Background:  The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The CA (capsid) protein represents a potential new target: CA is sufficient to form mature HIV-1 cores in vitro and extensive structure-function mutational analysis of CA has shown that the proper assembly, morphology and stability of the mature capsid core are essential for the infectivity of HIV-1 virions.

Methods:  An in vitro capsid assembly assay was developed based on association of CA on immobilized oligonucleotides and used to screen compound libraries. Inhibition of both WT and drug-resistant HIV-1 replication was studied in C8166 T lymphocytes. Compound binding to CA was determined by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. The effect of compounds on viral maturation was determined by Western blotting and electron microscopy (EM).

Results:  Compound library screens using the CA assembly assay yielded several different clusters of structurally related compounds (chemotypes) that inhibited capsid assembly. Optimization of 2 chemotypes resulted in compounds with potent antiviral activity against WT and drug-resistant HIV-1 (EC₅₀≤100 nM; cytotoxicity ≥14 mM). NMR and X-ray crystallography showed that both chemotypes bound to the N-terminal domain of CA by inducing the formation of a pocket, not present in the apo-protein structure, within a-helices 1, 2, 4, and 7, which overlaps with the binding site for the previously reported CAP inhibitors. Passage of virus in the presence of these inhibitors produced several resistance mutations, mostly in highly conserved residues in the N-terminal domain of CA in or near the inhibitor binding pocket (e.g. K30, V36, T58), but also within the C-terminal domain of CA. The resistance mutations selected by the 2 chemotypes differed, consistent with differences in their detailed interactions, within the pocket. Most of the resistance mutations also impaired virus replicative capacity. EM studies showed that treatment of virus-producing cells with compounds from the 2 chemotypes resulted in profound, although different, effects on the morphology of progeny virions: either preventing formation of virions or drastically affecting core morphology.

Conclusions:  Compounds binding to CA N-terminal domain have potent antiretroviral activity by affecting viral core morphology; this demonstrates that CA is a good target for discovery of new anti-HIV drugs.