Small molecule inhibitors of a Candida albicans histone modifying enzyme

描述（由申请人提供）：白色念珠菌是一种广泛存在的人类真菌病原体，在全身感染期间导致高死亡率，对免疫功能低下的艾滋病患者尤其危险。因为真菌如白色念珠菌是真核生物，开发对人类无毒的抗真菌疗法往往具有挑战性。最近，RU109被鉴定为在出芽酵母Saccharomyces cerevisiae中催化组蛋白H3赖氨酸56乙酰化的酶。缺乏Rtt109的突变体是可以存活的，但生长缓慢，对基因毒性药物极其敏感。值得注意的是，在真菌物种之外没有发现RTT109基因的同源物，RTT109蛋白不含有其他组蛋白乙酰转移酶（HAT）酶家族中发现的特征残基。因此，我们假设我们可以确定抑制Rtt109功能的小分子，而不会对其他HAT酶产生实质性影响。此外，由于Rtt109同源物仅限于真菌，它们对哺乳动物宿主的毒性最小，是小分子治疗干预的有希望的靶点。在这次修订的提案中，我的目的是阐明Rtt109在白色念珠菌发病中的作用，并发现体内有效的Rtt109抑制化合物。我已经确认了白色念珠菌Rtt109酶的功能守恒，因为它对H3K56乙酰化和对基因毒性药物的抗性至关重要。我将在体外测试白色念珠菌rtt109-/-突变体对巨噬细胞的敏感性是否增加，以及在已建立的小鼠念珠菌病模型中是否具有致病性。其次，我将筛选一个小分子文库，用于Rtt109体外抑制组蛋白乙酰化。为此，我们开发了一种高通量测定方法，通过纯化的重组Rtt109，用抗h3k56 -乙酰基抗体检测，可以定量评估组蛋白乙酰化。最后，我将开始表征对哺乳动物细胞无毒的候选化合物，因为它们对念珠菌组蛋白修饰的影响，以及对小鼠发病机制的影响。公共卫生相关性：白色念珠菌是一种致病性真菌，对免疫功能低下的个体，包括艾滋病患者尤其危险。最近，人们发现了一种对真菌正常生长很重要的新酶。我建议研究这种酶如何促进白色念珠菌的生长和毒力。我还将确定抑制这种酶的化合物，目的是开发新的治疗方法来对抗真菌感染。

DESCRIPTION (provided by applicant): Candida albicans is a widespread human fungal pathogen that causes high rates of mortality during systemic infections, and is particularly dangerous for immunocompromised AIDS patients. Because fungi such as C. albicans are eukaryotes, development of antifungal therapeutics that is non-toxic to humans is often challenging. Recently, RU109 was identified as the enzyme that catalyzes acetylation of histone H3 lysine 56 in the budding yeast, Saccharomyces cerevisiae. Mutants lacking Rtt109 are viable, but is slow growing and extremely sensitive to genotoxic agents. Notably, no close homologs of RTT109 genes are found outside of fungal species, and Rtt109 proteins do not contain signature residues found in the other families of histone acetyltransferase (HAT) enzymes. Therefore, we hypothesize that we can identify small molecules that inhibit Rtt109 function without substantial effect on other HAT enzymes. Furthermore, as Rtt109 homologs are restricted to fungi, they represent promising targets for small molecule therapeutic intervention with minimal toxicity for mammalian hosts. In this revised proposal, I aim to elucidate the role of Rtt109 in pathogenesis by C. albicans and to discover Rtt109 inhibitory compounds that are efficient in vivo. I have confirmed the functional conservation of the C. albicans Rtt109 enzyme, because it is essential for H3K56 acetylation and for resistance to genotoxic agents. I will test whether C. albicans rtt109-/- mutants display increased sensitivity to macrophages in vitro and whether they are pathogenic in the established murine candidiasis model. Second, I will screen a library of small molecules for inhibition of histone acetylation by Rtt109 in vitro. To do this, we have developed a high-throughput assay which will allow quantitative assessment of histone acetylation by purified, recombinant Rtt109, detected with an anti-H3K56-acetyl antibody. Finally, I will begin to characterize candidate compounds that are non-toxic to mammalian cells for their effects on histone modification in Candida, and on pathogenesis in mice. PUBLIC HEALTH RELEVANCE: Candida albicans is a pathogenic fungus that is particularly dangerous to immunocompromised individuals, including AIDS patients. Recently, a new enzyme was discovered that is important for normal growth of fungi. I propose to study how this enzyme contributes to growth and virulence of Candida albicans. I will also identify compounds that inhibit this enzyme, with the goal of developing new therapeutic approaches to combat fungal infections.