Covalent anti-HIV compound that induces HIV-1 capsid multimerization and degradation, decomposes the viral core

Covalent inhibitors form strong, essentially irreversible interactions with their targets, offering enhanced structural selectivity within the target cavity and prolonged target engagement even at low doses. Here, we report that ACAi-001, an HIV-1 capsid (CA)-targeting compound identified through in silico screening of the hydrophobic cavity in the CA N-terminal domain (CA-NTD), inhibits HIV-1 replication in cell-based assays. ACAi-001 covalently binds CA to induce aberrant CA multimerization and degradation in vitro, a pharmacological property distinct from that of existing CA inhibitors such as lenacapavir. This mechanism was confirmed by Western blotting (WB), size exclusion chromatography (SEC), differential scanning fluorimetry (DSF), and liquid chromatography-mass spectrometry (LC-MS). These results suggest that ACAi-001, with two putative covalent interactions in its chemical structure, binds directly to Ser16 in the N-terminal domain targeting cavity in addition to Cys198 or Cys218 in the C-terminal domain of CA. The treatment of viral particles with ACAi-001 results in the loss of intact, conical HIV-1 cores, as directly visualized by transmission electron microscopy (TEM), establishing the HIV-1 core as the direct target of ACAi-001. These findings demonstrated that ACAi-001 may be a promising next-generation anti-HIV-1 CA inhibitor with unique mechanisms of action, offering new insight into HIV-1 core assembly and disassembly.