Multidrug-resistant tuberculosis (MDR-TB) continues to spread worldwide and remains one of the leading causes of death among infectious diseases. The enoyl-acyl carrier protein reductase (InhA) belongs to FAS-II family and is essential for the formation of the Mycobacterium tuberculosis cell wall. Recent years, InhA direct inhibitors have been extensively studied to overcome MDR-TB. However, there are still no inhibitors that have entered clinical research. Here, the ensemble docking-based virtual screening along with biological assay were used to identify potent InhA direct inhibitors from Chembridge, Chemdiv, and Specs. Ultimately, 34 compounds were purchased and first assayed for the binding affinity, of which four compounds can bind InhA well with K_D values ranging from 48.4 to 56.2 µM. Among them, compound 9,222,034 has the best inhibitory activity against InhA enzyme with an IC₅₀ value of 18.05 µM. In addition, the molecular dynamic simulation and binding free energy calculation indicate that the identified compounds bind to InhA with “extended” conformation. Residue energy decomposition shows that residues such as Tyr158, Met161, and Met191 have higher energy contributions in the binding of compounds. By analyzing the binding modes, we found that these compounds can bind to a hydrophobic sub-pocket formed by residues Tyr158, Phe149, Ile215, Leu218, etc., resulting in extensive van der Waals interactions. In summary, this study proposed an efficient strategy for discovering InhA direct inhibitors through ensemble docking-based virtual screening, and finally identified four active compounds with new skeletons, which can provide valuable information for the discovery and optimization of InhA direct inhibitors.

耐多药结核病（MDR-TB）继续在全球范围内传播，仍然是传染病中导致死亡的主要原因之一。烯酰基载体蛋白还原酶 (InhA) 属于 FAS-II 家族，对于结核分枝杆菌细胞壁的形成至关重要。近年来，InhA直接抑制剂已被广泛研究以克服耐多药结核病。但目前尚无抑制剂进入临床研究。在这里，基于整体对接的虚拟筛选和生物测定被用来鉴定来自 Chembridge、Chemdiv 和 Specs 的有效 InhA 直接抑制剂。最终，购买了 34 种化合物并首先进行了结合亲和力测定，其中 4 种化合物可以很好地结合 InhA，K _D值范围为 48.4 至 56.2 µM。其中，化合物9,222,034对InhA酶的抑制活性最好，IC ₅₀值为18.05 µM。此外，分子动力学模拟和结合自由能计算表明，所鉴定的化合物以“延伸”构象与InhA结合。残基能量分解表明，Tyr158、Met161 和 Met191 等残基在化合物的结合中具有较高的能量贡献。通过分析结合模式，我们发现这些化合物可以与Tyr158、Phe149、Ile215、Leu218等残基形成的疏水亚袋结合，从而产生广泛的范德华相互作用。综上所述，本研究提出了通过基于集成对接的虚拟筛选发现InhA直接抑制剂的有效策略，最终鉴定出4种具有新骨架的活性化合物，可为InhA直接抑制剂的发现和优化提供有价值的信息。