Article

Involvement of the Second Extracellular Loop and Transmembrane Residues of CCR5 in Inhibitor Binding and HIV-1 Fusion: Insights into the Mechanism of Allosteric Inhibition

Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
Journal of Molecular Biology (Impact Factor: 3.96). 09/2008; DOI: 10.1016/j.jmb.2008.06.041

ABSTRACT C-C chemokine receptor 5 (CCR5), a member of G-protein-coupled receptors, serves as a coreceptor for human immunodeficiency virus type 1 (HIV-1). In the present study, we examined the interactions between CCR5 and novel CCR5 inhibitors containing the spirodiketopiperazine scaffolds AK530 and AK317, both of which were lodged in the hydrophobic cavity located between the upper transmembrane domain and the second extracellular loop (ECL2) of CCR5. Although substantial differences existed between the two inhibitors—AK530 had 10-fold-greater CCR5-binding affinity (Kd = 1.4 nM) than AK317 (16.7 nM)—their antiviral potencies were virtually identical (IC50 = 2.1 nM and 1.5 nM, respectively). Molecular dynamics simulations for unbound CCR5 showed hydrogen bond interactions among transmembrane residues Y108, E283, and Y251, which were crucial for HIV-1-gp120/sCD4 complex binding and HIV-1 fusion. Indeed, AK530 and AK317, when bound to CCR5, disrupted these interhelix hydrogen bond interactions, a salient molecular mechanism enabling allosteric inhibition. Mutagenesis and structural analysis showed that ECL2 consists of a part of the hydrophobic cavity for both inhibitors, although AK317 is more tightly engaged with ECL2 than AK530, explaining their similar anti-HIV-1 potencies despite the difference in Kd values. We also found that amino acid residues in the β-hairpin structural motif of ECL2 are critical for HIV-1-elicited fusion and binding of the spirodiketopiperazine-based inhibitors to CCR5. The direct ECL2-engaging property of the inhibitors likely produces an ECL2 conformation, which HIV-1 gp120 cannot bind to, but also prohibits HIV-1 from utilizing the “inhibitor-bound” CCR5 for cellular entry—a mechanism of HIV-1's resistance to CCR5 inhibitors. The data should not only help delineate the dynamics of CCR5 following inhibitor binding but also aid in designing CCR5 inhibitors that are more potent against HIV-1 and prevent or delay the emergence of resistant HIV-1 variants.

0 Followers
 · 
62 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Membrane fusion between the viral envelope and plasma membranes of target cells has previously been correlated with HIV-1 infection. Lipids in the plasma membrane, including sphingomyelin (SM), may be crucially involved in HIV-1 infection; however, the role of lipid-metabolic enzymes in membrane fusion remains unclear. In the present study, we examined the roles of SM synthase (SMS) in HIV-1 Env-mediated membrane fusion using a cell-cell fusion assay with HIV-1 mimetics and their target cells. We employed reconstituted cells as target cells that stably express SMS1 or SMS2 in SMS-deficient cells. Fusion susceptibility was approximately five-fold higher in SMS2-expressing cells (not in SMS1-expressing cells) than in SMS-deficient cells. The enhancement of fusion susceptibility observed in SMS2-expressing cells was reversed and reduced by SMS2 knockdown. We also found that catalytically non-active SMS2 promoted membrane fusion susceptibility. Moreover, SMS2 co-localized and was constitutively associated with the HIV receptor/co-receptor complex in the plasma membrane. In addition, HIV-1 Env treatment resulted in a transient increase in nonreceptor tyrosine kinase (Pyk2) phosphorylation in SMS2- and catalytically non-active SMS2-expressing cells. We observed that F-actin polymerization in the region of membrane fusion was more prominent in SMS2-expressing cells than SMS-deficient cells. Taken together, our research provides insight into a novel function of SMS2: regulation of HIV-1 Env-mediated membrane fusion via actin rearrangement.
    Journal of Biological Chemistry 09/2014; 289(44). DOI:10.1074/jbc.M114.574285 · 4.60 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cenicriviroc is a potent antagonist of the chemokine coreceptors 5 and 2 (CCR5/CCR2) and blocks HIV-1 entry. The CCR5 inhibitor maraviroc has been shown in tissue culture to be able to repel cell-free virions from the cell surface into extracellular space. We hypothesized that cenicriviroc might exhibit a similar effect, and tested this using clinical samples from the Phase IIb study 652-2-202, by measuring rates of intracellular DNA decline. We also monitored viral RNA levels in culture fluids. We infected PM-1 cells with CCR5-tropic HIV-1 BaL in the presence or absence of inhibitory concentrations of cenicriviroc (20 nM) or maraviroc (50 nM) or controls. Viral load levels and p24 were measured by ELISA, quantitative PCR and quantitative real-time reverse transcription PCR at 4 h post-infection. Frozen PBMC DNA samples from 30 patients with virological success in the Phase IIb study were studied, as were early and late reverse transcript levels. Docking studies compared binding between cenicriviroc/CCR5 and maraviroc/CCR5. Unlike maraviroc, cenicriviroc did not cause an increase in the amount of virus present in culture fluids at 4 h compared with baseline. The use of cenicriviroc did, however, result in lower levels of intracellular viral DNA after 4 h. Structural modelling indicates that cenicriviroc binds more deeply than maraviroc to the hydrophobic pocket of CCR5, providing an explanation for the absence of viral rebound with cenicriviroc. In contrast to maraviroc, cenicriviroc does not repel virus back into extracellular space. Differences in results may be due to superior binding of cenicriviroc to CCR5 compared with maraviroc. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
    Journal of Antimicrobial Chemotherapy 11/2014; 70(3). DOI:10.1093/jac/dku451 · 5.44 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The cellular entry of HIV-1 to CD4(+) T-cells requires ordered interactions of HIV-1-envelope glycoprotein with CXCR4 receptors. However, such interactions, which should be critical for rational structure-based discovery of new CXCR4 inhibitors, remain poorly understood. Herein, we first determined the effects of amino acid substitutions in CXCR4 on HIV-1NL4-3-glycoprotein-elicited fusion events using site-directed mutagenesis-based fusion assays and identified eleven potentially key amino acid substitutions, including D97A and E288A, which caused a >30% reduction of the fusion. We subsequently carried out computational search of a screening library containing ∼604,000 compounds in order to identify potential CXCR4 inhibitors. The computational search used the shape of IT1t, a known CXCR4 inhibitor, as a reference and employed various algorithms including shape similarity, isomer generation, and docking against a CXCR4 crystal structure. Sixteen small-molecules were identified for biological assays based on their high shape similarity to IT1t and their putative binding modes formed hydrogen bond interactions with amino acids identified above. Three compounds, having a piperidinylethanamine core, showed activity and were resynthesized. One molecule, designated CX6, was proven to significantly inhibit X4-HIV-1NL4-3-glycoprotein-elicited fusion (IC50: 1.9 μM), inhibit SDF-1α-elicited Ca(2+) flux (IC50:92 nM), and exert anti-HIV-1 activity (IC50:1.5 μM). Structural modeling demonstrated that CX6 bound to CXCR4 through hydrogen-bond interactions with Asp97 and Glu288. Our study suggests that targeting CXCR4 residues important for HIV-1-envelope glycoprotein-elicited fusion should be a useful and practically feasible approach in identifying novel CXCR4 inhibitors and sheds important insights into the mechanism by which small-molecule CXCR4 inhibitors exert their anti-HIV-1 activity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Antimicrobial Agents and Chemotherapy 01/2015; 59(4). DOI:10.1128/AAC.04654-14 · 4.45 Impact Factor