Novel Inhibitors of Cruzain and Trypanosoma brucei Cathpesin B as Potential Drug Candidates for the Treatment of African Trypanosomiasis and Chagas Disease

项目摘要 由于既没有疫苗，也没有耐受性良好的治疗方法，锥虫属引起 使人衰弱的高发病率的人类疾病，迫切需要有效的药物。 克氏锥虫在中美洲和南美洲引起恰加斯病（CD）， 每年有5万人死亡，德克萨斯州最近报告的病例超过40例。布氏锥虫罗德西亚亚种 和T.冈比亚布鲁氏菌引起非洲昏睡病（HAT），影响超过6万人 世界范围内。CD和HAT都包含未满足的医疗需求，有效药物可以 不存在.人感染T. cruzi和T.布氏 分别需要相关的半胱氨酸蛋白酶cruzain和TbCat B。的目的 一项提议是鉴定cruzain和TbCatB的新型可逆共价抑制剂（RCI）， 在T. cruzi和T. 布鲁氏菌属，并且在锥虫病的鼠模型中是有疗效的。我们的方法建立在 先前的化合物如K11777，一种含有亲电子乙烯基的三肽类似物， 砜部分（图1 -特定目的）。K11777是一种不可逆共价灭活剂， cruzain和TbCatB与的活性位点半胱氨酸形成稳定的共价加合物 原生动物半胱氨酸蛋白酶。K11777在动物模型中治愈恰加斯病， 进行人体临床试验。然而，最近的焦点已经从不可逆酶转移到 可逆共价抑制剂（RCI）的灭活剂，因为后者具有较少的毒性问题， 对目标的选择性更强我们将研究2-乙烯基的引入- 在选择性肽支架中的杂环基团，取代乙烯基砜的“弹头”， K11777，因为我们相信这样的部分将为cruzain和TbCatB提供合适的RCI。到 迄今为止，我们已经制备了拟肽2-乙烯基杂环抑制剂（P2 VHI），其包括 与乙烯基共轭的嘧啶、恶唑或噻唑基团。这些P2 VHI显示时间- 依赖性，可逆性抑制cruzain（Ki = 7-20 nM），并杀死细胞培养物中的锥虫。 我们提出：（a）乙烯基的亲电性可以通过取代乙烯基来“调节”。 杂环基团，和（B）在乙烯基上添加酶促半胱氨酸残基将 容易逆转。在本提案的R21阶段，我们将推进这一概念， 一系列P2 VHI，其表现出（a）对cruzain的改善的效力，（B）对TbCat B的抑制，（c） 含有对原生动物蛋白酶具有选择性的肽支架，和（d）在 低微摩尔浓度。我们将确定是否形成可逆的共价加合物， 这些目标的活性位点半胱氨酸。在R33阶段，我们将开发一个化学模型， 2-乙烯基杂环，使我们能够探测和优化硫醇的可逆反应性 向P2 VHI发展我们将生产一系列第二代P2 VHI， 候选药物的性质、毒性和安全性特征。我们将评估这些化合物， 人类感染的细胞模型，然后在小鼠模型中进行概念验证评估， CD和帽子

PROJECT SUMMARY With neither vaccines nor well-tolerated therapies available, the genus Trypanosoma causes debilitating human diseases of high morbidity for which effective drugs are urgently needed. Trypanosoma cruzi causes Chagas Disease (CD) in Central and South America, resulting in 50,000 annual deaths, with >40 recent cases reported in Texas. Trypanosoma brucei rhodesiense and T. brucei gambiense cause African sleeping sickness (HAT), affecting over 60,000 people world-wide. Both CD and HAT comprise unmet medical needs for which effective medicines do not exist. The establishment and maintenance of human infection by both T. cruzi and T. brucei requires, respectively, the related cysteine proteases cruzain and TbCat B. The objective of this proposal is to identify novel reversible-covalent inhibitors (RCIs) of cruzain and TbCatB, that exert potent and selective enzyme inhibition and trypanocidal activity in cell cultures of T. cruzi and T. brucei spp., and are curative in murine models of trypanosomiasis. Our approach builds on precedent compounds such as K11777, a tri-peptide analogue that contains an electrophilic vinyl- sulfone moiety (Fig. 1 – SPECIFIC AIMS). K11777 is an irreversible covalent inactivator of cruzain and TbCatB which forms a stable covalent adduct with the active-site cysteines of protozoal cysteine proteases. K11777 cures Chagas disease in animal models, and has progressed to human clinical trials. However, recent focus has shifted from irreversible enzyme inactivators to reversible covalent inhibitors (RCIs), as the latter have fewer toxicity issues and are more selective for their intended targets. We will examine the introduction of a 2-vinyl- heterocyclic group in a selective peptide scaffold, replacing the vinyl-sulfone “warhead” of K11777, as we believe such a moiety would provide suitable RCIs for cruzain and TbCatB. To date we have made peptidomimetic 2-vinyl heterocycle inhibitors (P2VHIs) which include a pyrimidine, oxazole or thiazole group conjugated to the vinyl group. These P2VHIs exhibit time- dependent, reversible inhibition of cruzain (Ki = 7-20 nM), and kill trypanosomes in cell culture. We propose that (a) the electrophilicity of the vinyl group may be “tuned” by substitution of the heterocyclic group, and that (b) addition of an enzymatic cysteine residue on the vinyl group will be readily reversible. In the R21 phase of this proposal, we will advance this concept and produce a series of P2VHIs that exhibit (a) improved potency for cruzain, (b) inhibition of TbCatB, (c) contain peptide scaffolds which are selective for protozoan proteases, and (d) be trypanocidal at low micromolar concentrations. We will determine if a reversible covalent adduct is formed with the active-site cysteines of these targets. In the R33 phase, we will develop a chemical model for the 2-vinyl heterocycles that allows us to probe and optimize the reversible reactivity of thiols toward P2VHIs. We will produce a series of second-generation P2VHIs with the physicochemical properties, toxicity and safety profiles of drug candidates. We will evaluate these compounds in cellular models of human infection, followed by proof-of-concept assessment in murine models of CD and HAT.