Briganti Lorenzo, Annamalai Arun S, Bester Stephanie M, Wei Guochao, Andino-Moncada Jonathan R, Singh Satya P, Kleinpeter Alex B, Tripathi Meghna, Nguyen Binh, Radhakrishnan Rajalingam, Singh Parmit K, Greenwood Juliet, Schope Lauren I, Haney Reed, Huang Szu-Wei, Freed Eric O, Engelman Alan N, Francis Ashwanth C, Kvaratskhelia Mamuka
Division of Infectious Diseases, University of Colorado - Anschutz Medical Campus, Aurora, Colorado, USA.
Department of Biological Science and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida, USA.
mBio. 2025 May 14;16(5):e0361324. doi: 10.1128/mbio.03613-24. Epub 2025 Apr 15.
Lenacapavir (LEN) is the first-in-class viral capsid protein (CA) targeting antiretroviral for treating multi-drug-resistant HIV-1 infection. Clinical trials and cell culture experiments have identified resistance-associated mutations (RAMs) in the vicinity of the hydrophobic CA pocket targeted by LEN. The M66I substitution conferred by far the highest level of resistance to the inhibitor compared to other RAMs. Here we investigated structural and mechanistic bases for how the M66I change affects LEN binding to CA and viral replication. The high-resolution X-ray structure of the CA(M66I) hexamer revealed that the β-branched side chain of Ile66 induces steric hindrance specifically to LEN, thereby markedly reducing the inhibitor binding affinity. By contrast, the M66I substitution did not affect the binding of Phe-Gly (FG)-motif-containing cellular cofactors CPSF6, NUP153, or SEC24C, which engage the same hydrophobic pocket of CA. In cell culture, the M66I variant did not acquire compensatory mutations. Analysis of viral replication intermediates revealed that HIV-1 predominantly formed correctly matured viral cores, which were more stable than their wild-type counterparts. The mutant cores stably bound to the nuclear envelope but failed to penetrate inside the nucleus. Furthermore, the M66I substitution markedly altered HIV-1 integration targeting. Taken together, our findings elucidate mechanistic insights into how the M66I change confers remarkable resistance to LEN and affects HIV-1 replication. Moreover, our structural findings provide a powerful means for future medicinal chemistry efforts to rationally develop second-generation inhibitors with a higher barrier to resistance.IMPORTANCELenacapavir (LEN) is a highly potent and long-acting antiretroviral that works by a unique mechanism of targeting the viral capsid protein. The inhibitor is used in combination with other antiretrovirals to treat multi-drug-resistant HIV-1 infection in heavily treatment-experienced adults. Furthermore, LEN is in clinical trials for preexposure prophylaxis (PrEP) with interim results indicating 100% efficacy to prevent HIV-1 infections. However, one notable shortcoming is a relatively low barrier of viral resistance to LEN. Clinical trials and cell culture experiments identified emergent resistance mutations near the inhibitor binding site on capsid. The M66I variant was the most prevalent capsid substitution identified in patients receiving LEN to treat multi-drug-resistant HIV-1 infections. The studies described here elucidate the underlying mechanism by which the M66I substitution confers a marked resistance to the inhibitor. Furthermore, our structural findings will aid future efforts to develop the next generation of capsid inhibitors with enhanced barriers to resistance.
来那卡帕韦（LEN）是首个靶向病毒衣壳蛋白（CA）的抗逆转录病毒药物，用于治疗多重耐药的HIV-1感染。临床试验和细胞培养实验已在LEN靶向的疏水性CA口袋附近鉴定出耐药相关突变（RAMs）。与其他RAMs相比，M66I替代赋予抑制剂的耐药水平最高。在此，我们研究了M66I变化如何影响LEN与CA的结合及病毒复制的结构和机制基础。CA（M66I）六聚体的高分辨率X射线结构显示，Ile66的β-支链侧链对LEN产生空间位阻，从而显著降低抑制剂的结合亲和力。相比之下，M66I替代并不影响含苯丙氨酸-甘氨酸（FG）基序的细胞辅因子CPSF6、NUP153或SEC24C的结合，这些辅因子与CA的同一疏水性口袋结合。在细胞培养中，M66I变体未获得补偿性突变。对病毒复制中间体的分析表明，HIV-1主要形成正确成熟的病毒核心，这些核心比野生型对应物更稳定。突变核心稳定地结合在核膜上，但未能穿透进入细胞核。此外，M66I替代显著改变了HIV-1整合靶向。综上所述，我们的研究结果阐明了M66I变化如何赋予对LEN显著耐药性并影响HIV-1复制的机制见解。此外，我们的结构研究结果为未来药物化学研究提供了有力手段，以合理开发具有更高耐药屏障的第二代抑制剂。
重要性
来那卡帕韦（LEN）是一种高效长效的抗逆转录病毒药物，通过独特的靶向病毒衣壳蛋白机制发挥作用。该抑制剂与其他抗逆转录病毒药物联合使用，用于治疗重度治疗经验的成年人中的多重耐药HIV-1感染。此外，LEN正在进行暴露前预防（PrEP）的临床试验，中期结果表明其预防HIV-1感染的疗效为100%。然而，一个显著的缺点是病毒对LEN的耐药屏障相对较低。临床试验和细胞培养实验在衣壳上抑制剂结合位点附近鉴定出了新出现的耐药突变。M66I变体是接受LEN治疗多重耐药HIV-1感染的患者中最常见的衣壳替代突变。本文所述研究阐明了M66I替代赋予对该抑制剂显著耐药性的潜在机制。此外，我们的结构研究结果将有助于未来开发具有更高耐药屏障的下一代衣壳抑制剂。
