AIDS Opportunistic Pathogens: Targeting the Methyl Citrate Cycle

描述（申请人提供）：光念珠菌是HIV血清阳性和HIV血清阴性人群中重要的条件致病菌。在美国，近15年的大多数临床系列研究表明，光滑假丝酵母占粘膜和弥散性念珠菌病的15-20%，光滑假丝酵母对三唑类抗真菌药物具有先天耐药性，需要开发新的化疗策略来治疗光滑假丝酵母弥散性和粘膜感染。甲基柠檬酸循环代表了一个极好的潜在靶点，为治疗干预的光棘草。甲基柠檬酸循环对丙酸和丙酰辅酶a进行解毒，丙酸和丙酰辅酶a是C. glabrata作为蛋白质降解副产物内源性产生的。该循环由Cit3、Pdh1和Icl2酶催化，分三步将丙酸酯降解为丙酮酸酯。我们已经证明，整个甲基柠檬酸循环在巨噬细胞吞噬后被强烈诱导。我们还表明，PDH1或ICL2的破坏使C. glabrata对丙酸盐非常敏感，可能是由于代谢毒素甲基柠檬酸盐的积累。我们建议首先分析通过甲基柠檬酸循环的通量是如何控制的；我们特别想知道，在C. glabrata中，丙酸盐是如何转化为丙酰辅酶a的，这是进入甲基柠檬酸循环的入口点。我们假设这种活性是由于ACS1和/或ACS2基因编码的乙酰和丙炔辅酶a合成酶的双重功能。我们将测试这一假设，并确定Acs活性是否通过途径控制通量并增强丙酸毒性。我们还设计了一个全细胞实验来筛选抑制Pdh1或Icl2的化合物。我们建议筛选NCI DTP化合物收集中针对这两种酶的化合物。我们将从几个方面描述初始撞击。首先，我们将在体外确定化合物是否抑制Pdh1或Icl2的活性；其次，我们将测试阳性化合物对白色念珠菌或烟曲霉的同源酶的活性，以评估对其他重要真菌病原体的活性。这是一个高风险的项目，也是我实验室的一个新的研究领域；鉴定抑制该循环的化合物将在未来允许在感染的弥散性和粘膜模型中表征甲基柠檬酸循环。活性化合物也可能为人类治疗的未来发展提供一个起点。公共卫生相关性：光念珠菌是hiv阳性和hiv阴性个体感染的重要原因。我们正在研究枸橼酸甲基化循环作为新药的潜在靶点。我们建议筛选来自NIH的大量化合物，以确定抑制该途径的化合物；其中一些可能最终导致念珠菌和其他真菌感染的新治疗方法的发展。

DESCRIPTION (provided by applicant): Candida glabrata is an important opportunistic pathogen in HIV seropositive and HIV seronegative populations. In the United States, most clinical series over the last 15 years show that C. glabrata accounts for 15-20% of mucosal and disseminated candidiasis, C. glabrata is innately resistant to triazole antifungals, and there is a need for development of new chemotherapeutic strategies for treatment of C. glabrata disseminated and mucosal infections. The methylcitrate cycle represents an excellent potential target for therapeutic intervention in C. glabrata. The methylcitrate cycle carried out detoxification of propionate and propionyl-CoA, produced endogenously by C. glabrata as a byproduct of the degradation of protein. The cycle degrades propionate to pyruvate in three steps, catalyzed by the Cit3, Pdh1 and Icl2 enzymes. We have shown that the entire methylcitrate cycle is strongly induced following phagocytosis by macrophages. We have also shown that disruption of PDH1 or ICL2 renders C. glabrata exquisitely sensitive to propionate, likely due to the accumulation of the metabolic toxin methylcitrate. We propose first to analyze how flux through the methylcitrate cycle is controlled; specifically we would like to know how, in C. glabrata, propionate is converted to propionyl-CoA, the entry point into the methylcitrate cycle. We hypothesize that this activity is due to dual function acetyl- and propionyl-CoA synthetases encoded by the ACS1 and/or ACS2 genes. We will test this hypothesis and determine if Acs activity controls flux through the pathway and potentiates propionate toxicity. We also have designed a whole cell assay to screen for compounds that inhibit Pdh1 or Icl2. We propose to screen the NCI DTP compound collection for compounds that target either of these enzymes. We will characterize initial hits in several ways. First, we will determine in vitro if the compounds inhibit Pdh1 or Icl2 activity; second we will test positive compounds against the orthologous enzymes of C. albicans or A. fumigatus to assess activity against other important fungal pathogens. This is a high risk project, and a new area of investigation for my lab; identification of compounds that inhibit this cycle will in the future allow characterization of the methylcitrate cycle during infection in both disseminated and mucosal models of infection. Active compounds may also provide a starting point for future development of human therapeutics. PUBLIC HEALTH RELEVANCE: Candida glabrata is an important cause of infection in HIV-positive and HIV-negative individuals. We are studying the methylcitrate cycle in C. glabrata as a potential target for new drugs. We propose screening a large collection of compounds available from the NIH to identify compounds that inhibit this pathway; some of these may ultimately lead to development of new treatments of Candida and other fungal infections.