Hijacking Plasmodium ubiquitin-proteasome system to defeat drug resistance

项目概要/摘要 到2020年，将有超过2.5亿例衰弱病例和50多万例死亡，其中大部分是幼儿， 疟疾是一个持续的全球健康危机。引起疟疾的寄生虫恶性疟原虫（Pf） 已对大多数已部署的抗疟药物产生抗药性， （艺术）。抗逆转录病毒疗法及其半合成类似物被认为是疟疾治疗的关键。艺术是 前药在寄生虫体内被激活，形成一个反应性自由基， 蛋白质、脂质和其他细胞成分。抗逆转录病毒疗法耐药性在东南亚普遍存在， 据报道，在非洲。ART联合疗法（ACT）是治疗疟疾的主要手段，但其 当两种药物的组合成为事实上的单一疗法时，疗效可能会偏离轨道。此外，委员会认为， Pf长期暴露于ACTs诱导多药耐受。我们最近发现抑制剂 特异于Pf蛋白酶体（Pf 20 S）在Pf生命周期的每个阶段杀死Pf并与ART协同作用， 克服抗逆转录病毒疗法该提议建立在我们的发现基础上，即ART的共价杂交 类似物和Pf 20 S抑制剂，我们称之为artezlatin（ATZ），可以增强ART作用，并克服 它的每一个组成部分的阻力。我们已经合成了更有效的Pf 20 S抑制剂ATZ 而不是它们的组分Pf 20 S抑制剂。它们不仅能杀死野生型和抗ART（K13突变体）Pf， 具有蛋白酶体突变，赋予对Pf 20 S抑制剂的抗性，但也杀死表达Pf的Pf。 ART耐药和PI耐药突变。我们提出以下机制，ATZ 克服对Pf 20 S抑制剂的耐药性：我们发现， 在寄生虫中，ART成分结合Pf蛋白，就像激活的ART本身一样。Pf泛素蛋白酶体 系统将ATZ结合的蛋白质翻译成寡肽，其中一些显示Pf抑制剂 ATZ的组成部分。我们假设，ATZ携带肽在细胞内的长期接触， Pf 20 S活性位点增强了ATZ的Pf 20 S抑制剂组分的结合，克服了ATZ抑制剂组分的结合。 降低结合，否则由Pf 20 S点突变赋予。因此，ATZ可以克服 它的每一个组成部分的阻力。在疟疾的小鼠模型中，ATZ将伯氏疟原虫驱赶到 伯氏疟原虫ART耐药K13突变体的检测限和抑制复发， 在本提案的目标1中，我们将进行铅优化，以提高ATZ的 效力、选择性和ATZ的药代动力学性质。在目标2中，我们将探索ATZ的机制 尝试选择ATZ抗性寄生虫;确定 耐药性，如果有的话;并研究抗疟药的抗疟活性，在阶段的Pf生命周期时，单独的艺术 是无效的。目的3将测试ATZ在小鼠中的功效，包括感染Pf的人源化小鼠。 .

Project Summary/Abstract In 2020, with over 250 million debilitating cases and over half a million deaths, mostly in young children, malaria is a persistent global health crisis. The malaria-causing parasite Plasmodium falciparum (Pf) has developed resistance to most antimalarial drug deployed, including the backbone artemisinins (ARTs). ART and its semi-synthetic analogs are considered essential for malaria treatment. ARTs are prodrugs that are activated within the parasites to form a reactive radical that covalently attacks proteins, lipids and other cellular constituents. ART resistance is widespread in Southeast Asia and has been reported in Africa. ART combination therapy (ACT) is a mainstay for treatment of malaria, but its efficacy can be derailed when a two-drug combination becomes de facto monotherapy. Moreover, extended exposure of Pf to ACTs induces multidrug tolerance. We recently showed that inhibitors specific for the Pf proteasome (Pf20S) kill Pf in each stage of its life cycle and synergize with ART, overcoming ART resistance. This proposal builds on our discovery that a covalent hybrid of an ART analogue and a Pf20S inhibitor that we call an artezomib (ATZ) can enhance ART action and overcome resistance to each of its components. We have synthesized ATZs that are more potent Pf20S inhibitors than their component Pf20S inhibitor. They not only kill wild type and ART-resistant (K13 mutant) Pf, Pf with proteasome mutations that confer resistance to the Pf20S inhibitor, but also kill Pf that expresses both ART-resistant and PI-resistant mutations. We propose the following mechanism by which ATZs overcome resistance to the Pf20S inhibitor within them: We found that upon activation of ATZ in the parasites, the ART component binds Pf proteins, like activated ART itself. The Pf ubiquitin proteasome system digests ATZ-bound proteins into oligopeptides, some of which display the Pf inhibitor component of the ATZ. We hypothesize that extended contact of ATZ-bearing peptides within the Pf20S active site augments the binding of the Pf20S inhibitor component of the ATZ, overcoming the decreased binding otherwise conferred by Pf20S point mutations. Thus, an ATZ can overcome resistance to each of its components. In mouse models of malaria, an ATZ drove P. berghei below the limit of detection and suppressed recrudescence of a P. berghei ART-resistant K13 mutant and doing so better than ART. In Aim 1 of this proposal, we will conduct lead optimization to improve ATZs' potency, selectivity and ATZs' pharmacokinetic properties. In Aim 2, we will explore ATZs' mechanism of action; attempt to select for ATZ-resistant parasites; determine the frequency and mechanism of resistance, if any; and study antimalarial activity of ATZs in stages of the Pf life cycle when ART alone is ineffective. Aim 3 will test the efficacy of ATZs in mice, including humanized mice infected with Pf. .