Assay Development for Identification of HIV-1 Protease Autoprocessing Specific Inhibitors

HIV-1 蛋白酶自动加工特异性抑制剂鉴定的检测方法开发

• 批准号: 9332334
• 负责人: CHAOPING CHEN
• 金额: $ 37.86万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9332334
• 关键词: Adverse effects; Affinity; Allosteric Site; Alpha Cell; Anti-HIV Agents; Antiviral Agents; Binding; Biochemical; Biological Assay; Catalytic Domain; Cells; Chemicals; Collection; Complement; Development; Diversity Library; Drug Design; Drug resistance; Enzyme-Linked Immunosorbent Assay; Enzymes; FDA approved; Genetic; Goals; HIV; HIV Protease; HIV-1; HIV-1 protease; Lead; Life; Light; Longevity; Mammalian Cell; Maps; Mediating; Modeling; Molecular Bank; Patients; Peptide Hydrolases; Performance; Pharmaceutical Preparations; Phenotype; Process; Production; Protease Inhibitor; RNA-Directed DNA Polymerase; Reaction; Resistance; Sagittaria; Structure; Technology; Therapeutic; Time; Treatment outcome; Update; Viral; Virion; analog; assay development; base; drug development; experimental study; follow-up; high throughput screening; improved; inhibitor/antagonist; novel; novel therapeutic intervention; novel therapeutics; pol Gene Products; resistant strain; response; screening; small molecule; therapeutic target; therapy outcome

Assay Development for Identification of HIV-1 Protease Autoprocessing Specific Inhibitors Project Summary This proposal is in response to PAR-13-364: Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic Discovery. The proposed study aims to establish a multiplex primary assay and develop and evaluate follow-up assays that will enable identification of small molecules selectively targeting HIV- 1 protease precursor autoprocessing, an essential viral-specific process that has not been previously exploited for anti-HIV drug development. In the infected cell, HIV protease is initially synthesized as part of the Gag-Pol polyprotein precursor. During the late stage of virion production, the precursor self-catalyzes the cleavage reactions that lead to liberation of the free mature PR. The currently available FDA-approved protease inhibitors (PIs) primarily target the catalytic site of the mature PR. These PIs are significantly less effective at suppressing precursor autoprocessing, suggesting that these two forms of HIV-1 protease are enzymatically different. This proposed study will develop and establish a platform to enable HTS campaigns to search for novel autoprocessing inhibitors with action modes different from the current PIs. This next generation of therapeutic probes, when used in combination with the current PIs, will implement a new therapeutic approach: targeting a vital enzyme (HIV-1 protease) at two distinct functional states (procure and mature PR) and at different regions (non-catalytic site and catalytic site) at the same time. Such a strategy is expected to drastically increase difficulty (genetic barrier) for HIV to evolve viable strains simultaneously resistant to inhibitors from both classes. We have recently established a cell-based assay for the study of HIV-1 protease autoprocessing mechanism. This assay has for the first time made it possible to screen for autoprocessing inhibitors by AlphaLISA (amplified luminescent proximity homogeneous assay ELISA) technology. Our pilot screens of 23,000 small molecule compounds displayed a Z' factor >0.5 and an S/N ratio >15, and identified 35 highly active hits displaying inhibition activities approaching the positive controls (Figure 7). Built upon this platform, we here propose to further develop the primary assay into a multiplex format to enable normalized quantification of autoprocessing efficiency to the numbers of transfected cells (Aim1a). This updated multiplex primary assay will be used to screen a collection of 20,000 life diversity compounds with a new fusion precursor that mimics an autoprocessing phenotype observed with a proviral model (Aim 1b). The highly active hits found from Aim 1b and our previous pilot screen will be verified with fresh stock compounds in serial dilutions (Aim 1c) and the confirmed high priority hits will be subjected to a battery of secondary and tertiary assays to be established to examine their possible action mechanisms (Aims 2a-d). In particular, we hope to identify compounds that inhibit precursor autoprocessing by interacting with a region(s) beyond the known catalytic site of the mature PR (i.e., allosteric inhibitors). The novel autoprocessing inhibitors will be further evaluated for their inhibitory effects on replication of HIV-1 strains (Aim 2f). Successful execution of these proposed experiments will establish a framework for identification of novel autoprocessing specific inhibitors through large scale screens. Further characterization of the identified compounds will aid in the development of a new class of therapeutic drugs to complement the current PIs. Biochemical and structural examination of these new drugs may also shed light on the mechanism of precursor autoprocessing.

鉴定HIV-1蛋白酶自动加工特异性抑制剂的试验发展