Extreme Resistance to Mitochondrial Inhibitors in Plasmodium falciparum

恶性疟原虫对线粒体抑制剂的极度耐药性

• 批准号: 8624359
• 负责人: DENNIS E KYLE
• 金额: $ 21.25万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8624359
• 关键词: Admission activity; Amino Acid Substitution; Antimalarials; Atovaquone resistance; Cessation of life; Chemicals; Chloroguanide; Clinical; Clinical Research; Combined Modality Therapy; Culicidae; Cytochromes b; Data; Development; Diagnostic and Statistical Manual of Mental Disorders; Dihydroorotate Dehydrogenase Inhibitor; Dose; Drug Combinations; Drug Targeting; Drug resistance; Exposure to; Failure; Future; Gene Amplification; Genes; Genotype; Goals; Health; Human; In Vitro; Infection; Knowledge; Malaria; Malaria prevention; Mitochondria; Molecular; Mutation; Parasite resistance; Parasites; Parasitic Diseases; Patients; Pharmaceutical Preparations; Phase; Phase II Clinical Trials; Phenotype; Plasmodium falciparum; Positioning Attribute; Prevention; Pyrazoles; Pyridones; Pyrimethamine; Quinolones; Reagent; Recrudescences; Regimen; Reporting; Resistance; Risk; Series; Single Nucleotide Polymorphism; Testing; Thailand; Treatment Failure; atovaquone; base; clinically relevant; drug discovery; drug testing; effective therapy; in vivo; inhibitor/antagonist; molecular marker; mutant; next generation sequencing; novel; phase 2 study; pre-clinical; pressure; public health relevance; resistance mechanism; success; transmission process

DESCRIPTION (provided by applicant): Malaria is a major health problem worldwide, with over 300 million people becoming infected and up to one million deaths annually. The emergence and spread of resistance to most anti-malarial drugs has made the effective treatment of malaria difficult and there is an urgent need for new anti-malarial drugs or drug combinations. Atovaquone is a safe, effective drug that is used in combination with proguanil for the treatment and prevention of malaria. Previous studies have shown that clinical resistance to atovaquone is conferred by single nucleotide polymorphisms (SNPs) in the mitochondrial encoded cytochrome b gene of P. falciparum. Although multiple non-synonymous SNPs can be selected under drug pressure in vitro, thus far clinical resistance is limited to amino acid substitutions at position 268 (e.g., Y268S). From phase II studies of atovaquone in Thailand, we have characterized multiple isolates of P. falciparum that were collected from patients that failed treatment with atovaquone alone (various dose regimens) or in combination with either proguanil or pyrimethamine. Interestingly, we observed a broad range of resistance to atovaquone, from 5 to >10,000 fold in these isolates. The low-grade resistant isolates possessed no cytochrome b mutations, whereas the moderate to extreme resistant isolates all had aa268 mutations. Given the broad range of resistance observed, more than just the Y268S mutation seemed likely to be the sole basis for resistance. By using a series of inhibitors we found extremely atovaquone resistant parasites that are highly resistant to all drugs tested that target the mitochondria. These include the normally potent 4(1H)-quinolones and pyridones, and dihydroorotate dehydrogenase (DHODH) inhibitors. The major goals of this project are to identify the molecular markers of resistance, that along with cytochrome b mutations convey an extreme resistance phenotype. We will clone P. falciparum from admission and recrudescence isolates from the atovaquone Phase 2 clinical studies to obtain reference clones with low, moderate, and extreme resistance to mitochondrial inhibitors. We will use next generation sequencing to identify novel SNPs or CNVs associated with resistance; this effort will be aided by comparing the resistant clones with P. falciparum clones from the same patient prior to treatment. In addition, we will test the hypothesis that extreme resistance was selected in vivo by simultaneous exposure to atovaquone and pyrimethamine. Finally we will assess the potential for transmission of resistance genotypes to mosquitos. The results of these studies will provide critical knowledge about the resistance risks and potential for spread of extreme resistance to mitochondrial inhibitors in the field.

描述（由申请人提供）：疟疾是世界范围内的一个主要健康问题，每年有超过 3 亿人受到感染，多达 100 万人死亡。大多数抗疟疾药物耐药性的出现和蔓延，使得疟疾的有效治疗变得困难，迫切需要新的抗疟疾药物或药物组合。阿托伐醌是一种安全有效的药物，与氯胍联合用于治疗和预防疟疾。先前的研究表明，恶性疟原虫线粒体编码的细胞色素b基因中的单核苷酸多态性（SNP）赋予了阿托伐醌的临床耐药性。尽管在体外药物压力下可以选择多个非同义SNP，但迄今为止临床耐药性仅限于268位的氨基酸取代（例如Y268S）。从泰国阿托伐醌的 II 期研究中，我们对从失败的患者中收集的多种恶性疟原虫分离株进行了表征 单独使用阿托伐醌（不同剂量方案）或与氯胍或乙胺嘧啶联合治疗。有趣的是，我们观察到这些分离株对阿托伐醌具有广泛的耐药性，从 5 倍到 >10,000 倍。低级耐药菌株不具有细胞色素b突变，而中度至极端耐药菌株均具有aa268突变。鉴于观察到的耐药性范围广泛，似乎不仅仅是 Y268S 突变是耐药性的唯一基础。通过使用一系列抑制剂，我们发现了对阿托伐醌极其耐药的寄生虫，它们对所有测试的针对线粒体的药物都具有高度耐药性。这些包括通常有效的 4(1H)-喹诺酮类和吡啶酮类药物，以及二氢乳清酸脱氢酶 (DHODH) 抑制剂。 该项目的主要目标是确定耐药性分子标记，这些分子标记与细胞色素 b 突变一起传达极端耐药表型。我们将从阿托伐醌 2 期临床研究的入院和复发分离株中克隆恶性疟原虫，以获得对线粒体抑制剂具有低、中和极端耐药性的参考克隆。我们将使用下一代测序来识别与耐药性相关的新 SNP 或 CNV；通过将耐药克隆与治疗前来自同一患者的恶性疟原虫克隆进行比较，将有助于这项工作。此外，我们将测试通过同时暴露于阿托伐醌和乙胺嘧啶在体内选择极端耐药性的假设。最后，我们将评估抗性基因型向蚊子传播的可能性。这些研究的结果将为线粒体抑制剂的耐药性风险和极端耐药性在该领域传播的可能性提供重要知识。