Characterization of human immunodeficiency virus (HIV-1) envelope glycoprotein variants selected for resistance to a CD4-mimetic compound

Abstract

Binding to host cell receptors, CD4 and CCR5/CXCR4, triggers conformational changes in the human immunodeficiency virus (HIV-1) envelope glycoprotein (Env) trimer that promote virus entry. CD4 binding allows the gp120 exterior Env to bind CCR5/CXCR4 and induces a pre-hairpin intermediate conformation in the gp41 transmembrane Env. Small-molecule CD4-mimetic compounds (CD4mcs) bind within the conserved Phe-43 cavity of gp120, near the binding site for CD4. CD4mcs inhibit HIV-1 infection by competing with CD4 and by prematurely activating Env, leading to irreversible inactivation. BNM-III-170 is a CD4mc that inhibits the infection of approximately 70% of HIV-1 strains at micromolar concentrations. We selected and analyzed variants of the primary HIV-1AD8 strain resistant to BNM-III-170. Two changes (S375N and I424T) in gp120 residues that flank the Phe-43 cavity each conferred ∼5-fold resistance to BNM- III-170 with minimal fitness cost. A third change (E64G) in Layer 1 of the gp120 inner domain resulted in ∼100-fold resistance to BNM-III-170, ∼2-3-fold resistance to soluble CD4-Ig, and a moderate decrease in viral fitness. The gp120 changes additively or synergistically contributed to BNM-III-170 resistance. The sensitivity of the Env variants to BNM-III-170 inhibition of virus entry correlated with their sensitivity to BNM-III-170- induced Env activation and shedding of gp120. The S375N and I424T changes, but not the E64G change, conferred resistance to BMS-806, a potent HIV-1 entry inhibitor that blocks Env conformational transitions. These studies identify pathways whereby HIV-1 can develop resistance to CD4mcs and BMS-806 conformational blockers, two classes of entry inhibitors that target the conserved gp120 Phe-43 cavity.IMPORTANCECD4-mimetic compounds (CD4mcs) and BMS-806 are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. Although CD4mcs and BMS-806 inhibit HIV-1 entry by different mechanisms, they both target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Our study identifies changes near this pocket that can confer various levels of resistance to the antiviral effects of both a CD4mc and BMS-806. We relate the antiviral potency of a CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate changes in Env conformation and to induce the shedding of the gp120 exterior Env from the spike. These findings will guide efforts to improve the potency and breadth of small-molecule HIV-1 entry inhibitors.

Indoline CD4-mimetic Compounds Mediate Potent and Broad HIV-1 Inhibition and Sensitization to Antibody-dependent Cellular Cytotoxicity

“…In this case, two changes in the Phe 43 cavity (I424T and S375N) and a change (E64G) in Layer 1 of the gp120 inner domain individually contributed to BNM-III-170 resistance, and acted additively to increase resistance to BNM-III-170 (39).…”

“…The inhibition of infection was diminished for the pairwise combinations of the gp120 changes; however, variants containing all three permutations of the pairwise mutations were still inhibited by the indoline CD4mcs 19, 26 and 30 with IC50 values less than 75 µM. In all cases, the indoline CD4mcs were able to inhibit these resistance-selected HIV-1AD8 mutants more effectively than BNM-III-170 (39). Thus, although the gp120 changes that determine resistance to our current lead indane CD4mc, BNM-III-170, also result in relative resistance to the indoline CD4mcs, the increased potency of the indoline CD4mcs allows measurable inhibition of HIV-1 variants that are resistant to BNM-III-170.…”

“…Previously, CD4mc-resistant variants of HIV-1AD8 were selected by incubating infected cultures with increasing concentrations of BNM-III-170 (39). After selection, the derived mutant HIV-1AD8 130-C was completely resistant to entry inhibition by concentrations of BNM-III-170 up to 300 µM.…”

“…(1). HIV-1 passaged in the presence of BNM-III-170 develops resistance to this CD4mc as a result of Env changes in the Phe 43 cavity and in a gp120 element that allosterically modulates CD4 binding (39).…”

“…Second, the nitrogen at N3 would be expected to limit conformational freedom of the 5-membered ring, thereby decreasing the number of conformational states that the oxalamide (at C1) and methyl guanidinium (at C2) can adopt. Only one state has been observed by X-ray for the indane CD4mc complexes with gp120 (24,37,39), but an entropic penalty would be expected in selecting this pose upon complexation. Lastly, the rigidified 5-membered ring of the indolines may lead to different geometries of the substituents at the N3 position, compared to the reported indane congeners (40).…”