“Pseudosubstrate\nEnvelope”/Free Energy\nPerturbation-Guided Design and Mechanistic Investigations of Benzothiazole\nHIV Capsid Modulators with High Ligand Efficiency

Based on our proposed “pseudosubstrate envelope”\nconcept, 25 benzothiazole-bearing HIV capsid protein (CA) modulators\nwere designed and synthesized under the guidance of free energy perturbation\ntechnology. The most potent compound, <b>IC-1k</b>, exhibited\nan EC<sub>50</sub> of 2.69 nM against HIV-1, being 393 times more\npotent than the positive control PF74. Notably, <b>IC-1k</b> emerged as the highest ligand efficiency (LE = 0.32) HIV CA modulator,\nsurpassing that of the approved drug lenacapavir (LE = 0.21). Surface\nplasmon resonance assay and crystallographic analysis confirmed that <b>IC-1k</b> targeted HIV-1 CA within the chemical space of the “pseudosubstrate\nenvelope”. Further mechanistic studies revealed a dual-stage\ninhibition profile: <b>IC-1k</b> disrupted early-stage capsid–host-factor\ninteractions and promoted late-stage capsid misassembly. Preliminary\npharmacokinetic evaluations demonstrated significantly improved metabolic\nstability in human liver microsomes for <b>IC-1k</b> (<i>T</i><sub>1/2</sub> = 91.3 min) compared to PF74 (<i>T</i><sub>1/2</sub> = 0.7 min), alongside a favorable safety profile.\nOverall, <b>IC-1k</b> presents a promising lead compound for\nfurther optimization.