Exploring the potential and identifying <i>Withania somnifera</i> alkaloids as novel dihydrofolate reductase (DHFR) inhibitors by the AlteQ method

There is an urgent need to discover and develop novel drugs to combat <i>Mycobacterium tuberculosis</i>, the causative agent of tuberculosis (TB) in humans. Alkaloids have been shown to have wide-ranging therapeutic application and could be ideal candidates for drug development, and research is underway to develop new anti-tubercular drugs from natural sources. In this regard, the current research deals with finding novel lead compounds from the <i>Withania somnifera</i> (WS) plant. Broad health benefits of WS are due to the presence of diverse chemical constituents which include anaferine and anahygrine and which belong to the alkaloid family. In the present study, these two compounds have been theoretically studied to understand their electronic properties using the density functional theory (DFT) at the B3LYP/6-311 + G (d,p) level. HOMO and LUMO properties and molecular electrostatic potential (MEP) surface were calculated. Further, to understand the mechanism of action of these compounds and to identify their putative drug target, molecular docking and dynamics studies were employed against <i>Mycobacterium tuberculosis</i> dihydrofolate reductase (DHFR). It was determined that NADP⁺ affects stability of the complexes by reducing fluctuations of residues 14–23 and 117–126. It was also found that Ile5 and Gln28 play an important role in complexation. Electron density analysis (using the AlteQ method) of the intermolecular region, analyzing both the anaferin-NADP⁺ and anahygrin-NADP⁺ complexes showed that anaferin and anahygrin complexes are more stable in the presence of NADP⁺. It has been established that in most intermolecular contacts the contribution of the ligand to the electron density is greater than that of NADP⁺. The present study thus provides an excellent way to analyze the effect of anaferine and anahygrine in essential processes of <i>M. tuberculosis</i>. Communicated by Ramaswamy H. Sarma当前亟需发现并开发新型药物，以对抗引发人类结核病（TB）的结核分枝杆菌（*Mycobacterium tuberculosis*）。生物碱已被证实具有广泛的治疗应用潜力，可成为药物开发的理想候选化合物，目前学界正致力于从天然来源开发新型抗结核药物。有鉴于此，本研究旨在从睡茄（*Withania somnifera*，简称WS）中筛选新型先导化合物。睡茄具有广泛的保健功效，这源于其体内含有多种属于生物碱类的化学成分，其中包括阿法里宁（anaferine）和阿那吉林（anahygrine）。本研究采用密度泛函理论（Density Functional Theory，DFT），在B3LYP/6-311+G(d,p)水平下对这两种化合物进行了理论计算，以解析其电子性质。研究计算了最高占据分子轨道（Highest Occupied Molecular Orbital，HOMO）、最低未占据分子轨道（Lowest Unoccupied Molecular Orbital，LUMO）性质以及分子静电势（Molecular Electrostatic Potential，MEP）表面。此外，为阐明这两种化合物的作用机制并预测其潜在药物靶点，本研究针对结核分枝杆菌二氢叶酸还原酶（*Mycobacterium tuberculosis* dihydrofolate reductase，DHFR）开展了分子对接与分子动力学模拟研究。研究发现，烟酰胺腺嘌呤二核苷酸磷酸（Nicotinamide Adenine Dinucleotide Phosphate，NADP+）可通过降低14~23号与117~126号氨基酸残基的波动幅度，增强复合物的稳定性。同时研究证实，异亮氨酸5位（Ile5）与谷氨酰胺28位（Gln28）在复合物形成过程中发挥关键作用。通过AlteQ方法对阿法里宁-NADP+与阿那吉林-NADP+两种复合物的分子间区域进行电子密度分析后发现，在NADP+存在的条件下，阿法里宁与阿那吉林的复合物稳定性更强。研究证实，在多数分子间相互作用中，配体的电子密度贡献占比高于NADP+。综上，本研究为解析阿法里宁与阿那吉林在结核分枝杆菌生命活动关键过程中的作用提供了可行方法。由Ramaswamy H. Sarma转交。