CHARACTER COVALENT INHIBITORS OF SERINE, CYSTEINE & ASPARTYL PROTEINASE: HIV

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们对质谱学核心设备的使用将支持我们在发现、表征和设计酶抑制剂方面的努力。我们实验室研究了四类作为潜在治疗靶点的蛋白酶：丝氨酸(膜型丝氨酸蛋白酶1，卡波西肉瘤疱疹病毒蛋白水解酶和颗粒酶)、半胱氨酸(克鲁赞，SARS主要蛋白酶)、天冬氨酸(HIV蛋白酶和组织蛋白酶E)和谷氨酸(Eqolisin)。我们的工作包括建立强大的表达系统、酶和生化特性、确定多肽底物的专一性、合成有效的荧光底物、发展高通量筛选方法、筛选候选抑制物库、合成新的抑制物，并从动力学和结构上表征这些抑制物的作用机理。我们也有一个非常积极的计划，为几个相同的蛋白酶靶标开发高度选择性的大分子抑制剂。质谱学核心设施的使用是我们努力的组成部分。质谱学提供了一种正交的和确定的方法来确认给定的抑制剂以预期的方式与被研究的蛋白水解酶形成共价加合物。它对于确定异源表达系统中产生的重组蛋白酶和抑制物的真实性也是必不可少的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Our use of the mass spectrometry core facility will be to support our efforts in proteinase inhibitor discovery, characterization, and design. Our lab studies four classes of proteinases as potential therapeutic targets: serine (membrane type serine proteinase 1, kaposi's sarcoma herpes virus proteinase, and the granzymes), cysteine (cruzain, SARS major proteinase), aspartyl (HIV proteinase and Cathepsin E) and glutamic (eqolisin). 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. We also have a very active program on developing highly selective macromolecular inhibitors for several of the same protease targets.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. It is also essential for determining the authenticity of the recombinant proteases and inhibitors that are produced in heterologous expression systems.