Mechanism of Trypanosoma Cruzi's Transsialidase in Chagas' Disease

描述（由申请人提供）：南美锥虫病（美洲锥虫病）影响拉丁美洲1600 - 1800万人。它在感染者中的死亡率为25-30%，每年造成45，000至50，000人死亡。鞭毛虫原生动物寄生虫克氏锥虫是南美锥虫病的病原体，通过吸血的“刺客虫”叮咬传播给人类。最常见的病原体是Triatoma infestans（阿根廷的vinchucas或巴西的barbeiros）。 克氏锥虫中的转唾液酸酶催化唾液酸从宿主糖缀合物转移到其他寄生虫糖缀合物，并且在克氏锥虫（恰加斯病的病原体）的毒力中具有关键作用。克氏锥虫转唾液酸酶（TcTS）是一种经验证的抑制靶标，具有治愈这种致命慢性疾病的治疗可能性。TcTS也是令人感兴趣的，因为它在序列和结构上与严格的水解酶，兰氏锥虫唾液酸酶（TrSA）非常相似。两者的广泛比较将揭示唾液酸转移酶活性的结构要求，并且也是将来将唾液酸酶转化为具有合成价值的转唾液酸酶的努力的指导。本理论研究将集中在TcTS和TrSA的水解反应和TcTS的唾液酸转移反应的整个酶结构上使用混合（混合量子和经典，QM/MM）方法的反应机理。有一个方法开发部分，这个建议与两个不同的QM/MM实现，一个本地的程序琥珀色，和一个链接琥珀色与QM程序高斯，通过我们设计的一个程序名为瞳孔。还将对对应于反应路径上不同点的酶的各种X射线晶体结构进行分子动力学（MD）模拟。通过对野生型和突变型酶的分子动力学模拟和比较，揭示了唾液酸转移反应的关键活性位点相互作用和动力学性质。我们还将使用对接，自由能计算和基于机制的线索相结合，对TcTS的抑制剂设计进行广泛的研究。 公共卫生相关性：本研究的一个更广泛的影响是对理解催化的一般原则的贡献。从合成的角度来看，转唾液酸酶可用于进行具有工业和药用价值的O-连接和S-连接的糖缀合物的复杂合成。更深入地了解酶的机制可能是整个家族的抑制剂设计研究的指导。

DESCRIPTION (provided by applicant): Chagas' disease (American Trypanosomiasis) affects 16-18 million people in Latin America. It has a 25-30% death rate among infected people and causes between 45,000 and 50,000 deaths a year. The flagellate protozoan parasite Trypanosoma cruzi, the causative agent of Chagas' disease is transmitted to humans by bites of blood-feeding "Assassin bugs". The most common agent is Triatoma infestans (vinchucas in Argentina, or barbeiros in Brazil). The enzyme trans-sialidase in T.cruzi catalyzes the transfer of sialic acids from host aloglycoconjugates to other parasite glycoconjugates and has a critical role in virulence of T.cruzi, the etiological agent of Chagas' disease. T.cruzi trans-sialidase (TcTS) is a validated target for inhibition with therapeutic possibilities for the cure for this lethal chronic disease. TcTS is also of interest due to its strong similarity in sequence and structure to a strict hydrolase, Trypanosoma rangeli sialidase (TrSA). An extensive comparison of the two will reveal structural requirements for sialyl-transferase activity and also be a guide for future efforts to transform sialidases into trans-sialidases that are of synthetic value. This theoretical study will focus on the reaction mechanisms for hydrolysis reactions of TcTS and TrSA and sialyl-transfer reaction of TcTS using hybrid (mixed quantum and classical, QM/MM) methods on entire enzyme structures. There is a methods development part of this proposal associated with two different QM/MM implementations, one native to the program Amber, and one that links Amber with the QM program Gaussian, via a program we designed name Pupil. Molecular dynamics (MD) simulations on various X-ray crystal structures of the enzymes which correspond to different points on the reaction paths will also be performed. Critical active site interactions and dynamical properties for sialyl-transfer reaction will be uncovered analyzing MD simulations of wild type and mutant enzymes and comparing the results. We will also perform extensive research into inhibitor design for TcTS, using a combination of docking, free energy calculations and mechanism-based leads. Public Health Relevance: A broader impact of the present research is the contribution to the understanding of the general principles of catalysis. From a synthetic point of view, trans-sialidases can be used to perform complex syntheses of O-linked and S-linked glycoconjugates of industrial and medicinal value. A deeper understanding of the mechanisms of the enzymes could be a guide for inhibitor design studies of the entire family.