Center for Sickle Cell Disease, Howard University, Washington, DC, United States of America.
PLoS One. 2012;7(6):e39481. doi: 10.1371/journal.pone.0039481. Epub 2012 Jun 29.
HIV-1 Tat protein recruits host cell factors including CDK9/cyclin T1 to HIV-1 TAR RNA and thereby induces HIV-1 transcription. An interaction with host Ser/Thr protein phosphatase-1 (PP1) is critical for this function of Tat. PP1 binds to a Tat sequence, Q(35)VCF(38), which resembles the PP1-binding "RVxF" motif present on PP1-binding regulatory subunits. We showed that expression of PP1 binding peptide, a central domain of Nuclear Inhibitor of PP1, disrupted the interaction of HIV-1 Tat with PP1 and inhibited HIV-1 transcription and replication. Here, we report small molecule compounds that target the "RVxF"-binding cavity of PP1 to disrupt the interaction of PP1 with Tat and inhibit HIV-1 replication. Using the crystal structure of PP1, we virtually screened 300,000 compounds and identified 262 small molecules that were predicted to bind the "RVxF"-accommodating cavity of PP1. These compounds were then assayed for inhibition of HIV-1 transcription in CEM T cells. One of the compounds, 1H4, inhibited HIV-1 transcription and replication at non-cytotoxic concentrations. 1H4 prevented PP1-mediated dephosphorylation of a substrate peptide containing an RVxF sequence in vitro. 1H4 also disrupted the association of PP1 with Tat in cultured cells without having an effect on the interaction of PP1 with the cellular regulators, NIPP1 and PNUTS, or on the cellular proteome. Finally, 1H4 prevented the translocation of PP1 to the nucleus. Taken together, our study shows that HIV- inhibition can be achieved through using small molecules to target a non-catalytic site of PP1. This proof-of-principle study can serve as a starting point for the development of novel antiviral drugs that target the interface of HIV-1 viral proteins with their host partners.
HIV-1 Tat 蛋白招募宿主细胞因子，包括 CDK9/ cyclin T1，与 HIV-1 TAR RNA 结合，从而诱导 HIV-1 转录。与宿主丝氨酸/苏氨酸蛋白磷酸酶-1（PP1）的相互作用对 Tat 的这一功能至关重要。PP1 与 Tat 序列 Q(35)VCF(38)结合，该序列类似于存在于 PP1 结合调节亚基上的 PP1 结合“RVxF”基序。我们表明，表达 PP1 结合肽，即核抑制因子 PP1 的中心结构域，破坏了 HIV-1 Tat 与 PP1 的相互作用，并抑制了 HIV-1 的转录和复制。在这里，我们报告了靶向 PP1 的“RVxF”-结合腔的小分子化合物，以破坏 PP1 与 Tat 的相互作用并抑制 HIV-1 复制。我们使用 PP1 的晶体结构，虚拟筛选了 30 万个化合物，并鉴定出 262 个小分子，这些小分子被预测与 PP1 的“RVxF”-容纳腔结合。然后，这些化合物在 CEM T 细胞中被检测抑制 HIV-1 转录的活性。其中一种化合物 1H4 在非细胞毒性浓度下抑制 HIV-1 的转录和复制。1H4 可阻止 PP1 介导的含有 RVxF 序列的底物肽的去磷酸化。1H4 还在没有影响 PP1 与细胞调节剂 NIPP1 和 PNUTS 的相互作用或细胞蛋白质组的情况下，破坏了 PP1 与 Tat 的关联。最后，1H4 阻止了 PP1 向核内的易位。总之，我们的研究表明，通过使用小分子靶向 PP1 的非催化位点，可以实现 HIV-抑制。这项初步研究可以作为开发靶向 HIV-1 病毒蛋白与其宿主伴侣界面的新型抗病毒药物的起点。
