Chemokine receptor-5 (CCR5) is a receptor for the HIV entry inhibitor peptide T (DAPTA)
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.
References (60)
- et al.
The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates
Cell
(1996) Beyond receptor expression: the influence of receptor conformation, density, and affinity in HIV-1 infection
Virology
(2000)- et al.
A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors
Cell
(1996) - et al.
Effects of [d-Ala1] peptide T-NH2 and HIV envelope glycoprotein gp120 on cyclic AMP dependent protein kinases in normal and psoriatic human fibroblasts
J. Invest. Dermatol.
(1998) - et al.
gp120 and neurotoxicity in vivo
Trends Pharmacol. Sci.
(1995) Chemokine receptors: keys to AIDS pathogenesis
Cell
(1998)- et al.
Cloning and characterization of a novel murine macrophage inflammatory protein-1alpha receptor
J. Biol. Chem.
(1996) - et al.
Enhanced expression, native purification, and characterization of CCR5, a principal HIV-1 coreceptor
J. Biol. Chem.
(1999) - et al.
Interactions among HIV gp120, CD4, and CXCR4: dependence on CD4 expression level, gp120 viral origin, conservation of the gp120 COOH- and NH2-termini and V1/V2 and V3 loops, and sensitivity to neutralizing antibodies
Virology
(1998) - et al.
Co-receptors for HIV-1 entry
Curr. Opin. Immunol.
(1997)
Antiviral and immunological benefits in HIV patients receiving intranasal peptide T (DAPTA)
Peptides
Molecular cloning and functional characterization of a novel human CC chemokine receptor (CCR%) for RANTES, MIP-1β, and MIP-1α
J. Biol. Chem.
Peptide T blocks GP120/CCR5 chemokine receptor-mediated chemotaxis
Clin. Immunol.
Peptide T[4–8] is core HIV envelope sequence required for CD4 receptor attachment (letter)
Lancet
Peptide T inhibits HIV-1 infection mediated by the chemokine receptor-5 (CCR5)
Antiviral Res.
HIV envelope-CD4 interaction not inhibited by synthetic octapeptides (letter)
Lancet
The HIV protein, GP120, activates nuclear protein kinase C in nuclei from lymphocytes and brain
Biochem. Biophys. Res. Commun.
CC CKR5: a RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1
Science
The LD78beta isoform of MIP-1alpha is the most potent CC-chemokine in inhibiting CCR5-dependent human immunodeficiency virus type 1 replication in human macrophages
J. Virol.
A small-molecule, nonpeptide CCR5 antagonist with highly potent and selective anti-HIV-1 activity
Proc. Natl. Acad. Sci. U.S.A.
HIV entry and tropism: the chemokine receptor connection
AIDS
Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease
Annu. Rev. Immunol.
HIV-1-induced cell fusion is mediated by multiple regions within both the viral envelope and the CCR-5 co-receptor
EMBO J.
Peptide T sequences prevent neuronal cell death produced by the envelope protein (gp120) of the human immunodeficiency virus
Drug Dev. Res.
Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide
Nature
Identification of RANTES, MIP-1 alpha, and MIP-1 beta as the major HIV-suppressive factors produced by CD8+ T cells (see comments)
Science
Cloning and functional expression of CC CKR5, a human monocyte CC chemokine receptor selective for MIP-1(alpha), MIP-1(beta), and RANTES
J. Leukoc. Biol.
Change in coreceptor use coreceptor use correlates with disease progression in HIV-1—infected individuals
J. Exp. Med.
Identification of a major co-receptor for primary isolates of HIV-1
Nature
Use of a gp120 binding assay to dissect the requirements and kinetics of human immunodeficiency virus fusion events
J. Virol.
Cited by (29)
GPR75: An exciting new target in metabolic syndrome and related disorders
2022, BiochimieCitation Excerpt :This further confirms that not Gi/Go but Gq was involved in the stimulation of excitatory pathways following treatment with CCL5, which is known to bind with CCR5, CCR3, and CCR1 [29]. The inhibitor of these receptors are already known and include D-alapeptide T-amide, an inhibitor of CCR5 [30], BX471, an inhibitor of CCR1 [31], and SB328437, an inhibitor of CCR3 [32]. Interestingly, it was observed that the CCL5-induced induction of p-AKT was not blocked by the pretreatment with these antagonists alone or in combination in SH-SY5Y cells (neuroblastoma cell line) [29].
The progress of chemokines and chemokine receptors in autism spectrum disorders
2021, Brain Research BulletinAdvances of CCR5 antagonists: From small molecules to macromolecules
2020, European Journal of Medicinal ChemistryDAPTA, a C-C chemokine receptor 5 (CCR5) antagonist attenuates immune aberrations by downregulating Th9/Th17 immune responses in BTBR T<sup>+</sup> Itpr3tf/J mice
2019, European Journal of PharmacologyCitation Excerpt :The BTBR T+ Itpr3tf/J (BTBR) mouse model is increasingly utilized to examine the underlying mechanisms for ASD behaviors. We studied the effects of D-Ala-peptide T-amide (DAPTA), a selective CCR5 chemokine receptor antagonist (Polianova et al., 2005), in BTBR mice. Previously, DAPTA has been shown to effectively diminish astrocyte and microglia activation in the hippocampus (Rosi et al., 2005).
Neuropathic pain inhibitor, RAP-103, is a potent inhibitor of microglial CCL1/CCR8
2018, Neurochemistry InternationalPeptide regulation of cofilin activity in the CNS: A novel therapeutic approach for treatment of multiple neurological disorders
2017, Pharmacology and TherapeuticsCitation Excerpt :One peptide was further modified to enhance cell binding and peptide stability. This peptide, d-ala-Peptide T-Amide (DAPTA), an octapeptide with the sequence D-ala-STTTNYT-amide, was found to be an antagonist of G-protein-coupled chemokine receptors CCR2 and CCR5 (Padi et al., 2012; Polianova, Ruscetti, Pert, & Ruff, 2005) used by HIV to gain cell entry. Activation of many chemokine receptors leads to a rapid dephosphorylation of cofilin and a corresponding decline in phalloidin stained F-actin (Cameron et al., 2010), whereas inhibition of cofilin activation through stimulation of the Rac1, LIMK, cofilin pathway blocks entry of CCR5-tropic HIV-1 viruses (Anand, Zhao, Nagaraja, Robinson, & Ganju, 2013).