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[例如，利什曼原虫属、疟原虫属、锥虫属]通过催化同型半胱氨酸（Hcy）和腺苷（Ado）的代谢在控制胞内Hcy水平中起关键作用。通过控制胞内α-Hcy水平，这种酶在调节S-腺苷-L-甲硫氨酸（α-Met）依赖性甲基转移酶中起关键作用，所述甲基转移酶对哺乳动物细胞和寄生虫的生存力至关重要（例如，寄生虫中的CIMET依赖性甲基转移酶参与mRNA加帽和反式剪接）。当这些生物化学过程被抑制，通过升高细胞内的α-Hcy水平，使用已知的抑制剂的人α-Hcy水解酶，抗寄生虫作用，在体外和体内观察。这些结果表明，如果可以设计出寄生虫HCY水解酶的特异性抑制剂，它们将具有作为抗寄生虫剂的临床潜力。这些化合物的临床成功的关键是它们对人α-Hcy水解酶缺乏抑制作用，因此，使对哺乳动物细胞的毒性作用最小化。最近，我们的实验室已经克隆并过表达了来自杜氏利什曼原虫（L）和克氏锥虫（T）的β-Hcy水解酶，以及与人酶相比它们与NAD +结合的差异（即，NAD+与人β-Hcy水解酶结合的Kd为120 nM，而寄生虫酶的Kd值约为1-2 μ M。NAD+亲和力的这些差异解释了为什么3 '-脱氧腺苷（3'-脱氧-Ado）是杜氏乳杆菌和克氏锥虫酶的有效抑制剂，但它对人β-Hcy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与杜氏乳杆菌、克氏锥虫以及恶性疟原虫（P）的NAD+结合位点结合的结构特征，（ii）阐明NAD +与人类和寄生虫酶结合差异的结构基础，阐明人与寄生虫酶的结构、催化活性和对抑制的敏感性之间的关系及其差异。这项研究计划的结果应该是确定寄生虫的同型半胱氨酸水解酶的特异性抑制剂，作为抗寄生虫药的临床潜力。

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.