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，由医学系、化学系和生物化学系的研究人员合作进行。由博士组成的科学家小组。Buckner（生物学），Gelb（化学）和Verlinde（基于结构的药物设计）作为一个团队在抗寄生虫药物开发方面工作了大约15年，在该领域共发表了100多篇文章。这项工作将使用最先进的设施和设备，以及专家研究人员，确保一个富有成效和成功的研究计划。拟议研究的长期目标是开发用于治疗恰加斯病的甾醇14a-去甲基化酶（14DM）酶抑制剂。克氏锥虫是恰加斯病的病原，在拉丁美洲广泛传播，可引起心肌病和病理性胃肠道扩张。目前对这种疾病的治疗方法由于疗效差和毒性而不足。在之前的资助期内，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很强的效力。具体来说，与抗癌药物tipfarnib相关的一系列化合物被发现对甾醇生物合成途径中的关键酶（14DM）具有高活性。需要更多的研究来确定对恰加斯病活性优化的替法尼的类似物。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个蒂法尼类似物。我们的一位合作者在2010年解决了T. cruzi 14DM的晶体结构，将用于指导新的蒂法尼类似物的设计。肝微粒体培养的代谢产物的理化性质和分析将有助于指导合成计划，使生物可利用的化合物具有良好的代谢稳定性。2)测试替法尼类似物的疗效、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，并根据预先确定的标准进行进展或终止。所有化合物将在克氏T. 14DM酶的结合试验中进行测试，以抑制克氏T.无尾线虫，以及对哺乳动物细胞的细胞毒性。后续研究将测试其水溶性、在肝微粒体中的稳定性以及对人类CYP450酶的抑制作用。接下来将测试化合物在啮齿动物体内的药代动力学特性，并在已建立的克氏弓形虫感染小鼠模型中测试最佳化合物的功效。选定的化合物将用于hERG通道抑制、人类受体谱分析和遗传毒性研究。3)筛选协同伙伴药物。从2000种化合物的筛选中，我们已经确定了60种FDA批准的对克氏T.无尾链球菌具有抑制活性的药物。其中8种已被证明与tipfarnib类似物（JK-11）联合时具有协同活性。在本研究中，我们将从体外试验转向在克氏锥虫感染小鼠模型中测试这些组合。我们希望找到至少一种FDA批准的口服药物，可以提高tipfarnib类似物的体内疗效。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，这种方案比目前60-90天的疗程要短，并能阻止潜在耐药性的发展。

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. PUBLIC HEALTH RELEVANCE: The proposed research will help develop new treatments for Chagas disease, a parasitic disease common in Latin America that also infects >300,000 individuals in the USA. The existing drugs are limited by poor efficacy and toxicity. New drugs are desperately needed to help prevent deaths from damage to the heart and intestinal system caused by the etiologic agent of Chagas disease (Trypanosoma cruzi).