Rational development of anti-Trypanosoma cruzi drugs

描述（由申请人提供）：本研究项目将在华盛顿大学进行，作为医学、化学和生物化学系研究人员之间的合作。由Buckner博士（生物学）、Gelb博士（化学）和Verlinde博士（基于结构的药物设计）组成的科学家小组已经作为一个团队在抗寄生虫药物开发方面工作了大约15年，在该领域发表了超过100篇文章。这项工作将使用最先进的设施和设备以及专家研究人员来完成，确保研究计划富有成效且成功。 拟议研究的长期目标是开发用于治疗恰加斯病的甾醇14 α-脱甲基酶（14 DM）抑制剂。克氏锥虫是南美锥虫病的病原体，广泛分布于拉丁美洲，可引起心肌病和胃肠道病理性扩张。目前对这种疾病的治疗是不够的，因为疗效差和毒性。在之前的资助期间，我们的研究表明，甾醇生物合成抑制剂在体外和克氏锥虫感染的动物模型中具有高度效力。具体而言，发现与癌症药物替吡法尼相关的一系列化合物对甾醇生物合成途径中的关键酶（14 DM）具有高度活性。需要更多的研究来确定优化的抗恰加斯病活性的替吡法尼类似物。在下一个资助期内，该小组建议追求以下具体目标：1）使用合理的设计方法，每年设计和合成约100种替吡法尼类似物。T. cruzi 14 DM在2010年解决了我们的合作者之一将用于指导新的tipifarnib类似物的设计。对肝微粒体代谢产物的理化性质和分析将有助于指导合成计划，以制备具有良好代谢稳定性的生物可利用化合物。 2)测试替吡法尼类似物的疗效、药理学和毒性。新合成的化合物将在一系列研究中进行测试，并根据预定义的标准进行升级或终止。所有化合物将在T细胞上进行结合试验。cruzi 14 DM酶抑制T. Cruzi无鞭毛体，以及对哺乳动物细胞的细胞毒性。后续研究将检测水溶性、在肝微粒体中的稳定性和对人CYP 450酶的抑制。接下来将测试化合物在啮齿动物中的药代动力学性质，并且将测试最佳化合物在已建立的T.克氏感染将对选定的化合物进行hERG通道抑制、人受体分析和遗传毒性研究。 3)筛选协同合作伙伴药物。从2000个化合物的筛选中，我们已经鉴定出60个FDA批准的药物对T。克鲁兹无鞭毛体。其中8种已被证明在与替吡法尼类似物（JK-11）组合时具有协同活性。在这个目标中，我们将从体外试验转移到在T.克氏感染我们希望确定至少一种FDA批准的口服药物，可以提高tipifarnib类似物的体内功效。通过确定一种合作药物，我们设想了一种抗恰加斯病药物方案的可能性，该方案比目前60-90天的治疗疗程短，并阻止了潜在耐药性的发展。

DESCRIPTION (provided by applicant): This research project will be performed at the University of Washington as a collaboration between investigators in the Departments of Medicine, Chemistry, and Biochemistry. The group of scientists consisting of Drs. Buckner (biology), Gelb (chemistry), and Verlinde (structure-based drug design) has worked as a team on antiparasitic drug development for ~15 years with collectively >100 published articles in the field. The work will be done using state-of-the-art facilities and equipment, as well as expert research staff, ensuring a productive and successful research program. The long-term objective of the proposed research is to develop inhibitors of the sterol 14a-demethylase (14DM) enzyme for treating Chagas disease. Trypanosoma cruzi, the etiologic agent of Chagas disease, is widespread in Latin America and causes cardiomyopathy and pathological dilations of the gastrointestinal tract. The currently available treatments for this disease are inadequate because of poor efficacy and toxicity. In the previous funding period, our research showed that inhibitors of sterol biosynthesis are highly potent in vitro and in animal models of Trypanosoma cruzi infection. Specifically, a series of compounds related to a cancer drug, tipifarnib, was discovered to be highly active against a key enzyme (14DM) in the sterol biosynthesis pathway. More research is needed to identify analogs of tipifarnib that are optimized for activity against Chagas disease. In the next funding period, the group proposes to pursue the following specific aims: 1) Design and synthesize of ~100 tipifarnib analogs per year using rational design methods. A crystal structure of the T. cruzi 14DM solved in 2010 by one of our collaborators will be used to guide design of new tipifarnib analogs. Physicochemical properties and analysis of metabolites from liver microsome incubations will help direct the synthesis plans to make bioavailable compounds with good metabolic stability. 2) Test the efficacy, pharmacology, and toxicity of tipifarnib analogs. The newly synthesized compounds will be tested in a series of studies and advanced or terminated by pre-defined criteria. All compounds will be tested in binding assays on the T. cruzi 14DM enzyme, for inhibition of T. cruzi amastigotes, and for cytotoxicity against mammalian cells. Follow up studies will test aqueous solubility, stability in liver microsomes, and inhibition of human CYP450 enzymes. Compounds will next be tested for pharmacokinetic properties in rodents and the best compounds will be tested for efficacy in the mouse model of established T. cruzi infection. Selected compounds will be tested for hERG channel inhibition, human receptor profiling, and genotoxicity studies. 3) Screen for synergy partner drugs. From a screen of 2000 compounds, we have identified 60 FDA approved drugs with inhibitory activity on T. cruzi amastigotes. Eight have been shown to have synergistic activity when combined with the tipifarnib analog (JK-11). In this Aim, we will move from in vitro assays to testing the combinations in the mouse model of T. cruzi infection. We hope to identify at least one FDA approved, orally administered drug that boosts the in vivo efficacy of the lead tipifarnib analog. By identifying a partner drug, we envision the possibility of an anti-Chagas drug regimen that is shorter than current treatment courses of 60-90 days and stems development of potential drug resistance.