Chemical profiling of HIV-1 capsid-targeting antiviral PF74.

The capsid protein (CA) of HIV-1 plays essential roles in multiple steps of the viral replication cycle by assembling into functional capsid core, controlling the kinetics of uncoating and nuclear entry, and interacting with various host factors. Targeting CA represents an attractive yet underexplored antiviral approach. Of all known CA-targeting small molecule chemotypes, the peptidomimetic PF74 is particularly interesting because it binds to the same pocket used by a few important host factors, resulting in highly desirable antiviral phenotypes. However, further development of PF74 entails understanding its pharmacophore and mitigating its poor metabolic stability. We report herein the design, synthesis, and evaluation of a large number of PF74 analogs aiming to provide a comprehensive chemical profiling of PF74 and advance the understanding on its detailed binding mechanism and pharmacophore. The analogs, containing structural variations mainly in the aniline domain and/or the indole domain, were assayed for their effect on stability of CA hexamers, antiviral activity, and cytotoxicity. Selected analogs were also tested for metabolic stability in liver microsomes, alone or in the presence of a CYP3A inhibitor. Collectively, our studies identified important pharmacophore elements and revealed additional binding features of PF74, which could aid in future design of improved ligands to better probe the molecular basis of CA-host factor interactions, design strategies to disrupt them, and ultimately identify viable CA-targeting antiviral leads.

Toward Structurally Novel and Metabolically Stable HIV-1 Capsid-Targeting Small Molecules.

HIV-1 capsid protein (CA) plays an important role in many steps of viral replication and represents an appealing antiviral target. Several CA-targeting small molecules of various chemotypes have been studied, but the peptidomimetic PF74 has drawn particular interest due to its potent antiviral activity, well-characterized binding mode, and unique mechanism of action. Importantly, PF74 competes against important host factors for binding, conferring highly desirable antiviral phenotypes. However, further development of PF74 is hindered by its prohibitively poor metabolic stability, which necessitates the search for structurally novel and metabolically stable chemotypes. We have conducted a pharmacophore-based shape similarity search for compounds mimicking PF74. We report herein the analog synthesis and structure-activity relationship (SAR) of two hits from the search, and a third hit designed via molecular hybridization. All analogs were characterized for their effect on CA hexamer stability, antiviral activity, and cytotoxicity. These assays identified three active compounds that moderately stabilize CA hexamer and inhibit HIV-1. The most potent analog (10) inhibited HIV-1 comparably to PF74 but demonstrated drastically improved metabolic stability in liver microsomes (31 min vs. 0.7 min t1/2). Collectively, the current studies identified a structurally novel and metabolically stable PF74-like chemotype for targeting HIV-1 CA.

Novel HIV-1 capsid-targeting small molecules of the PF74 binding site.

The PF74 binding site in HIV-1 capsid protein (CA) is a compelling antiviral drug target. Although PF74 confers mechanistically distinct antiviral phenotypes by competing against host factors for CA binding, it suffers from prohibitively low metabolic stability. Therefore, there has been increasing interest in designing novel sub-chemotypes of PF74 with similar binding mode and improved metabolic stability. We report herein our efforts to explore the inter-domain interacting indole moiety for designing novel CA-targeting small molecules. Our design includes simple substitution on the indole ring, and more importantly, novel sub-chemotypes with the indole moiety replaced with a few less electron-rich rings. All 56 novel analogs were synthesized and evaluated for antiviral activity, cytotoxicity, and impact on CA hexamer stability. Selected analogs were tested for metabolic stability in liver microsomes. Molecular modeling was performed to verify compound binding to the PF74 site. In the end, 5-hydroxyindole analogs (8,9 and 12) showed improved potency (up to 20-fold) over PF74. Of the novel sub-chemotypes, α- and ß-naphthyl analogs (33 and 27) exhibited sub micromolar antiviral potencies comparable to that of PF74. Interestingly, although only moderately inhibiting HIV-1 (single-digit micromolar EC50s), analogs of the 2-indolone sub-chemotype consistently lowered the melting point (Tm) of CA hexamers, some with improved metabolic stability over PF74.