Laboratory for Molecular Virology and Gene Therapy, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Kapucijnenvoer 33, VCTB +5, Bus 7001, 3000, Leuven, Flanders, Belgium.
The Francis Crick Institute, London, UK.
Retrovirology. 2018 Jan 12;15(1):5. doi: 10.1186/s12977-018-0389-2.
Combination antiretroviral therapy efficiently suppresses HIV replication in infected patients, transforming HIV/AIDS into a chronic disease. Viral resistance does develop however, especially under suboptimal treatment conditions such as poor adherence. As a consequence, continued exploration of novel targets is paramount to identify novel antivirals that do not suffer from cross-resistance with existing drugs. One new promising class of targets are HIV protein-cofactor interactions. Transportin-SR2 (TRN-SR2) is a β-karyopherin that was recently identified as an HIV-1 cofactor. It has been implicated in nuclear import of the viral pre-integration complex and was confirmed as a direct binding partner of HIV-1 integrase (IN). Nevertheless, consensus on its mechanism of action is yet to be reached.
Here we describe the development and use of an AlphaScreen-based high-throughput screening cascade for small molecule inhibitors of the HIV-1 IN-TRN-SR2 interaction. False positives and nonspecific protein-protein interaction inhibitors were eliminated through different counterscreens. We identified and confirmed 2 active compound series from an initial screen of 25,608 small molecules. These compounds significantly reduced nuclear import of fluorescently labeled HIV particles.
Alphascreen-based high-throughput screening can allow the identification of compounds representing a novel class of HIV inhibitors. These results corroborate the role of the IN-TRN-SR2 interaction in nuclear import. These compounds represent the first in class small molecule inhibitors of HIV-1 nuclear import.
联合抗逆转录病毒疗法能有效地抑制感染患者体内的 HIV 复制，使 HIV/AIDS 转变为慢性病。然而，病毒耐药性确实会产生，尤其是在治疗条件不理想的情况下，如依从性差。因此，继续探索新的靶点对于发现不易与现有药物产生交叉耐药的新型抗病毒药物至关重要。一种新的有前途的靶标是 HIV 蛋白共因子相互作用。转运蛋白-SR2（TRN-SR2）是一种 β-核孔蛋白，最近被鉴定为 HIV-1 辅助因子。它已被牵连到病毒前整合复合物的核输入中，并被证实为 HIV-1 整合酶（IN）的直接结合伴侣。然而，其作用机制尚未达成共识。
在这里，我们描述了一种基于 AlphaScreen 的高通量筛选级联技术的开发和应用，用于筛选 HIV-1 IN-TRN-SR2 相互作用的小分子抑制剂。通过不同的对照筛选消除了假阳性和非特异性蛋白质-蛋白质相互作用抑制剂。我们从最初的 25608 种小分子筛选中鉴定并确认了 2 个活性化合物系列。这些化合物显著减少了荧光标记的 HIV 颗粒的核输入。
基于 AlphaScreen 的高通量筛选可以鉴定出代表新型 HIV 抑制剂的化合物。这些结果证实了 IN-TRN-SR2 相互作用在核输入中的作用。这些化合物代表了 HIV-1 核输入的首个小分子抑制剂类药物。
