Elsevier

Antiviral Research

Volume 67, Issue 2, August 2005, Pages 83-92
Antiviral Research

Chemokine receptor-5 (CCR5) is a receptor for the HIV entry inhibitor peptide T (DAPTA)

https://doi.org/10.1016/j.antiviral.2005.03.007Get rights and content

Abstract

The chemokine receptor CCR5 plays a crucial role in transmission of HIV isolates, which predominate in the early and middle stages of infection, as well as those, which populate the brain and cause neuro-AIDS. CCR5 is therefore an attractive therapeutic target for design of entry inhibitors. Specific rapid filtration binding assays have been useful for almost 30 years both for drug discovery and understanding molecular mechanisms of drug action. Reported in 1986, prior to discovery of chemokine co-receptors and so thought to act at CD4, peptide T (DAPTA) appears to greatly reduce cellular viral reservoirs in both HAART experienced and treatment naïve patients, without toxicities. We here report that DAPTA potently inhibits specific CD4-dependent binding of gp120 Bal (IC50 = 0.06 nM) and CM235 (IC50 = 0.32 nM) to CCR5. In co-immunoprecipitation studies, DAPTA (1 nM) blocks formation of the gp120/sCD4 complex with CCR5. Confocal microscopic studies of direct FITC–DAPTA binding to CCR5+, but not CCR5−, cells show that CCR5 is a DAPTA receptor. The capability of DAPTA to potently block gp120–CD4 binding to the major co-receptor CCR5 explains its molecular and therapeutic mechanism of action as a selective antiviral entry inhibitor for R5 tropic HIV-1 isolates.

Introduction

High affinity binding of gp120 to CD4 receptor molecules induces conformational changes in both molecules (Myszka et al., 2000, Rizzuto and Sodroski, 2000, Lin et al., 2001), which results in enhanced binding of gp120 to one of several co-chemokine receptors (Trkola et al., 1996, Olson et al., 1999, Berger et al., 1999, Bieniasz et al., 1997, Littman, 1998).

The β-chemokine receptor CCR5 is the major co-receptor for macrophage-tropic (R5) strains, which are predominant during the asymptomatic stage of infection, and play a crucial role in the transmission of HIV-1 (Olson et al., 1999, Berger et al., 1999, Moore et al., 1997, Littman, 1998, Meyer et al., 1996, He et al., 1997). T-cell tropic viruses utilizing CXCR4 co-receptor (X4-tropic viral strains) usually emerge concomitant with the decline of CD4+ T-cells in the symptomatic stages of HIV-1 infection (Connor et al., 1997).

Molecules that specifically block HIV envelope protein (gp120) binding to CCR5 comprise a new class of receptor-based therapeutic agents for HIV-1 infection (Moore et al., 1997) and gp120-mediated pathogenesis (Brenneman et al., 1988b, Mulroney et al., 1998, Mankowski et al., 2002, Lipton et al., 1995). Several diverse inhibitors of CCR5 or CXCR4 co-receptors have been identified and include small molecules (Strizki et al., 1997, Baba et al., 1999), peptides (Kilby et al., 1998) chemokines and their modified analogs (Simmons et al., 1997, Aquaro et al., 2001) and antibodies (Wu et al., 1997a, Olson et al., 1999). Drugs which block entry can reduce viral burden in treatment experienced patients (Lalezari et al., 2003).

DAPTA, a non-toxic experimental antiviral entry inhibitor (Pert et al., 1986), which is derived from the V2 region, near the stem of HIV-1SF-2 env protein (amino acids 185–192) (Sanchez-Pescador et al., 1985) blocks infection of R5 and dual tropic (R5/X4) HIV-1 strains in monocyte-derived macrophages, microglia and primary CD4+ T-cells (Ruff et al., 2001) and is an antagonist of CCR5-mediated chemotaxis (Redwine et al., 1999). A pre-HAART era placebo-controlled trial showed that DAPTA caused cognitive improvements (Heseltine et al., 1998), and a later analysis of stored samples suggests a clinically significant reduction in plasma viral load (K. Goodkin, University of Miami, personal communication). A more recent uncontrolled clinical trial reports that DAPTA substantially suppressed virus in persistently infected cellular reservoirs (Pert et al., 1986) in both HAART experienced and naïve to treatment patients.

In order to determine possible clinical mechanisms we studied the receptor target of DAPTA action. We here describe CD4-dependent binding of two M-tropic gp120 molecules to CCR5 expressing cells that is potently inhibited by DAPTA. In an immunoprecipitation assay we show that DAPTA, at low concentrations, previously shown to block infection, prevents binding of solubilized gp120/sCD4 complex to both detergent solubilized and membrane bound CCR5 receptor. These data therefore indicate that the mechanism of DAPTA inhibition of R5-tropic HIV-1 infection is to prevent binding of the gp120/sCD4 complex to CCR5. The results implicate a functional role for the DAPTA epitope in gp120/sCD4 complex and CCR5 co-receptor interactions, and suggest clinical usefulness of DAPTA as an HIV entry inhibitor and gp120 antagonist by blocking envelope binding to the co-receptor CCR5.

Section snippets

Compounds

d-Ala1-peptide T-amide (d-A1STTTNYT-NH2) or “DAPTA” (MW 846) was synthesized under GMP conditions by Bachem (Torrence, CA), purified to >95% homogeneity and structure was verified by HPLC isolation, amino acid analysis and sequencing using ABI 470A gas-phase sequencer with on-line HPLC. 0.1 mM stock solutions in sterile water are stored at −20 °C.

Chemokine MIP-1β, recombinant soluble gp120 env protein from clade B HIV-1 Bal isolate, human HIVIg and an anti-CD4 polyclonal antibody T4–4 (R. Sweet

Specific association of gp120/sCD4 complexes to cellular CCR5

Fluorescently labeled gp120 (monomeric) proteins from the R5 tropic HIV-1 viruses, Bal (clade B) and CM235 (clade E) were used in binding reactions conducted with CD4-negative canine thymocyte cells (Cf2Th), or those cells expressing high levels of CCR5 (Cf2Th/synR5). GHOST CD4 cells and GHOST CD4·CCR5, which express CD4 and chemokine receptors at level comparable to the levels expressed in PBMC's (Trkola et al., 1999) were also studied.

Fluorescently labeled gp120 (FITC–gp120) efficiently bound

Discussion

DAPTA was initially reported to inhibit infection of an uncharacterized early passage patient viral isolate when tested in primary PBMCs (Pert et al., 1986). Although the antiviral effect was not observed for lab adapted HIV isolates grown in T cell lines (Sodroski et al., 1987), these studies were conducted prior to the identification of the chemokine entry co-receptors CXCR4 (Feng et al., 1996) and CCR5 (Choe et al., 1996, Deng et al., 1996, Olson et al., 1999, Berger, 1997, Bieniasz et al.,

Acknowledgments

We thank Jason T. Brenner for excellent technical assistance in the confocal analyses, Dan Bertolette for immunoprecipitation methods and Cari Sadowski for general technical support.

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