Asymmetric deactivation of HIV-1 gp41 following fusion inhibitor binding.

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.

Kinetic dependence to HIV-1 entry inhibition.

Infection by human immunodeficiency virus type 1 (HIV-1) involves the fusion of viral and cellular membranes mediated by formation of the gp41 trimer-of-hairpins. A designed protein, 5-Helix, targets the C-terminal region of the gp41 ectodomain, disrupting trimer-of-hairpins formation and blocking viral entry. Here we show that the nanomolar inhibitory potency of 5-Helix (IC50 approximately 6 nm) is 4 orders of magnitude larger than its subpicomolar binding affinity (K(D) approximately 0.6 pm). This discrepancy results from the transient exposure of the 5-Helix binding site on gp41. As a consequence, inhibitory potency is determined by the association rate, not by binding affinity. For a series of 5-Helix variants with mutations in their gp41 binding sites, the IC50 and K(D) values poorly correlate. By contrast, an inverse relationship between IC50 values and association rate constants (k(on)) extends for over 2 orders of magnitude. The kinetic dependence to inhibition places temporal restrictions on an intermediate state of HIV-1 membrane fusion and suggests that access to the C-terminal region of the gp41 ectodomain is largely free from steric hindrance. Our results support the importance of association kinetics in the development of improved HIV-1 fusion inhibitors.

HIV-1 gp41 as a target for viral entry inhibition.

The recent success of the fusion inhibitor T-20 (enfuvirtide) in clinical studies has ushered in a new chapter in the development of anti-HIV-1 therapeutics. T-20 is the first FDA-approved drug that targets the viral transmembrane protein gp41. This protein, along with gp120, promotes viral entry through a coordinated cascade of conformational transitions that lead to the fusion of the HIV-1 and target cell membranes. The interaction of gp120 with CD4 and a chemokine receptor stimulates gp41 to extend and bridge the space between the virus and cell. Subsequently, gp41 collapses into a trimer-of-hairpins structure that brings the viral and cellular membranes into close proximity necessary for fusion. Enfuvirtide targets the gp41 amino-terminal region exposed in the transient extended state, blocking the ultimate collapse into the trimer-of hairpins and inhibiting membrane fusion. The vulnerability of this transient extended state has stimulated the development of new agents, ranging from small molecules to large proteins, that bind to gp41 and inhibit its structural transformations. The discovery and characterization of these inhibitors have not only led to new antiviral strategies, but have also shed light on the accessibility of gp41 epitopes that might play a role in HIV-1 vaccine development.