Targeting Trypanosoma brucei S-adenosylmethionine decarboxylase in Drug Discovery

描述(申请人提供)：人类非洲锥虫病(HAT)，俗称昏睡病，由单细胞寄生虫布氏锥虫引起，通过受感染的采采蝇传播给人类。锥虫病对撒哈拉以南非洲大量人口的健康产生深远影响。如果不进行治疗，它是致命的，目前的药物治疗由于毒性和新出现的耐药性而存在问题。多胺的生物合成途径是开发抗HAT药物的有效靶点，新的证据提供了一个令人信服的证据，S-腺苷甲硫氨酸脱羧酶(ADOMetDC)是开发新的抗锥虫药物的一个非常有前途的靶点。在腐胺生产多胺亚精胺的过程中，ADOMetDC催化限速步骤。哺乳动物的Adobe MetDC是一种同源二聚体，它使用丙酮酰基作为辅因子来稳定在Adobe Met脱羧基过程中形成的碳负离子中间体。最近，我们的实验室证明了锥虫Adobe MetDC是 由一种独特的机制调节，与催化死亡的ADOMetDC同系物形成异二聚体。这种蛋白质被命名为Prozyme，与Adobe MetDC形成高亲和力的异源二聚体，并将其活性提高1000倍。进一步证实了异源二聚体是体内的功能酶。我的主要目标是阐明酶调节ADOMetDC激活的机制，并鉴定和表征新的ADOMetDC抑制剂，这些抑制剂对于开发新的抗锥虫药物至关重要。为此，我首先将重点放在识别参与激活的关键氨基酸残基上，试图深入了解变构调节的机制。下一步，我将通过将布氏锥虫的ADOMetDC/Prozyme复合体与T.ruzi和L.donovani的同源物进行比较，来表征Prozyme激活ADOMetDC的物种差异。最后，我将对一个小分子化合物文库进行高通量筛选(HTS)，以确定布鲁氏毛滴虫的新型抑制剂，并测试它们对培养的寄生虫的活性。在这些研究完成后，我将确定并验证一些新型的ADOMetDC抑制剂，用于未来的先导优化研究，这些研究可能导致发现治疗HAT的新的、毒性较低的药物。项目简介：非洲人类锥虫病是一种昆虫传播的疾病，如果不治疗是致命的，但目前的药物治疗是有毒的，很难管理。这项提案中描述的工作描述了一种在寄生虫中具有独特功能的基本酶的特征，目的是开发新的化疗药物来治疗这种毁灭性的疾病。

DESCRIPTION (provided by applicant): Human African Trypanosomiasis (HAT), commonly called sleeping sickness, is caused by single-celled parasites, Trypanosoma brucei, which are transmitted to humans by infected tsetse flies. Trypanosomiasis has a profound impact on the health of a large number of people in sub-Saharan Africa. It is fatal if left untreated and current drug therapy is problematic because of toxicity and emerging resistance. The polyamine biosynthetic pathway is a validated target for the development of drugs against HAT and the emerging evidence provides a compelling case that S-adenosylmethionine decarboxylase (AdoMetDC) is a highly promising target for the development of new anti-trypanosomal agents. AdoMetDC catalyses the ratelimiting step in the production of the polyamine spermidine from putrescine. Mammalian AdoMetDC is a homodimer that uses a pyruvoyl group as a cofactor to stabilize the carbanion intermediate formed during the decarboxylation of AdoMet. Recently, our lab demonstrated that the trypanosomatid AdoMetDC is regulated by a unique mechanism, heterodimer formation with a catalytically dead AdoMetDC homolog. This protein, designated prozyme, forms a high-affinity heterodimer with AdoMetDC and increases its activity by >1,000-fold. Further we confirmed that the heterodimer is the functional enzyme in vivo. My primary goals are to elucidate the mechanisms that regulate the AdoMetDC activation by the prozyme and to identify and characterize novel AdoMetDC inhibitors essential for the development of new anti-trypanosomal drugs. In order to do that, I will first focus on the identification of crucial amino acid residues involved in the activation in an attempt to gain insights into the mechanism of allosteric regulation. Next I will characterize species differences in the activation of AdoMetDC by prozyme by comparing the T. brucei AdoMetDC/prozyme complex to the homologs from T. cruzi and L. donovani. Finally I will perform a high-throughput screen (HTS) of a small molecule compound library to identify novel inhibitors of T. brucei AdoMetDC and to test these for activity against cultured parasites. Upon completion of these studies I will have identified and validated a number of novel inhibitors of AdoMetDC for future lead optimization studies that may lead to the discovery of new, less toxic drugs for the treatment of HAT. Project Narrative: Human African Trypanosomiasis is a insect borne disease that is fatal if untreated, yet current drug therapies are toxic and difficult to administer. The work described in this proposal characterizes an essential enzyme that has unique features in the parasite, with the goal of developing new chemotherapeutic agents against this devastating disease.