Data Sheet 1_HIV subtype-specific gp140-CD4 binding, Temsavir efficacy, and identification of novel adhesion inhibitors against Chinese HIV strains.docx

IntroductionThe HIV epidemic in China is characterized by significant genetic diversity with multiple circulating recombinant forms (CRFs). The gp120-CD4 interaction, essential for viral cellular entry, exhibits subtype-dependent structural variations that compromise therapeutic efficacy. Although temsavir remains the only FDA-approved adhesion inhibitor, its activity against predominant HIV subtypes in China has not been systematically evaluated.
MethodsHIV subtyping of 472 clinical samples identified five major strains (B, CRF01_AE, CRF07_BC, CRF08_BC, and CRF55_01B). Recombinant gp140 proteins from these subtypes were expressed, purified, and analyzed via bio-layer interferometry (BLI) to characterize CD4 binding properties. Structural analyses compared hydrogen bonding and interface buried surface areas. Temsavir’s inhibitory efficacy was assessed, and virtual screening of 13,819 compounds combined with BLI validation was performed to identify novel inhibitors.
ResultsSubtype B demonstrated the strongest CD4 binding affinity (KD=79 pM), while CRF55_01Bshowed the weakest binding (KD=8.76 nM). CRF variants exhibited reduced hydrogen bonding and smaller interface buried surface areas, correlating with diminished binding affinity. Temsavir inhibition was subtype-dependent, achieving 35.7% inhibition for subtype B versus <1.3% for CRF01_AE and CRF55_01B, primarily due to steric hindrance induced by the S375H mutation. Five novel inhibitors targeting CRF55_01B were identified with inhibition rates 19%.
DiscussionThis study elucidates the molecular mechanisms of HIV-1 adhesion variation and provides specific candidate HIV adhesion inhibitors for prevalent CRF subtypes in China. Subsequent efforts will focus on preclinical validation and structure–activity relationship optimization of these candidates, laying the groundwork for developing personalized therapeutic strategies against region-specific strains.

引言
中国的HIV流行疫情以显著的遗传多样性为特征，存在多种流行重组型（circulating recombinant forms，CRFs）。gp120与CD4的相互作用是病毒侵入宿主细胞的关键环节，其结构会随病毒亚型不同而发生变化，进而削弱治疗效果。尽管替沙韦（temsavir）仍是唯一获美国食品药品监督管理局（Food and Drug Administration，FDA）批准的HIV黏附抑制剂，但针对中国主要HIV亚型的活性尚未得到系统评估。

材料与方法
本研究对472份临床样本进行HIV亚型分型，共鉴定出5种主要毒株：B型、CRF01_AE、CRF07_BC、CRF08_BC以及CRF55_01B。针对这些亚型的重组gp140蛋白经表达、纯化后，通过生物层干涉测量法（bio-layer interferometry，BLI）进行分析，以表征其与CD4的结合特性。结构分析对比了各毒株的氢键作用及界面埋藏表面积。本研究评估了替沙韦的抑制效果，并对13819种化合物进行虚拟筛选，结合生物层干涉测量法验证，以筛选新型抑制剂。

结果
B型毒株的CD4结合亲和力最强（解离常数KD=79 pM），而CRF55_01B的结合亲和力最弱（KD=8.76 nM）。流行重组型毒株的氢键作用更少，界面埋藏表面积更小，这与其结合亲和力降低相关。替沙韦的抑制作用具有亚型依赖性：B型毒株的抑制率达35.7%，而CRF01_AE与CRF55_01B的抑制率均低于1.3%，这主要是由S375H突变引发的空间位阻所致。本研究筛选出5种针对CRF55_01B的新型抑制剂，其抑制率可达19%。

讨论
本研究阐明了HIV-1黏附变异的分子机制，并为中国流行的HIV CRF亚型提供了针对性的候选黏附抑制剂。后续研究将聚焦于这些候选抑制剂的临床前验证及构效关系优化，为开发针对区域特异性毒株的个性化治疗策略奠定基础。