NOVEL INHIBITORS OF FUNGAL ASPARTIC PROTEINASES

描述：免疫功能低下的患者容易感染 通常由正常的宿主免疫系统清除的生物体。念珠菌 白色念珠菌(CA)感染导致几个中度到危及生命的或 播散性临床疾病。可以预防这种感染的药物将 帮助减轻接受移植、化疗、 和抗击艾滋病。CA分泌的蛋白酶有助于穿透 细胞外基质传播感染。这些蛋白水解酶被认为是 毒力因子，是天冬氨酸蛋白酶家族的成员。它是 已知胃抑素，一种天冬氨酸蛋白酶的通用抑制剂，将 抑制CA的生长。我们最近已经确定了三维 一种与真菌相关的酵母酶A(YprA)的结构 它的天然蛋白质抑制剂IA-3。这个新的综合体展示了 我们建议在这个项目中进一步探索的抑制。IA-3 IS 完全在溶液中展开，正如我们通过核磁共振分析所确定的那样。通过 了解YprA和IA-3之间的相互作用，我们计划设计新的 针对CA密切相关的蛋白水解酶的抑制剂。我们将实现 这一目标通过四个具体目标实现。在目标1中，我们将创建突变体 IA-3帮助研究螺旋-线圈转变和物理 与酶的相互作用通过荧光能量转移和 连续流微混合程序。在目标2中，突变体将被设计成 确定相互作用的关键点，这可能会导致简化 抑制物结构。基于突变体与YprA的相互作用， 我们将在AIMS 1和AIMS 2中选择一些用于核磁共振研究，以确定 未结合状态的动力学和结合时结构的变化。在AIM 3中 我们将在结构上进行改变，以允许结合和抑制 具有相似结构和功能的白色念珠菌蛋白酶。具体而言 目标4我们将利用IA-3/YprA综合体提供的新信息来 协助设计小分子抑制剂，通过 新的约束机制。通过这一计划开发的化合物将是 在培养中对白色念珠菌进行了检测。

DESCRIPTION: Immunocompromised patients are susceptible to infection by organisms that are typically cleared by the normal host immune system. Candida albicans (CA) infection leads to several moderate to life threatening or disseminated clinical diseases. Agents that could prevent this infection would help relieve suffering in patients undergoing transplantation, chemotherapy, and fighting AIDS. Proteases secreted by CA aid in penetration through the extracellular matrix to spread infection. These proteases are believed to be virulence factors, and are members of the aspartic proteinase family. It is known that pepstatin, a general inhibitor of aspartic proteinases, will suppress growth of CA. We have recently determined the three-dimensional structure of a related fungal enzyme, yeast proteinase A (YprA) in complex with its natural protein inhibitor, IA-3. This new complex reveals novel features of the inhibition that we proposed to explore further in this project. IA-3 is completely unfolded in solution, as we have established by NMR analysis. By understanding the interactions between YprA and IA-3, we plan to design new inhibitors targeted to the closely related protease from CA. We will achieve this objective through four specific aims. In Aim 1, we will create mutants of the IA-3 to aid in studies of the helix-coil transition and the physical interaction with enzyme through fluorescence energy transfer and continuous-flow micro-mixing procedures. In Aim 2, mutants will be designed to determine critical points of interaction, which could result in simplification of the inhibitor structure. Based on the interactions of the mutants with YprA, we will select some for studies by NMR in both Aims 1 and 2 to determine the dynamics of the unbound state and changes in structure upon binding. In Aim 3 we will make changes in the structure to permit binding to and inhibition of the Candida albicans protease of similar structure and function. In Specific Aim 4 we will exploit the new information provided by the IA-3/YprA complex to aid in design of small molecule inhibitors that achieve specificity through a new mechanism of binding. Compounds developed through this program will be tested against Candida albicans in culture.