Distinct Effects of Two HIV-1 Capsid Assembly Inhibitor Families That Bind the Same Site within the N-Terminal Domain of the Viral CA Protein

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding newHIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to formmature HIV-1 capsids in vitro, and extensive structure-function andmutational analyses of CA have shown that the proper assembly, morphology, and stability of the mature capsid core are essential for the infectivity of HIV-1 virions. Here we describe the development of an in vitro cap-sid assembly assay based on the association of CA-NC subunits on immobilized oligonucleotides. This assay was used to screen a compound library, yielding several different families of compounds that inhibited capsid assembly. Optimization of two chemi-cal series, termed the benzodiazepines (BD) and the benzimidazoles (BM), resulted in compounds with potent antiviral activity against wild-type and drug-resistant HIV-1. Nuclear magnetic resonance (NMR) spectroscopic and X-ray crystallographic analy-ses showed that both series of inhibitors bound to the N-terminal domain of CA. These inhibitors induce the formation of a pocket that overlaps with the binding site for the previously reported CAP inhibitors but is expanded significantly by these new, more potent CA inhibitors. Virus release and electronmicroscopic (EM) studies showed that the BD compounds prevented vi-rion release, whereas the BM compounds inhibited the formation of the mature capsid. Passage of virus in the presence of the inhibitors selected for resistance mutations that mapped to highly conserved residues surrounding the inhibitor binding pocket