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）：疟疾是全球一个主要的健康问题，每年有超过3亿人被感染，最多可达100万人死亡。对大多数抗疟疾药物的抗药性的出现和传播使疟疾的有效治疗变得困难，并且迫切需要新的抗疟疾药物或药物组合。 Atovaquone是一种安全有效的药物，与Proguanil结合使用，以治疗和预防疟疾。先前的研究表明，对恶性疟原虫的线粒体编码的细胞色素B基因，单核苷酸多态性（SNP）赋予了对阿托夸酮的临床耐药性。尽管可以在体外药物压力下选择多个非同义SNP，但迄今为止，临床耐药性仅限于位置268的氨基酸取代（例如Y268S）。从泰国阿托瓦夸酮的第二阶段研究中，我们表征了多种恶性疟原虫的分离株，这些分离株是从失败的患者中收集的 单独使用Atovaquone（各种剂量方案）或与前甲基或乙胺碱进行治疗。有趣的是，我们观察到了对阿托瓦夸酮的广泛抗性，这些分离株中从5到10,000倍。低度抗性分离株没有细胞色素B突变，而中度至极端抗性分离株都具有AA268突变。鉴于观察到的广泛电阻范围，不仅仅是Y268S突变似乎是抗药性的唯一基础。通过使用一系列抑制剂，我们发现极度抗抗性寄生虫对靶向线粒体的所有药物具有高度耐药性。其中包括正常有效的4（1H） - 喹诺酮和吡啶酮，以及二氢易能酸酯脱氢酶（DHODH）抑制剂。 该项目的主要目标是识别抗性的分子标记物，并与细胞色素B突变一起传达出极端的抗性表型。我们将从Atovaquone 2阶段临床研究中从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的参考克隆。我们将使用下一代测序来识别与电阻相关的新型SNP或CNV。在治疗前将抗性克隆与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，我们将测试以下假设：通过同时接触Atovaquone和乙胺胺，在体内选择了极端耐药性。最后，我们将评估将电阻基因型传播到蚊子的潜力。这些研究的结果将提供有关阻力风险和对线粒体抑制剂在现场的极端抗性的潜力的关键知识。