CHARACTERIZATION COVALENT INHIBITORS OF SERINE, CYSTEINE & ASPARTYL PROTEINASE

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们使用的质谱核心设施将支持我们的努力，蛋白酶抑制剂的发现，表征和设计。我们的实验室研究了三种主要的蛋白酶作为潜在的治疗靶点：丝氨酸（膜型丝氨酸蛋白酶1，卡波西肉瘤疱疹病毒蛋白酶和颗粒酶），半胱氨酸（cruzain，SARS主要蛋白酶，falcipain），和乙酰基蛋白酶（eqolysin和HIV蛋白酶）。我们的工作涉及开发强大的表达系统，酶和生化表征，确定肽底物特异性，合成有效的荧光底物，开发高通量筛选试验，筛选候选抑制剂库，合成新型抑制剂，并表征这些抑制剂的作用机制动力学和结构。 使用质谱核心设施是我们努力的一部分。质谱法提供了一种正交和确定的方法，用于确认给定的抑制剂以预期的方式与研究中的蛋白水解酶形成共价加合物。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our use of the mass spectrometry core facility will be to support our efforts in proteinase inhibitor discovery, characterization, and design. Our lab studies three major classes of proteinases as potential therapeutic targets: serine (membrane type serine protienase 1, kaposi's sarcoma herpes virus proteinase, and the granzymes), cysteine (cruzain, SARS major proteinase, falcipain), and aspartyl proteases (eqolysin, and HIV proteinase). Our work involves developing robust expression systems, enzymatic and biochemical characterization, determining peptide substrate specificity, synthesizing effective fluorogenic substrates, developing high-throughput screening assays, screening candidate inhibitor libraries, synthesizing novel inhibitors, and characterizing the mechanism of action of these inhibitors kinetically and structurally. The use of the mass spectrometry core facility is integral to our efforts. Mass spectrometry provides an orthogonal and definitive way of confirming that a given inhibitor forms a covalent adduct in the expected manner with the proteolytic enzyme under investigation.