Extreme Resistance to Mitochondrial Inhibitors in Plasmodium falciparum

描述(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抗药性的出现和蔓延使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。阿托瓦酮是一种安全、有效的药物，与普罗瓜尼联合用于治疗和预防疟疾。以往的研究表明，恶性疟原虫对阿托瓦酮的临床耐药性是由线粒体编码的细胞色素b基因的单核苷酸多态(SNPs)引起的。虽然在体外药物压力下可以选择多个非同义SNP，但到目前为止，临床耐药仅限于268位氨基酸的替换(如Y268S)。从泰国阿托瓦酮的II期研究中，我们已经确定了从失败患者身上收集的多个恶性疟原虫分离株的特征。 阿托瓦酮单独治疗(不同剂量方案)或与普鲁瓜尼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瓦酮的耐药性范围很广，从5倍到10,000倍。低度耐药株无细胞色素b突变，而中至极耐药株均有aa268突变。考虑到观察到的广泛耐药性，Y268S突变似乎不仅仅是耐药性的唯一基础。通过使用一系列抑制剂，我们发现了对阿托瓦酮高度耐药的寄生虫，这些寄生虫对所有针对线粒体的测试药物都具有高度耐药性。这些药物包括通常有效的4(1H)-喹诺酮类和吡啶酮类，以及二氢橙酸脱氢酶(DHODH)抑制剂。这个项目的主要目标是识别抗性的分子标记，与细胞色素b突变一起传递极端抗性表型。我们将从阿托瓦酮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、中和极抗药性的参考克隆。我们将使用下一代测序来识别与耐药性相关的新SNPs或CNV；这一努力将通过在治疗前将耐药克隆与同一患者的恶性疟原虫克隆进行比较而得到帮助。此外，我们将检验这样一种假设，即在体内通过同时接触阿托瓦酮和乙胺嘧啶来选择极端抗药性。最后，我们将评估耐药基因对蚊子传播的可能性。这些研究的结果将提供有关该领域对线粒体抑制剂极端耐药的抗药性风险和传播潜力的关键知识。

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.