil motif (YGGIEKKIEAIEKKIEAIEKKIEAIEKKIEA (SEQ ID NO:29)), or a modified form thereof;

(b) the IZ coiled-coil motif (IKKEIEAIKKEQEAIKKKIEAIEK (SEQ ID NO:31)), or a modified form thereof;

(d) the SZ coiled-coil motif (IEKKIEAIEKKIEAIEKKIEAIEK SEQ ID NO: 112), or a modified form thereof.

6. The chimeric peptide of claim 5 , wherein the scaffold portion of said peptide comprises the IZ coiled-coil motif.

7. The chimeric peptide of claim 2 , wherein the cysteine portion of said peptide is located at the NH 2 -terminus of the α-helical domain of said peptide.

8. The chimeric peptide of claim 7 , wherein said cysteine portion is separated from said α-helical domain by a spacer region.

9. The chimeric peptide of claim 2 , wherein the cysteine portion is located at the COOH-terminus of the α-helical domain of said peptide.

10. The chimeric peptide of claim 7 , wherein the cysteine portion comprises three consecutive cysteine residues.

11. The chimeric peptide of claim 10 , wherein said cysteine portion is separated from said α-helical domain by a spacer region.

12. The chimeric peptide of claim 10 , wherein the NH 2 -terminal cysteine residue has a protected thiol group.

13. A soluble chimeric peptide comprising an amino acid sequence selected from the group consisting of

(a) SEQ ID NO:2, designated as CCIZN17;

(b) SEQ ID NO:7, designated as CCEZN17;

(d) SEQ ID NO:9, designated as CCEZN17Ala4;

(e) SEQ ID NO:13, designated as CCIZN23;

(f) SEQ ID NO:15, designated as CCEZN23;

(g) SEQ ID NO:12, designated as Biotin-CCIZN23;

(h) SEQ ID NO:21, designated as SCCIZN17;

(i) SEQ ID NO:98, designated as CCSZN17;

(j) SEQ ID NO:100, designated as CCS17N17;

(k) SEQ ID NO:109, designated as CCE10N17;

(l) SEQ ID NO:116, designated as CCE17N17;

(m) SEQ ID NO:120, designated as CCE17GluN17; and,

(n) SEQ ID NO:18, designated as CCIZN36.

14. A covalently-stabilized, trimeric coiled-coil that mimics all or a portion of the internal, trimeric coiled-coil motif of HIV gp41 comprising three chimeric peptides, wherein each chimeric peptide comprises:

(a) a soluble α-helical domain fused in helical phase to all or a portion of the HIV gp41 NH 2 -terminal heptad repeat domain; and,

(b) a cysteine portion comprising at least two cysteine residues located outside of said α-helical domain at the NH 2 -terminus of said α-helical domain;

wherein said chimeric peptides are covalently-stabilized as a coiled-coil via disulfide bonds between said cysteine residues of individual peptides.

15. A covalently-stabilized, trimeric coiled-coil that mimics all or a portion of the internal, trimeric coiled-coil motif of HIV gp41 which comprises:

(a) one chimeric peptide comprising:

a soluble α-helical domain fused in helical phase to all or a portion of the HIV gp41 NH 2 -terminal heptad repeat domain; and,

(ii) a cysteine portion comprising at least two cysteine residues located outside of the α-helical domain of said peptide at the NH 2 -terminus of said α-helical domain; and,

(b) two chimeric peptides comprising a soluble α-helical domain fused in helical phase to all or a portion of the HIV gp41 NH 2 -terminal heptad repeat domain, each derivatized with an electrophilic moiety at the NH 2 -terminus of said α-helical domain present in said two chimeric peptides;

wherein a nucleophilic sulfhydryl of each cysteine residue in the chimeric peptide in (a) forms a thioether bond with the electrophilic moiety of each derivatized chimeric peptide in (b).

16. A covalently-stabilized, trimeric coiled-coil that mimics all or a portion of the internal, trimeric coiled-coil motif of HIV-1 gp41 selected from the group consisting of:

(a) (CCIZN17) 3 ([SEQ ID NO:2] 3 );

(b) (CCEZN17) 3 ([SEQ ID NO:7] 3 );

(d) (CCEZN17Ala4) 3 ([SEQ ID NO:9] 3 );

(e) (CCIZN23) 3 ([SEQ ID NO:13] 3 );

(f) (CCEZN23) 3 ([SEQ ID NO:15] 3 );

(g) (Biotin-CCIZN23) 3 ([SEQ ID NO:12] 3 );

(h) (SCCIZN17) 3 ([SEQ ID NO:21] 3 );

(i) C(thiolZN17) 3 ;

(j) C(thioS17N17) 3 ;

(k) C(thioE10N17) 3 ;

(l) C(thioEZN17) 3 ;

(m) C(thioE17N17) 3 ;

(n) C(thioE17GluN17) 3 ; and,

(o) (CCIZN36) 3 ([SEQ ID NO:18] 3 ).

