Small molecule inhibitors of a Candida albicans histone modifying enzyme

描述(申请人提供)：白色念珠菌是一种广泛传播的人类真菌病原体，在全身感染期间会导致高死亡率，对免疫功能低下的艾滋病患者尤其危险。由于白色念珠菌等真菌是真核生物，开发对人类无毒的抗真菌疗法往往具有挑战性。最近，RU109被鉴定为酿酒酵母中催化组蛋白H3赖氨酸56乙酰化的酶。缺乏Rtt109的突变体是可行的，但生长缓慢，对遗传毒物极其敏感。值得注意的是，在真菌物种之外没有发现RTT109基因的紧密同源基因，并且Rtt109蛋白不包含在其他组蛋白乙酰转移酶(HAT)酶家族中发现的特征残基。因此，我们假设我们可以识别出抑制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同系物仅限于真菌，它们是小分子治疗干预的有希望的靶点，对哺乳动物宿主的毒性最小。在这个修订的提案中，我的目的是阐明Rtt109在白念珠菌致病机制中的作用，并发现在体内有效的Rtt109抑制化合物。我已经证实了白色念珠菌Rtt109酶的功能保守，因为它对于H3K56乙酰化和抵抗遗传毒性药物是必不可少的。我将在体外测试白色念珠菌rtt109-/-突变株是否对巨噬细胞表现出更高的敏感性，以及在已建立的小鼠念珠菌病模型中，它们是否具有致病性。其次，我将在体外筛选Rtt109抑制组蛋白乙酰化的小分子文库。为了做到这一点，我们开发了一种高通量的分析方法，它将允许通过纯化的重组Rtt109来定量评估组蛋白乙酰化，并用抗H3K56-乙酰基抗体进行检测。最后，我将开始描述候选化合物的特征，这些化合物对哺乳动物细胞无毒，因为它们对念珠菌组蛋白修饰和小鼠的发病机制有影响。公共卫生相关性：白色念珠菌是一种致病真菌，对包括艾滋病患者在内的免疫功能受损的人特别危险。最近，一种新的酶被发现，它对真菌的正常生长很重要。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，目标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.