Extreme Resistance to Mitochondrial Inhibitors in Plasmodium falciparum

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3亿人感染，多达100万人死亡。对大多数抗疟疾药物的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基因的单核苷酸多态性（SNP）赋予了对阿托伐醌的临床耐药性。尽管可以在体外药物压力下选择多种非同义SNP，但迄今为止临床抗性仅限于位置268处的氨基酸取代（例如，Y268S）。从阿托伐醌在泰国的II期研究中，我们已经表征了从失败的患者中收集的多个恶性疟原虫分离株， 单独用阿托伐醌（各种剂量方案）或与氯胍或乙胺嘧啶组合治疗。有趣的是，我们观察到对阿托伐醌的广泛耐药性，在这些分离株中从5倍到> 10，000倍。低度耐药菌株无细胞色素B突变，而中度至极端耐药菌株均具有aa 268突变。鉴于观察到的广泛耐药性，不仅仅是Y268 S突变似乎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具有极强抗性的寄生虫，它们对所有靶向线粒体的药物都具有高度抗性。这些包括通常有效的4（1H）-喹诺酮和吡啶酮，以及二氢乳清酸脱氢酶（DHODH）抑制剂。 该项目的主要目标是鉴定抗性的分子标记，即沿着细胞色素B突变传递极端抗性表型。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、中和极端抗性的参考克隆。我们将使用下一代测序来鉴定与耐药性相关的新型SNP或CNV;这一努力将通过将耐药性克隆与治疗前来自同一患者的恶性疟原虫克隆进行比较来帮助。此外，我们将测试的假设，极端的电阻选择在体内同时暴露于阿托伐醌和乙胺嘧啶。最后，我们将评估抗性基因型传播给蚊子的潜力。这些研究的结果将提供关于耐药风险和在该领域传播对线粒体抑制剂极端耐药性的潜力的关键知识。

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.