School of Systems Biology, George Mason University, Manassas, Virginia 20110, United States.
Department of Chemistry and Biochemistry, George Mason University, Manassas, Virginia 20110, United States.
J Phys Chem B. 2023 Apr 13;127(14):3175-3186. doi: 10.1021/acs.jpcb.3c00429. Epub 2023 Mar 31.
Although Venezuelan equine encephalitis virus (VEEV) is a life-threatening pathogen with a capacity for epidemic outbreaks, there are no FDA-approved VEEV antivirals for humans. VEEV cytotoxicity is partially attributed to the formation of a tetrameric complex between the VEEV capsid protein, the nuclear import proteins importin-α and importin-β, and the nuclear export protein CRM1, which together block trafficking through the nuclear pore complex. Experimental studies have identified small molecules from the CL6662 scaffold as potential inhibitors of the viral nuclear localization signal (NLS) sequence binding to importin-α. However, little is known about the molecular mechanism of CL6662 inhibition. To address this issue, we employed all-atom replica exchange molecular dynamics simulations to probe, in atomistic detail, the binding mechanism of CL6662 ligands to importin-α. Three ligands, including G281-1485 and two congeners with varying hydrophobicities, were considered. We investigated the distribution of ligand binding poses, their locations, and ligand specificities measured by the strength of binding interactions. We found that G281-1485 binds nonspecifically without forming well-defined binding poses throughout the NLS binding site. Binding of the less hydrophobic congener becomes strongly on-target with respect to the NLS binding site but remains nonspecific. However, a more hydrophobic congener is a strongly specific binder and the only ligand out of three to form a well-defined binding pose, while partially overlapping with the NLS binding site. On the basis of free energy estimates, we argue that all three ligands weakly compete with the viral NLS sequence for binding to importin-α in an apparent compromise to preserve host NLS binding. We further show that all-atom replica exchange binding simulations are a viable tool for studying ligands binding nonspecifically without forming well-defined binding poses.
尽管委内瑞拉马脑炎病毒（VEEV）是一种具有流行爆发能力的危及生命的病原体，但目前尚无获得美国食品和药物管理局（FDA）批准的用于人类的 VEEV 抗病毒药物。VEEV 的细胞毒性部分归因于 VEEV 衣壳蛋白、核输入蛋白 importin-α 和 importin-β 以及核输出蛋白 CRM1 之间形成四聚体复合物，该复合物共同阻止核孔复合物的运输。实验研究已经从 CL6662 支架中鉴定出小分子作为病毒核定位信号（NLS）序列与 importin-α 结合的潜在抑制剂。然而，对于 CL6662 抑制的分子机制知之甚少。为了解决这个问题，我们采用全原子复制交换分子动力学模拟方法，从原子细节上探测 CL6662 配体与 importin-α 的结合机制。考虑了三种配体，包括 G281-1485 和两种疏水性不同的同系物。我们研究了配体结合构象的分布、它们的位置以及通过结合相互作用强度测量的配体特异性。我们发现，G281-1485 没有形成明确的结合构象，而是非特异性地结合，没有结合到整个 NLS 结合位点。与 less hydrophobic 同系物的结合变得与 NLS 结合位点非常紧密，但仍是非特异性的。然而，一个更疏水的同系物是一个强烈的特异性结合物，是三种配体中唯一一个形成明确结合构象的配体，同时与 NLS 结合位点部分重叠。根据自由能估计，我们认为三种配体都与 importin-α 与 NLS 序列的结合弱竞争，以维持宿主 NLS 结合。我们进一步表明，全原子复制交换结合模拟是研究非特异性结合而不形成明确结合构象的配体的一种可行工具。
