While HIV capsid (CA) plays an essential role in multiple stages of the viral life cycle, it remains an unexplored target for antiretroviral (ARV) therapy. Here, we report the discovery of a novel class of exquisitely potent and metabolically stable HIV capsid inhibitors (CAIs) that exhibit pharmacokinetic (PK) profiles suitable for slow-release parenteral administration.
In vitro CA binding and assembly assays, together with X-ray co-crystal structures of CAIs with cross-linked CA hexamers, were used to optimize compounds for high binding affinity to CA. Medicinal chemistry approaches were employed to optimize the antiretroviral activity and drug-like properties using a cytopathic antiviral assay in conjunction with extensive metabolism and pharmacokinetic profiling. CAI resistance-associated mutations were identified by in vitro resistance selections. CAI mode-of-action was defined by inhibitor time-of-addition, virion electron microscopy and viral DNA quantification.
GS-CA1, an exemplified member of a novel class of CAIs, is a highly potent inhibitor of HIV-1 replication in T cell lines (EC50 = 0.24 nM) and displays similar potency against multiple HIV-1 clinical isolates from all major clades in human PBMCs. Identified CAIs bind to a broadly conserved site at the interface of two adjacent monomers within a CA hexamer and accelerate CA assembly in vitro. The identified CAIs maintain full activity against HIV-1 mutants resistant to licensed ARVs and select for HIV CA variants L56I, M66I, Q67H or N74D with an attenuated in vitro replication phenotype. Mechanistic studies revealed a dual mode of action targeting both the late-stage virion maturation and post-entry CA functions. GS-CA1 shows high in vitro metabolic stability and favorable PK profiles in multiple preclinical species with low systemic drug clearances (0.08–0.33 L/hr/kg) and long half-lives (7.2–18.7 hr). Low aqueous solubility provides for an extended-release preclinical PK profile following subcutaneous administration of a solid depot formulation.
We have identified novel HIV-1 capsid inhibitors with uniquely potent antiviral activity and a favorable resistance profile orthogonal to existing ARVs. The high metabolic stability and low aqueous solubility of this new inhibitor class should enable the development of an extended-release parenteral formulation with the potential to be used as a novel long-acting antiretroviral treatment.