Molecular Dynamics Simulations of the Free and Inhibitor-Bound Cruzain Systems in Aqueous Solvent: Insights on the Inhibition Mechanism in Acidic pH

The major cysteine protease of <i>Trypanosoma cruzi</i>, cruzain (CRZ), has been described as a therapeutic target for Chagas’ disease, which affects millions of people worldwide. Thus, a series of CRZ inhibitors has been studied, including a new competitive inhibitor, Nequimed176 (NEQ176). Nevertheless, the structural and dynamic basis for CRZ inhibition remains unclear. Hoping to contribute to this ever-growing understanding of timescale dynamics in the CRZ inhibition mechanism, we have performed the first study using 100 ns molecular dynamics (MD) simulations of two CRZ systems in an aqueous solvent under pH 5.5: CRZ in the apo form (ligand free) and CRZ complexed to NEQ176. According to the MD simulations, the enzyme adopts an open conformation in the apo form and a closed conformation in the NEQ176-CRZ complex. We also suggest that this closed conformation is related to the hydrogen-bonding interactions between NEQ176 and CRZ, which occurs through key residues, mainly Gly66, Met68, Asn69, and Leu160. In addition, the cross-correlation analysis shows evidence of the correlated motions among Ala110-Asp140, Leu160-Gly189, and Glu190-Gly215 sub-domains, as well as, the movements related to Ala1-Thr59 and Asp60-Pro90 regions seem to be crucial for CRZ activity.

克氏锥虫（Trypanosoma cruzi）的主要半胱氨酸蛋白酶cruzain（CRZ）已被证实为恰加斯病（Chagas’ disease）的治疗靶点，该病在全球范围内影响数百万人群。为此，学界已对一系列CRZ抑制剂展开研究，其中包括新型竞争性抑制剂Nequimed176（NEQ176）。然而，CRZ抑制作用的结构与动态基础仍未阐明。为推动对CRZ抑制机制中时间尺度动态特性的研究进展，我们开展了首项相关研究：针对pH 5.5的水溶剂体系中的两种CRZ体系进行100纳秒分子动力学（Molecular Dynamics, MD）模拟，分别为无配体形式（apo form）的CRZ，以及与NEQ176结合的CRZ复合物。MD模拟结果表明，无配体形式的CRZ采取开放构象，而NEQ176-CRZ复合物则呈现闭合构象。我们进一步提出，该闭合构象与NEQ176和CRZ之间的氢键相互作用密切相关，此类相互作用通过关键残基介导，主要包括Gly66、Met68、Asn69及Leu160。此外，互相关分析结果显示，Ala110-Asp140、Leu160-Gly189以及Glu190-Gly215亚结构域间存在协同运动；同时，Ala1-Thr59与Asp60-Pro90区域的运动对于CRZ的活性具有关键作用。