A Covalent Inhibitor Targeting an Intermediate Conformation of the Fusogenic Subunit of the HIV-1 Envelope Complex

Amy Jacobs,Omar Quraishi,Xicai Huang,Nathalie Bousquet-Gagnon,Geneviève Nault,Nicholas Francella,W Gregory Alvord,Nga Pham,Chantal Soucy,Martin Robitaille,Dominique Bridon,Robert Blumenthal

doi:10.1074/jbc.m705577200

Abstract

Peptide inhibitors corresponding to sequences in the six helix bundle structure of the fusogenic portion (gp41) of the HIV envelope glycoprotein have been successfully implemented in preventing HIV entry. These peptides bind to regions in HIV gp41 transiently exposed during the fusion reaction. In an effort to improve upon these entry inhibitors, we have successfully designed and tested peptide analogs composed of chemical spacers and reactive moieties positioned strategically to facilitate covalent attachment. Using a temperature-arrested state prime wash in vitro assay we show evidence for the trapping of a pre-six helix bundle fusion intermediate by a covalent reaction with the specific anti-HIV-1 peptide. This is the first demonstration of the trapping of an intermediate conformation of a viral envelope glycoprotein during the fusion process that occurs in live cells. The permanent specific attachment of the covalent inhibitor is projected to improve the pharmacokinetics of administration in vivo and thereby improve the long-term sustainability of peptide entry inhibitor therapy and help to expand its applicability beyond salvage therapy.

Highlights

The human immunodeficiency virus (HIV)3 envelope protein complex consisting of gp41 and gp120 are non-covalently associated on the virus surface [1,2,3,4]
Using a temperature-arrested state prime wash in vitro assay (29 –31), we report a novel inhibition strategy of HIV that is based upon the design of compounds composed of two parts: a binding element responsible for conferring selectivity and high affinity for the HIV-1 envelope glycoprotein, and a reactive group responsible for the formation of a covalent bond between the affinity probe and the virus
HPLC Analysis of Covalent Entry Inhibitors Added to N36— The C-terminal helical region (CHR) peptide analogs were designed to bind with high affinity to the N-terminal helix of gp41 (Table 1)

Summary

A Covalent HIV Entry Inhibitor

Esized to be optimally aligned when placed onto or near the site normally occupied by Asp632 of the CHR peptide. Lys574 has been found to be an important target for small molecule inhibitors and has been determined by mutational analysis to be critical for six helix bundle formation and stability [33]. From this model, we have chemically linked the reactive groups to the ⑀-NH of a lysine residue inserted within the amino acid sequence of C34 normally occupied by Asp632 or onto the N terminus of the peptide using the ␣-NH of Trp628 (Fig. 1). Our results are strong evidence for the notion that gp intermediates exist transiently during the HIV fusion process and can be permanently trapped

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION