Hijacking Plasmodium ubiquitin-proteasome system to defeat drug resistance

项目摘要/摘要 2020年，有超过2.5亿例致人衰弱的病例和超过50万人死亡，其中大部分是幼儿， 疟疾是一种持久的全球健康危机。引起疟疾的寄生虫恶性疟原虫 已对部署的大多数抗疟疾药物产生抗药性，包括主干青蒿素 (艺术)。ART及其半合成类似物被认为是治疗疟疾所必需的。艺术是 在寄生虫体内被激活以形成共价攻击的反应性自由基的前药物 蛋白质、脂类和其他细胞成分。艺术抵抗在东南亚很普遍，已经 在非洲也有报道。ART联合疗法(ACT)是治疗疟疾的支柱，但其 当两种药物联合成为事实上的单一疗法时，疗效可能会脱轨。此外， PF长期暴露于ACTs可诱导多药耐受。我们最近证明了抑制剂 针对PF蛋白酶体(Pf20S)在PF生命周期的每个阶段杀死PF并与ART协同作用， 克服艺术阻力。这一提议建立在我们的发现之上，即一种艺术的共价杂交 类似物和Pf20S抑制剂，我们称为artezomib(ATZ)，可以增强ART的作用并克服 对它的每一个组成部分都有抗性。我们已经合成了ATZ，它们是更有效的Pf20S抑制剂 而不是它们的成分Pf20S抑制剂。它们不仅杀死野生型和抗逆转录病毒(K13突变体)Pf，Pf 蛋白酶体突变使人对Pf20S抑制剂产生抗药性，但也会杀死表达Pf的Pf 既有抗ART突变，也有抗PI突变。我们提出了以下机制，通过这些机制，ATZ 克服体内对Pf20S抑制剂的抵抗力：我们发现，当激活ATZ时， 寄生虫，ART组分结合PF蛋白，就像激活的ART本身一样。PF泛素蛋白酶体 系统将ATZ结合的蛋白质消化成寡肽，其中一些显示PF抑制剂 ATZ的组件。我们假设携带ATZ的多肽在体内的延长接触 Pf20S活性位点增加了ATZ的Pf20S抑制物组分的结合，克服了 Pf20S点突变导致的结合减少。因此，ATZ可以克服 对它的每一个组成部分都有抗性。在疟疾的小鼠模型中，ATZ将P.berghei驱赶到 伯氏疟原虫抗药性K13突变体的检测限和抑制复发 比艺术好多了。在本提案的目标1中，我们将进行领先优化，以改善ATZ的 药效、选择性和ATZ的药代动力学特性。在目标2中，我们将探索ATZ的机制 作用；试图选择抗ATZ寄生虫；确定频率和机制 抗药性，如果有的话；并研究在PF生命周期的不同阶段，单独使用ART时ATZ的抗疟疾活性 是无效的。目的3将在小鼠身上测试ATZ的效果，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. .