NOVEL INHIBITORS OF FUNGAL ASPARTIC PROTEINASES

真菌天冬氨酸蛋白酶的新型抑制剂

• 批准号: 6626411
• 负责人: Ben M. Dunn
• 金额: $ 21.4万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6626411
• 关键词: Candida albicans; aspartic endopeptidases; chemical structure function; enzyme activity; enzyme inhibitors; fluorescence resonance energy transfer; fungal proteins; microorganism culture; mutant; nuclear magnetic resonance spectroscopy

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.

描述：免疫功能低下的患者容易受到感染 通常被正常宿主免疫系统清除的生物体。念珠菌属 白色念珠菌 (CA) 感染会导致多种中度至危及生命或 传播性临床疾病。可以预防这种感染的药剂 帮助减轻接受移植、化疗的患者的痛苦， 和抗击艾滋病。 CA 分泌的蛋白酶有助于渗透 细胞外基质传播感染。这些蛋白酶被认为是 毒力因子，并且是天冬氨酸蛋白酶家族的成员。这是 已知胃酶抑素（天冬氨酸蛋白酶的通用抑制剂）会 抑制CA的生长。我们最近确定了三维 相关真菌酶的结构，酵母蛋白酶 A (YprA) 与 其天然蛋白质抑制剂 IA-3。这个新的综合体揭示了新的特征 我们建议在这个项目中进一步探索抑制作用。 IA-3 是 正如我们通过 NMR 分析所确定的，在溶液中完全展开。经过 了解 YprA 和 IA-3 之间的相互作用，我们计划设计新的 针对与 CA 密切相关的蛋白酶的抑制剂。我们将实现 这一目标通过四个具体目标实现。在目标 1 中，我们将创建以下突变体 IA-3 有助于研究螺旋-螺旋转变和物理 通过荧光能量转移与酶相互作用 连续流动微混合程序。在目标 2 中，突变体将被设计为 确定交互的关键点，这可能会导致简化 抑制剂的结构。基于突变体与 YprA 的相互作用， 我们将在目标 1 和 2 中选择一些进行 NMR 研究，以确定 未结合状态的动态以及结合时结构的变化。目标 3 我们将改变结构以允许结合和抑制 具有相似结构和功能的白色念珠菌蛋白酶。具体来说 目标 4 我们将利用 IA-3/YprA 综合体提供的新信息来 帮助设计小分子抑制剂，通过 新的绑定机制。通过该计划开发的化合物将 在培养物中针对白色念珠菌进行了测试。

项目成果

Laminar-flow fluid mixer for fast fluorescence kinetics studies.

用于快速荧光动力学研究的层流流体混合器。

• 发表时间: 2002-11
• 作者: Pabit, Suzette A;Hagen, Stephen J
• 通讯作者: Hagen, Stephen J

Smaller and faster: the 20-residue Trp-cage protein folds in 4 micros.

更小更快：20 个残基的色氨酸笼蛋白在 4 微米内折叠。

• 发表时间: 2002-11-06
• 影响因子: 15
• 作者: Qiu, Linlin;Pabit, Suzette A;Roitberg, Adrian E;Hagen, Stephen J
• 通讯作者: Hagen, Stephen J

Design of small volume HX and triple-resonance probes for improved limits of detection in protein NMR experiments.

设计小体积 HX 和三重共振探针，以提高蛋白质 NMR 实验中的检测限。

• 发表时间: 2003-09
• 作者: Li, Yu;Logan, Timothy M;Edison, Arthur S;Webb, Andrew
• 通讯作者: Webb, Andrew