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

Abstract

SummaryThe 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 (SDP) scaffold, 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 (K D : 1.4 nM) than AK317 (16.7 nM), their antiviral potency was virtually identical (IC 50 : 2.1 and 1.5 nM, respectively). Molecular dynamics simulations for inhibitor-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 inter-helix 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 potency despite the difference in K D values. We also found that amino acid residues in the β-hairpin structural motif of ECL2 are critical for HIV-1-elicited fusion and the binding of the SDP-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.

New Insights into the Mechanisms whereby Low Molecular Weight CCR5 Ligands Inhibit HIV-1 Infection

“…In support of this assumption, we have observed that both inhibitors interfere with the binding of the anti-CCR5 mAb 45531 to the distal half of ECL2 (but not with the binding of the anti-CCR5 mAbs 2D7 and CTC5 to the proximal half of ECL2 and the receptor N-terminal tail, respectively). These findings are in fact fully consistent with recent structural data on related CCR5 inhibitors indicating that their binding to the receptor disrupts interhelix interactions and interactions between helices and ECL2 (27).…”

Multiple CCR5 Conformations on the Cell Surface Are Used Differentially by Human Immunodeficiency Viruses Resistant or Sensitive to CCR5 Inhibitors

“…Whether MAb 45531 binds to a signaling-active conformation of CCR5 remains to be determined, although there is some indirect support for this scenario. The binding of small-molecule inhibitors interferes with the binding of only a few CCR5 MAbs, a prominent example being 45531 (19,30,31,52,54). We found that the CCR5 binding of 45531, but not other MAbs, such as 2D7 and PA14, was significantly reduced when VVC was added (see Fig.…”

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4 + T Cells

“…This resistance pathway often, but not always, results in a virus that is resistant to multiple CCR5 antagonists (22,33,39,53,56). The CCR5 antagonists that have been used clinically all seem to bind to a hydrophobic region in the transmembrane helices of CCR5 and induce conformational alterations in the extracellular domains of the coreceptor that inhibit recognition by gp120 (13,31,32,46,52). These conformational alterations are similar though not identical as judged by the ability of these compounds to affect the binding of various conformation-dependent monoclonal antibodies to CCR5.…”

“…Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sulfated tyrosine residues in the NЈ terminus of CCR5, and (ii) the crown of the V3 loop is thought to engage the extracellular loops (ECLs), particularly ECL2, of CCR5 (10-12, 14, 18, 28). Small-molecule CCR5 antagonists bind to a hydrophobic pocket in the transmembrane helices of CCR5 and exert their effects on HIV by altering the position of the ECLs, making them allosteric inhibitors of HIV infection (13,31,32,46,52). The conformational changes in CCR5 that are induced by CCR5 antagonists vary to some degree with different drugs, as evidenced by differential binding of antibodies and chemokines to various drug-bound forms of CCR5 (47,54).…”