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

Abstract

The emergence of resistance to existing classes of antiretroviral drugs necessitates finding new HIV-1 targets for drug discovery. The viral capsid (CA) protein represents one such potential new target. CA is sufficient to form mature HIV-1 capsids in vitro, and extensive structure-function and mutational 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 capsid 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 chemical 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 analyses 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 electron microscopic (EM) studies showed that the BD compounds prevented virion 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, but also to the C-terminal domain of CA. The resistance mutations selected by the two series differed, consistent with differences in their interactions within the pocket, and most also impaired virus replicative capacity. Resistance mutations had two modes of action, either directly impacting inhibitor binding affinity or apparently increasing the overall stability of the viral capsid without affecting inhibitor binding. These studies demonstrate that CA is a viable antiviral target and demonstrate that inhibitors that bind within the same site on CA can have distinct binding modes and mechanisms of action.

Characterization of the Mode of Action of a Potent Dengue Virus Capsid Inhibitor

“…Conversely, molecules of the BM series did not affect virus budding but prevented capsid maturation. Similar to HBV, these two compound series induced different biological phenotypes, although they both shared the same binding site within the N-terminal domain of the HIV capsid protein (64).…”

Host–rabies virus protein–protein interactions as druggable antiviral targets

“…Host assembly machines that have been optimized for the virus rather than for the host (e.g., by viral manipulation of host-signaling pathways) are promising antiviral targets that likely can be blocked without substantial toxicity; although they are composed of host proteins, their loss should not impair a host function, only propagation of the virus. Perhaps previous efforts to develop anti-capsid therapeutics have been disappointing precisely because they did not target the hypothesized host enzymes of capsid formation but rather attempted to block capsid protein interactions directly (17,18).…”

Roles of Capsid-Interacting Host Factors in Multimodal Inhibition of HIV-1 by PF74

“…Recent work discovered several novel small-molecule compounds that target CA (13)(14)(15). Among them, PF-3450074 (hereinafter abbreviated as PF74) has been extensively studied owing to its unique properties (5,13,(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26).…”