Asymmetric Deactivation of HIV-1 gp41 following Fusion Inhibitor Binding

Abstract

Both equilibrium and nonequilibrium factors influence the efficacy of pharmaceutical agents that target intermediate states of biochemical reactions. We explored the intermediate state inhibition of gp41, part of the HIV-1 envelope glycoprotein complex (Env) that promotes viral entry through membrane fusion. This process involves a series of gp41 conformational changes coordinated by Env interactions with cellular CD4 and a chemokine receptor. In a kinetic window between CD4 binding and membrane fusion, the N- and C-terminal regions of the gp41 ectodomain become transiently susceptible to inhibitors that disrupt Env structural transitions. In this study, we sought to identify kinetic parameters that influence the antiviral potency of two such gp41 inhibitors, C37 and 5-Helix. Employing a series of C37 and 5-Helix variants, we investigated the physical properties of gp41 inhibition, including the ability of inhibitor-bound gp41 to recover its fusion activity once inhibitor was removed from solution. Our results indicated that antiviral activity critically depended upon irreversible deactivation of inhibitor-bound gp41. For C37, which targets the N-terminal region of the gp41 ectodomain, deactivation was a slow process that depended on chemokine receptor binding to Env. For 5-Helix, which targets the C-terminal region of the gp41 ectodomain, deactivation occurred rapidly following inhibitor binding and was independent of chemokine receptor levels. Due to this kinetic disparity, C37 inhibition was largely reversible, while 5-Helix inhibition was functionally irreversible. The fundamental difference in deactivation mechanism points to an unappreciated asymmetry in gp41 following inhibitor binding and impacts the development of improved fusion inhibitors and HIV-1 vaccines. The results also demonstrate how the activities of intermediate state inhibitors critically depend upon the final disposition of inhibitor-bound states.

Complex interplay of kinetic factors governs the synergistic properties of HIV-1 entry inhibitors

“…In these experiments, we used HIV-1 pseudotyped with CXCR4-tropic Env HXB2 , U87-CD4-CXCR4 target cells, and CoRA AMD3100. The FI was C37, a well-characterized C-peptide with a binding site that overlaps that for T20 (38,52). As with maraviroc/T20 combinations in Fig.…”

“…Second, T20 activity was much more sensitive to maraviroc concentration than 5H WT activity; the impact on T20 potency appeared to saturate at ϳ1 nM maraviroc, whereas the impact on 5H WT potency did not saturate in the studied maraviroc concentration range. The differences likely reflect an asymmetry in exposure of the gp41 N-HR and C-HR regions and/or differences in the determinants of potency for FIs that target these sites (36,38).…”

“…2C). We next analyzed a mutant Env variant containing the V549E substitution known to disrupt C37-binding affinity by 5000-fold and reduce C37 inhibitory potency by 100-fold (Table 1) (38,53). The mutation did not alter the potency of AMD3100 (IC 50 ϳ26 nM), implying that the V549E substitution did not affect Env utilization of the coreceptor (Fig.…”

“…Because the gp41 N-HR and C-HR are only transiently exposed during the PHI, FI potency depends on kinetics of inhibitor binding and Env dynamics (16,17,19,(35)(36)(37)(38)(39)). An important determinant of FI activity is the lifetime of the PHI, a property that critically depends on Env interactions with chemokine receptors.…”

Polymeric Anti‐HIV Therapeutics

“…[83] Compared to the polysulfate functionalized PAMAM dendrimers, the polycarboxylate functionalized PAMAM dendrimers (19) were equally effective for HIV-1 but significantly less effective against strains of HIV-2. Analogous to dendrimers based on PAMAM, a 4 th generation poly(L-lysine) dendrimer modified with 32 3,6-disulfonaphthyl groups (20) has shown to be an effective inhibitor against infection of various clades of HIV-1. [84] Third, fourth and fifth generation glycodendrimers that display 16, 32 and 64 randomly sulfated galactose moieties (21) respectively, are very effective inhibitors of R5 and X4 tropic HIV-1.…”