17. A soluble chimeric peptide which comprises:

(a) a scaffold portion comprising a soluble, α-helical region capable of forming a trimeric coiled-coil; and,

(b) a N-peptide portion comprising all or a portion of the HIV gp41 NH 2 -terminal heptad repeat domain;

wherein said chimeric peptide is derivatized to incorporate an electrophilic moiety capable of forming a thioether bond,

wherein said scaffold portion in (a) is fused in helical phase to said N-peptide portion in (b), forming an α-helical domain, and said electrophilic moiety is located outside of said α-helical domain.

18. The chimeric peptide of claim 17 , wherein said electrophilic moiety is selected from the group consisting of an alkyl halide moiety and a Michael acceptor.

19. The chimeric peptide of claim 18 , wherein said alkyl halide moiety is a bromoacetyl moiety.

20. The covalently-stabilized trimeric coiled-coil of claim 16 that is (SEQ ID NO:18) 3 .

21. The chimeric peptide of claim 7 , wherein said scaffold portion comprises a sequence selected from the group consisting of:

SEQ ID NO: 29;

SEQ ID NO: 30;

SEQ ID NO: 31;

SEQ ID NO: 32;

SEQ ID NO: 35;

SEQ ID NO: 36;

SEQ ID NO: 37;

SEQ ID NO: 38;

SEQ ID NO: 39;

SEQ ID NO: 40;

SEQ ID NO: 112;

SEQ ID NO: 114; and

SEQ ID NO: 119.

22. The chimeric peptide of claim 21 , wherein said N-peptide portion comprises a sequence selected from the group consisting of:

SEQ ID NO: 44;

SEQ ID NO: 46;

SEQ ID NO: 48;

SEQ ID NO: 50;

SEQ ID NO: 51;

SEQ ID NO: 52; and

SEQ ID NO: 53.

23. The chimeric peptide of claim 22 , wherein said N-peptide portion consists of a sequence selected from the group consisting of:

SEQ ID NO: 44;

SEQ ID NO: 46;

SEQ ID NO: 48;

SEQ ID NO: 50;

SEQ ID NO: 51;

SEQ ID NO: 52; and

SEQ ID NO: 53.

24. The chimeric peptide of claim 23 , wherein said cysteine portion consists of the sequence of SEQ ID NO: 28.

25. The chimeric peptide of claim 7 , wherein said chimeric peptide consists of SEQ ID NO: 18.

26. A covalently-stabilized, trimeric coiled-coil that mimics all or a portion of a HIV gp41 N-peptide coiled-coil comprising three chimeric peptides, wherein each chimeric peptide is as described in claim 7 , wherein said three chimeric peptides are covalently-stabilized as a coiled-coil via disulfide bonds between cysteine residues present in said cysteine portion of each of said three chimeric peptides.

27. The trimeric coiled-coil of claim 26 , wherein said trimeric coiled-coil is homotrimeric.

28. The trimeric coiled-coil of claim 27 , wherein said scaffold portion of each chimeric peptide comprises a sequence selected from the group consisting of:

SEQ ID NO: 29;

SEQ ID NO: 30;

SEQ ID NO: 31;

SEQ ID NO: 32;

SEQ ID NO: 35;

SEQ ID NO: 36;

SEQ ID NO: 37;

SEQ ID NO: 38;

SEQ ID NO: 39;

SEQ ID NO: 40;

SEQ ID NO: 112;

SEQ ID NO: 114; and

SEQ ID NO: 119.

29. The trimeric coiled-coil of claim 28 , wherein said N-peptide portion of each chimeric peptide comprises a sequence selected from the group consisting of:

SEQ ID NO: 44;

SEQ ID NO: 46;

SEQ ID NO: 48;

SEQ ID NO: 50;

SEQ ID NO: 51;

SEQ ID NO: 52; and

SEQ ID NO: 53.

30. The trimeric coiled-coil of claim 29 , wherein said N-peptide portion of each chimeric peptide consists of a sequence selected from the group consisting of:

SEQ ID NO: 44;

SEQ ID NO: 46;

SEQ ID NO: 48;

SEQ ID NO: 50;

SEQ ID NO: 51;

SEQ ID NO: 52; and

SEQ ID NO: 53.

31. The trimeric coiled-coil of claim 30 , wherein said cysteine portion of each chimeric peptide consists of the sequence of SEQ ID NO: 28.

Assignments (4)

MERGER Recorded Aug 8, 2022

From: MERCK SHARP & DOHME CORP.

To: MERCK SHARP & DOHME LLC