AdoHcy Hydrolase Inhibitors as Antiparasitic Agents

描述（由申请人提供）：哺乳动物细胞和某些寄生虫（如利什曼原虫、疟原虫、锥虫）中的s -腺苷- l-同型半胱氨酸（AdoHcy）水解酶（EC 331 11）通过催化AdoHcy代谢为同型半胱氨酸（Hcy）和腺苷（Ado），在控制AdoHcy细胞内水平方面起关键作用。通过控制AdoHcy的细胞内水平，该酶在调节s-腺苷- l-蛋氨酸（AdoMet）依赖的甲基转移酶中起关键作用，这对哺乳动物细胞和寄生虫的生存至关重要（例如，寄生虫中AdoMet依赖的甲基转移酶参与mRNA盖帽和反式剪接）。当这些生化过程通过使用已知的人类AdoHcy水解酶抑制剂提高AdoHcy细胞内水平来抑制时，在体外和体内观察到抗寄生虫作用。这些结果表明，如果能够设计出寄生虫adhcy水解酶的特异性抑制剂，它们将具有作为抗寄生虫药物的临床潜力。对于这些化合物的临床成功至关重要的是它们对人类adhcy水解酶缺乏抑制作用，从而最大限度地减少对哺乳动物细胞的毒性作用。最近，我们实验室克隆并过表达了利什曼原虫(L) donovani和克氏锥虫(T)的AdoHcy水解酶，并观察了它们与人类酶结合NAD+的差异（即，NAD+与人类AdoHcy水解酶结合的Kd值为120 nM，而寄生虫酶的Kd值约为1-2 uM）。这些NAD+亲和力的差异解释了为什么3'-脱氧腺苷（3'-脱氧- ado）是L donovani和T cruzi酶的有效抑制剂，但对人类adhcy水解酶没有影响。基于这些重要的新发现，我们计划在下一个资助期内(i)优化3'-脱氧- ado与L donovani， T cruzi以及恶性疟原虫adhcy水解酶的NAD+结合位点的结构特征，（ii）阐明NAD+与人类和寄生虫酶结合差异的结构基础，以及（iii）阐明结构之间的关系。人与寄生虫酶的催化活性、抑制敏感性及其差异。这项研究计划的结果应该是鉴定寄生虫adhcy水解酶的特异性抑制剂，这些抑制剂具有临床抗寄生虫药物的潜力。

DESCRIPTION (provided by applicant): S-Adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3 3 1 1) in mammalian cells and certain parasites [e.g., Leishmania, Plasmodium, Trypansoma] plays a key role in controlling the intracellular levels of AdoHcy by catalyzing its metabolism to homocysteine (Hcy) and adenosine (Ado). By controlling intracellular levels of AdoHcy, this enzyme plays a pivotal role in regulating S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases which are crucial for the viability of mammalian cells and parasites (e.g., AdoMet-dependent methyltransferases in parasites are involved in mRNA capping and in trans-splicing). When these biochemical processes are inhibited by elevating intracellular levels of AdoHcy using known inhibitors of human AdoHcy hydrolase, antiparasitic effects were observed in vitro and in vivo. These results suggest that, if specific inhibitors of parasite AdoHcy hydrolases could be designed, they would have clinical potential as antiparasitic agents. Crucial for the clinical success of these compounds would be their lack of inhibitory effects on human AdoHcy hydrolase, thus, minimizing toxic effects to mammalian cells. Recently, our laboratory has cloned and overexpressed AdoHcy hydrolases from Leishmania (L) donovani and Trypansoma (T) cruzi and differences in their binding of NAD + compared to the human enzyme (i.e., the Kd for binding of NAD+ to human AdoHcy hydrolase is 120 nM compared to Kd values of approx 1-2 uM for the parasite enzymes) were observed. These differences in NAD+ affinity explain why 3'-deoxyadenosine (3'- deoxy-Ado) is a potent inhibitor of the L donovani and T cruzi enzymes but it has no effect on the human AdoHcy hydrolase. Based on these important new discoveries, we plan during the next grant period to (i) optimize the structural features of 3'-deoxy-Ado for binding to the NAD+ binding site of L donovani, T cruzi, as well as Plasmodium (P) falciparium, AdoHcy hydrolases, (ii) to elucidate the structural basis for the differences in the binding of NAD + to the human and parasite enzymes, and (iii) to elucidate the relationships of structure, catalytic activity and susceptibility to inhibition and their differences between the human and parasite enzymes. The outcome of this research program should be the identification of specific inhibitors of parasite AdoHcy hydiolases that have clinical potential as antiparasitic agents.