HIV-1 replication requires direct interaction between HIV-1 reverse transcriptase (RT) and cellular eukaryotic translation elongation factor 1A (eEF1A). Our previous work showed that disrupting this interaction inhibited HIV-1 uncoating, reverse transcription, and replication, indicating its potential as an anti-HIV-1 target. In this study, we developed a sensitive, live-cell split-luciferase complementation assay (NanoBiT) to quantitatively measure inhibition of HIV-1 RT interaction with eEF1A. We used this to screen a small molecule library and discovered small-molecule oxazole-benzenesulfonamides (C7, C8, and C9), which dose dependently and specifically inhibited the HIV-1 RT interaction with eEF1A. These compounds directly bound to HIV-1 RT in a dose-dependent manner, as assessed by a biolayer interferometry (BLI) assay, but did not bind to eEF1A. These oxazole-benzenesulfonamides did not inhibit enzymatic activity of recombinant HIV-1 RT in a homopolymer assay but did inhibit reverse transcription and infection of both wild-type (WT) and nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant HIV-1 in a dose-dependent manner in HEK293T cells. Infection of HeLa cells was significantly inhibited by the oxazole-benzenesulfonamides, and the antiviral activity was most potent against replication stages before 8 h postinfection. In human primary activated CD4+ T cells, C7 inhibited HIV-1 infectivity and replication up to 6 days postinfection. The data suggest a novel mechanism of HIV-1 inhibition and further elucidate how the RT-eEF1A interaction is important for HIV-1 replication. These compounds provide potential to develop a new class of anti-HIV-1 drugs to treat WT and NNRTI-resistant strains in people infected with HIV.IMPORTANCE Antiretroviral drugs protect many HIV-positive people, but their success can be compromised by drug-resistant strains. To combat these strains, the development of new classes of HIV-1 inhibitors is essential and a priority in the field. In this study, we identified small molecules that bind directly to HIV-1 reverse transcriptase (RT) and inhibit its interaction with cellular eEF1A, an interaction which we have previously identified as crucial for HIV-1 replication. These compounds inhibit intracellular HIV-1 reverse transcription and replication of WT HIV-1, as well as HIV-1 mutants that are resistant to current RT inhibitors. A novel mechanism of action involving inhibition of the HIV-1 RT-eEF1A interaction is an important finding and a potential new way to combat drug-resistant HIV-1 strains in infected people.

中文翻译：

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恶唑-苯磺酰胺衍生物抑制 HIV-1 逆转录酶与细胞 eEF1A 的相互作用并减少病毒复制。


HIV-1 复制需要 HIV-1 逆转录酶 (RT) 和细胞真核翻译延伸因子 1A (eEF1A) 之间的直接相互作用。我们之前的工作表明，破坏这种相互作用会抑制 HIV-1 脱壳、逆转录和复制，表明其作为抗 HIV-1 靶点的潜力。在这项研究中，我们开发了一种灵敏的活细胞分裂荧光素酶互补测定 (NanoBiT)，以定量测量 HIV-1 RT 与 eEF1A 相互作用的抑制作用。我们用它来筛选一个小分子库，发现了小分子恶唑苯磺酰胺（C7、C8 和 C9），它们剂量依赖性且特异性地抑制 HIV-1 RT 与 eEF1A 的相互作用。通过生物层干涉测量 (BLI) 测定评估，这些化合物以剂量依赖性方式直接与 HIV-1 RT 结合，但不与 eEF1A 结合。这些恶唑-苯磺酰胺在均聚物测定中不会抑制重组 HIV-1 RT 的酶活性，但在一定剂量下会抑制野生型 (WT) 和非核苷逆转录酶抑制剂 (NNRTI) 耐药的 HIV-1 的逆转录和感染HEK293T 细胞中的依赖性方式。恶唑苯磺酰胺显着抑制 HeLa 细胞的感染，并且在感染后 8 小时之前的复制阶段，抗病毒活性最为有效。在人类初级活化的 CD4+ T 细胞中，C7 在感染后长达 6 天的时间内抑制 HIV-1 的感染性和复制。这些数据表明了一种新的 HIV-1 抑制机制，并进一步阐明了 RT-eEF1A 相互作用对于 HIV-1 复制的重要性。这些化合物提供了开发一类新型抗 HIV-1 药物的潜力，用于治疗 HIV 感染者中的 WT 和 NNRTI 耐药菌株。重要性 抗逆转录病毒药物可以保护许多艾滋病毒阳性者，但其成功可能会受到耐药菌株的影响。为了对抗这些菌株，开发新型 HIV-1 抑制剂至关重要，也是该领域的优先事项。在这项研究中，我们鉴定了直接与 HIV-1 逆转录酶 (RT) 结合并抑制其与细胞 eEF1A 相互作用的小分子，我们之前已确定这种相互作用对 HIV-1 复制至关重要。这些化合物抑制细胞内 HIV-1 逆转录和 WT HIV-1 以及对当前 RT 抑制剂具有抗性的 HIV-1 突变体的复制。一种涉及抑制 HIV-1 RT-eEF1A 相互作用的新作用机制是一项重要发现，也是对抗感染者中耐药 HIV-1 菌株的潜在新方法。