Abstract：
Trypanosoma cruzi is a parasite that infects about 6-7 million people worldwide, mostly in Latin America, causing Chagas disease. Cruzain, the main cysteine protease of T. cruzi, is a validated target for developing drug candidates for Chagas disease. Thiosemicarbazones are one of the most relevant warheads used in covalent inhibitors targeting cruzain. Despite its relevance, the mechanism of inhibition of cruzain by thiosemicarbazones is unknown. Here, we combined experiments and simulations to unveil the covalent inhibition mechanism of cruzain by a thiosemicarbazone-based inhibitor (compound 1). Additionally, we studied a semicarbazone (compound 2), which is structurally similar to compound 1 but does not inhibit cruzain. Assays confirmed the reversibility of inhibition by compound 1 and suggested a two-step mechanism of inhibition. The Ki was estimated to be 36.3 μM and Ki* to be 11.5 μM, suggesting the pre-covalent complex to be relevant for inhibition. Molecular dynamics simulations of compounds 1 and 2 with cruzain were used to propose putative binding modes for the ligands. One-dimensional (1D) quantum mechanics/molecular mechanics (QM/MM) potential of mean force (PMF) and gas-phase energies showed that the attack of Cys25-S- on the C═S or C═O bond yields a more stable intermediate than the attack on the C═N bond of the thiosemicarbazone/semicarbazone. Two-dimensional (2D) QM/MM PMF revealed a putative reaction mechanism for compound 1, involving the proton transfer to the ligand, followed by the Cys25-S- attack at C═S. The ΔG and energy barrier were estimated to be -1.4 and 11.7 kcal/mol, respectively. Overall, our results shed light on the inhibition mechanism of cruzain by thiosemicarbazones.
摘要：
克氏锥虫是一种寄生虫，感染全球约6-7百万人，主要在拉丁美洲，导致查加斯病。Cruzain,克氏杆菌的主要半胱氨酸蛋白酶，是开发恰加斯病候选药物的有效目标。缩氨基硫酮是用于靶向Cruzain的共价抑制剂中最相关的弹头之一。尽管它的相关性，缩氨基硫脲抑制cruzain的机制尚不清楚。这里,我们将实验和模拟相结合，揭示了基于缩氨基硫脲的抑制剂（化合物1）对Cruzain的共价抑制机制。此外,我们研究了氨基脲（化合物2），在结构上与化合物1相似，但不抑制Cruzain。试验证实了化合物1抑制的可逆性,并提出了两步抑制机制。Ki估计为36.3μM，Ki*为11.5μM，表明前共价复合物与抑制有关。化合物1和2与cruzain的分子动力学模拟用于提出配体的推定结合模式。平均力（PMF）和气相能量的一维（1D）量子力学/分子力学（QM/MM）势表明，Cys25-S-对C=S或C=O键的攻击产生更稳定的中间体。二维（2D）QM/MMPMF揭示了化合物1的推定反应机理，涉及质子转移到配体，随后是C=S处的Cys25-S-攻击ΔG和能垒估计为-1.4和11.7kcal/mol，分别。总的来说,我们的研究结果揭示了缩氨基硫脲对cruzain的抑制机制。
