Design of C. parvum and T. gondii DHFR-TS Inhibitors

描述(由申请人提供)： 由微小隐孢子虫和弓形虫引起的机会性感染对免疫功能低下的患者、儿童和老年人来说是威胁生命的疾病。目前还没有治疗隐孢子虫病的有效方法，弓形虫病的治疗需要联合使用磺胺二恶英，这是一种许多患者都有严重不良反应的化合物。开发对病原体具有高度效力和高度选择性的新疗法是迫在眉睫的。新药靶标二氢叶酸还原酶胸苷合成酶(DHFR-TS)的晶体结构为针对这些靶标的基于结构的药物设计提供了必要的证据。在这项建议的第一个目标中，我们将设计甲氧普林类似物，以与微小隐孢子虫DHFRTS结构的物种选择元件良好地相互作用。物种选择性元件将通过比较致病性DHFR-TS和人DHFR的晶体结构来确定。新的抑制剂将被模拟到酶的结构中，同时考虑到配体诱导的构象变化，并预测结合模式。甲氧苄氨嘧啶类似物的晶体结构将在迭代周期中指导未来的设计。在第二个目标中，我们将利用弓形虫DHFR-TS与亲脂抑制剂结合的晶体结构信息，提高一种有前景的弓形虫DHFR-TS抑制剂的效力和选择性。改进弓形虫DHFR-TS抑制剂的设计将利用物种选择性元件。在第三个目标中，我们将阐明DHFR中乙胺胺抗性的结构基础，并设计一种新型的乙胺胺抗性弓形虫DHFR-TS抑制剂的效力和选择性。DHFR对乙胺胺和其他抗菌剂的耐药性是一个威胁到人们的问题，迫切需要能够抑制耐药酶的新疗法。在初步研究中，我们将解决乙胺嘧啶抗性DHFR-TS与一种新的、令人兴奋的抑制剂结合的晶体结构，该抑制剂对耐药酶的抑制常数为350 nM。利用结构信息，我们将阐明抗性的结构基础，并对新型缓蚀剂进行修饰，以获得更高的效力和选择性。

DESCRIPTION (provided by applicant): Opportunistic infections caused by Cryptosporidium parvum and Toxoplasma gondii represent life threatening diseases for immuno-compromised patients, children and the elderly. There are currently no effective treatments available for cryptosporidiosis and treatments for toxoplasmosis require the coadministration of sulfadioxine, a compound to which many patients have severe adverse reactions. The development of novel therapeutics that are highly potent and highly selective for the pathogen is of immediate importance. Crystal structures of the validated drug target, dihydrofolate reductase-thymidylate synthase (DHFR-TS), a bifunctional enzyme in protozoa, from C. parvum and T. gondii, provide essential evidence for structure-based drug design against these targets. In the first Aim of this proposal, we will design trimethoprim analogs to interact favorably with species-selective elements of the C. parvum DHFRTS structure. Species-selective elements will be determined by comparing crystal structures of pathogenic DHFR-TS and human DHFR. New inhibitors will be modeled into the structure of the enzyme, while accounting for ligand-induced conformational changes, and binding modes predicted. Crystal structures of trimethoprim analogs will guide future design in an iterative cycle. In the second Aim, we will improve the potency and selectivity of a promising T. gondii DHFR-TS inhibitor, using information from crystal structures of T. gondii DHFR-TS bound to lipophilic inhibitors. Designs for the improvement of T. gondii DHFR-TS inhibitors will take advantage of species-selective elements. In the third Aim, we will elucidate the structural basis of pyrimethamine resistance in DHFR and design potency and selectivity into a novel inhibitor of pyrimethamine-resistant T. gondii DHFR-TS. DHFR resistance to pyrimethamine and other antimicrobials is a threatening problem and novel therapeutics capable of inhibiting the resistant enzymes are desperately needed. We will solve a crystal structure of pyrimethamine-resistant DHFR-TS bound to a novel and exciting inhibitor that shows an inhibition constant of 350 nM against the resistant enzyme in preliminary studies. Using the structural information we will elucidate the structural basis of resistance and modify the novel inhibitor for greater potency and selectivity.