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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 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四类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），乙酰基（HIV蛋白酶和组织蛋白酶E）和谷氨酸（eqolisin）。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。我们也有一个非常积极的计划，开发针对几种相同蛋白酶靶标的高选择性大分子抑制剂。质谱核心设施的使用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。这对于确定在异源表达系统中产生的重组蛋白酶和抑制剂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.