Rational development of anti-Trypanosoma cruzi drugs

描述(由申请人提供)：这项研究项目将在华盛顿大学进行，作为医学系、化学系和生物化学系的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)组成的科学家小组在抗寄生虫药物开发方面作为一个团队工作了约15年，在该领域发表了100篇论文。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，以确保一个富有成效和成功的研究计划。这项拟议研究的长期目标是开发用于治疗恰加斯病的类固醇14a-脱甲基酶(14 DM)的抑制剂。克氏锥虫是恰加斯病的病原体，在拉丁美洲广泛流行，会引起心肌病和胃肠道病理性扩张。由于疗效和毒性较差，目前对这种疾病的治疗方法是不够的。在之前的资助期间，我们的研究表明，类固醇生物合成的抑制剂在体外和克氏锥虫感染的动物模型中都是高度有效的。具体地说，与抗癌药物tipifarnib有关的一系列化合物被发现对固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替法尼布的类似物，这些类似物对治疗恰加斯病的活性进行了优化。在下一个资金阶段，该小组建议追求以下具体目标：1)使用合理的设计方法设计和合成每年约100个替法尼布类似物。我们的一位合作者在2010年解决了T.CRUZI 14 DM的晶体结构，将用于指导新替法尼布类似物的设计。对肝微粒体培养的代谢物进行理化性质和分析将有助于指导合成计划，使生物可用化合物具有良好的代谢稳定性。2)检测替法尼类似物的药效、药理和毒性。新合成的化合物将在一系列研究中进行测试，并根据预先定义的标准进行推进或终止。所有化合物都将在克鲁兹旋毛虫14 DM酶上进行结合试验，以抑制克鲁兹旋毛虫的无鞭毛虫，并测试对哺乳动物细胞的细胞毒性。后续研究将测试其在水中的溶解性、在肝微粒体内的稳定性以及对人类细胞色素P450酶的抑制作用。接下来将测试化合物在啮齿动物身上的药代动力学特性，并将在已建立的克氏毛滴虫感染小鼠模型中测试最好的化合物的有效性。选定的化合物将进行HERG通道抑制、人类受体图谱和遗传毒性研究。3)筛选协同伙伴药物。从2000种化合物的筛选中，我们确定了FDA批准的60种对克氏毛滴虫无鞭毛虫具有抑制活性的药物。当与替法尼类似物(JK-11)结合时，已证明有8个具有协同活性。在这个目标中，我们将从体外测试转移到在小鼠感染毛滴虫模型中测试这些组合。我们希望确定至少一种FDA批准的口服药物，以增强替法尼布类似物的体内疗效。通过确定伙伴药物，我们设想了一种抗Chagas药物方案的可能性，该方案比目前60-90天的疗程更短，并阻止潜在的耐药性发展。

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.