Identification of Resistance Determinants for 1-Hydroxy-2-Alkyl-4(1)quinolones in Toxoplasma gondii

弓形虫中 1-羟基-2-烷基-4(1)喹诺酮类药物耐药性决定因素的鉴定

• 批准号: 174818595
• 负责人: Dr. Wolfgang Bohne
• 金额: --
• 依托单位: Institut für Medizinische Mikrobiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/174818595?language=en
• 关键词: Identification; Resistance; Determinants; Hydroxy; Alkyl

The increasing resistance of protozoan parasites against established drugs, particularly those of the malaria causative agent Plasmodium, emphasises the need for the identification of novel anti-parasitic compounds. We use Toxoplasma gondii as a model to elucidate the mode of action of anti-parasitic drugs in detail. The inhibition of the mitochondrial electron transport chain from apicomplexan parasites as T. gondii and P.falciparum is an effective anti-parasitic strategy. We recently identified 1-hydroxy-2-alkyl-4(1)quinolones, for example 1-hydroxy-2-dodecyl-4(1)quinolone (HDQ), as specific inhibitors of T. gondii oxidative phosphorylation. These compounds lead to a fast depolarization of the parasitic inner mitochondrial membrane potential and inhibit T. gondii and P. falciparum replication in the nano-molar range. HDQ possesses structural similarities to ubiquinone and it was shown by inhibition kinetics that HDQ is at least inhibiting the enzymatic activity of two enzymes, which possess ubiquinone binding sites (TgNDH2-I and PfDHODH). The T. gondii genome contains six ubiquinone reducing genes, which could be potential HDQ-targets. We propose to identify the relevant HDQ-target(s), whose inhibition is causing the replication inhibition, with the aid drug resistance T. gondii mutants. All six ubiquinone reducing genes will be amplified and sequenced from available HDQ resistant mutants, which were generated by chemical mutagenesis. Mutant alleles will be introduced into the wild type background in order to test whether a particular mutation is sufficient to confer resistance. Resistant mutants against two other HDQ derivatives will be generated and analyzed in the same way in order to investigate whether the individual 1-hydroxy-2-alkyl-4(1)quinolone derivatives target identical or different parasite molecules. Particular attention will be given to dihydroorotate dehydrogenase (DHODH) as a putative target, since we have preliminary evidence for a link between HDQ sensitivity and pyrimidine metabolism. We will uncouple pyrimidine de novo synthesis from respiratory chain function by expression of a ubiquinone independent DHODH, which uses fumarate instead of ubiquinone as an electron acceptor. Together, these studies will define relevant drug targets within the parasitic electron transport chain and will contribute to a detailed understanding on the mode of action of 1-hydroxy-2-alkyl-4(1) quinolones against T. gondii. Since P. falciparum possesses an identical set of ubiquinone reducing enzyme as T. gondii, these studies might also provide insights for novel drug targets in Plasmodium.

原生动物寄生虫对已有药物的抗药性日益增强，特别是那些引起疟疾的疟疾病原体的抗药性，强调了鉴定新的抗寄生虫化合物的必要性。我们以弓形虫为模型，详细阐述了抗寄生虫药物的作用机制。抑制弓形虫和恶性疟原虫等顶端复合体寄生虫的线粒体电子传递链是一种有效的抗寄生虫策略。我们最近发现1-羟基-2-烷基-4(1)喹诺酮类化合物，例如1-羟基-2-十二烷基-4(1)喹诺酮(HDQ)，是弓形虫氧化磷酸化的特异性抑制剂。这些化合物导致寄生线粒体膜电位的快速去极化，并在纳米摩尔范围内抑制弓形虫和恶性疟原虫的复制。HDQ在结构上与泛醌有相似之处，抑制动力学表明，HDQ至少抑制了两种酶的活性，这两种酶都具有泛醌结合位点(TgNDH2-I和PfDHODH)。弓形虫基因组包含6个泛醌还原基因，它们可能是潜在的HDQ靶标。我们建议用辅助耐药弓形虫突变体来鉴定相关的HDQ靶标(S)，它的抑制导致复制抑制。所有六个泛醌还原基因都将从现有的抗HDQ突变株中扩增和测序，这些突变株是通过化学诱变产生的。突变等位基因将被引入野生型背景中，以测试特定的突变是否足以产生抗药性。对另外两个HDQ衍生物的抗药性突变体将以相同的方式产生和分析，以调查单独的1-羟基-2-烷基-4(1)喹诺酮衍生物针对的是相同还是不同的寄生虫分子。由于我们有初步证据表明HDQ敏感性和嘧啶代谢之间的联系，因此将特别关注二氢罗酸脱氢酶(DHODH)作为假定的靶标。我们将通过表达一种不依赖泛醌的DHODH来将嘧啶的从头合成从呼吸链功能中分离出来，该DHODH使用富马酸而不是泛醌作为电子受体。总之，这些研究将确定寄生电子传递链中的相关药物靶点，并将有助于详细了解1-羟基-2-烷基-4(1)喹诺酮类药物对弓形虫的作用模式。由于恶性疟原虫与弓形虫具有相同的一组泛醌还原酶，这些研究也可能为疟原虫的新药物靶点提供见解。