DESIGN, SYNTHESIS AND CHARACTERIZATION OF FLUORINATED HIV PROTEASE INHIBITOR

氟化 HIV 蛋白酶抑制剂的设计、合成和表征

• 批准号: 6162253
• 负责人: R E LONDON
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162253
• 关键词: AIDS; HIV envelope protein gp120; HIV infections; active sites; antiAIDS agent; antiviral agents; aspirin; drug design /synthesis /production; drug screening /evaluation; enzyme activity; intermolecular interaction; nuclear magnetic resonance spectroscopy; pepsin; pharmacokinetics; phenylalanine analog; protease inhibitor; purine nucleoside phosphorylase; virus protein

Summary of Work: During the past year, studies on HIV protease were aimed primarily at determining the conformational selectivity of proteolytic cleavage, and its consequences. In particular, three of the eight cleavage sites on the Gag-Pol polyprotein which are the target sequences for HIV protease involve Araa-Pro bonds, where Araa corresponds to the aromatic amino acids tyrosine or phenylalanine. Since imide bonds formed with proline exhibit significant cis/trans isomerism, this leads to a question concerning the specificity of cleavage of the Araa-Pro bonds. This specificity can be determined under conditions in which the rate of cleavage greatly exceeds the isomerization rate - i.e., high enzyme concentrations and low temperature. In order to study the reaction, we have utilized two approaches: 1. A fluorogenic substrate peptide analog of the p17/p24 cleavage site of the gag polyprotein with the sequence: Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- Arg was used for HIV protease assays. Hydrolysis of the Tyr-Pro bond results in increased separation of the DABCYL fluorophore form the EDANS quencher, leading to an increase in fluorescence. 2. A fluorinated substrate: Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln, where FPhe = L-4- fluorophenylalanine, was used for NMR studies. The fluorine nucleus acts as a useful reporter group for both cis/trans isomerization and for cleavage, and in fact provides distinct signals depending on where the peptide is cleaved. As of this date, the 19F NMR studies were limited to the model aspartyl protease, pepsin, since HIV protease proved too unstable for use in the NMR experiments. Specificity for cleavage of the trans imide bond conformation was demonstrated in all cases. The conformational selectivity of proteolytic cleavage of Araa-Pro imide bonds by HIV protease provides a possible explanation for the accumulation of the cellular protein cyclophilin by HIV. In particular, the cyclophilin could act as an auxiliary enzyme for the protease, converting inactive, cis imide bonds into trans bonds which are substrates for the protease.

工作摘要：在过去的一年里，关于艾滋病毒蛋白酶的研究 主要目的是确定构象的选择性 蛋白水解性切割及其后果。特别是，其中三个 Gag-Pol多聚蛋白上的八个切割位点是靶点 HIV蛋白酶的序列涉及araA-Pro键，其中araA对应 到芳香族氨基酸酪氨酸或苯丙氨酸。由于酰亚胺键 与脯氨酸形成的化合物表现出显著的顺式/反式异构性，这导致 关于araA-Pro裂解特异性的一个问题 债券。这种专一性可以在下列条件下确定 裂解速率大大超过异构化速率--即高 酶浓度和低温。为了更好地研究 反应中，我们采用了两种方法：1.荧光底物 GAG多聚蛋白p17/p24裂解位点的多肽类似物 顺序：Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)- 精氨酸用于HIV蛋白水解酶的检测。酪氨酸-Pro键的水解性 结果增加了DABCyl荧光团与EDAN的分离 猝灭剂，导致荧光增强。2.氟化甲素 底物：SerGln-Asn-fPhe-Pro-Ile-Val-Gln，其中fPhe=L-4- 氟苯丙氨酸用于核磁共振研究。氟核作用于 作为顺式/反式异构化的有用报告基团 切割，实际上提供了不同的信号，这取决于 多肽被裂解。截至目前，~(19)F-核磁共振研究仅限于 模型天冬氨酸蛋白酶，胃酶，因为HIV蛋白酶也被证明 在核磁共振实验中使用时不稳定。裂解的特异性 在所有情况下都证明了反式亚胺键的构象。这个 AraA-Pro酰亚胺蛋白水解物的构象选择性 HIV蛋白酶的结合提供了一种可能的解释 HIV对细胞内亲环素的积累。特别是， 亲环素可以作为蛋白酶的辅助酶， 将不活跃的顺式亚胺键转化为反式键 该酶的底物。