Our proposal responds to the NIH program announcement """"""""Development of assays for high throughput drug screening,"""""""" which has the goal of offering public sector researchers opportunities to employ high throughput chemical screening (HTS) methods. The announcement asks applicants to propose development plans sufficient to show assay reproducibility; to test the assay with a small compound library; and to provide an outline for evaluating the significance of HTS hits. We describe a unique assay designed to identify compounds that inhibit HIV assembly. Our cell-based assay takes advantage of the fact that chimeric proteins can serve as sensitive, efficient, and reproducible reporters for virus assembly with signal-to-noise ratios that should be suitable for HTS. Our investigations will lead to the identification of compounds that inhibit transport, assembly and release of virions from virus-expressing cells. Such inhibitors will provide new HIV antivirals, will help unravel the complicated choreography of HIV assembly, and may reveal ways to block the replication of other pathogenic viruses. Thus, the results of our studies will have general applicability in understanding the HIV life cycle and in stopping virus infections. In keeping with program announcement guidelines, to achieve these goals, our specific aims are as follows: 1. Optimization of assembly assays for high throughput screening: Assembly assays will be modified to reduce assay-to-assay variability, improve signal-to-noise ratios, increase screening coefficient ratios, and adapt protocols for HTS formats. 2. Assay characterization using small chemical libraries: Optimized screening protocols will be employed to test small chemical libraries of diverse compound sets. Test screen data will be evaluated to assess statistical screening parameters, and results will be used to further refine methodologies for HTS. 3. Development of secondary screening procedures: Procedures for analysis and prioritization of primary hits will be developed and streamlined. Methods to rule out artifacts, and to discriminate specific assembly steps impacted by candidate inhibitors will be designed and tested. Through these efforts, the prospect of finding novel HIV inhibitors, and elucidating new assembly steps will be realized. ? Public Health Relevance: Our investigations are directly relevant to public health. We propose to develop a high throughput screen that will identify inhibitors of HIV assembly. Such inhibitors will serve as leads in the design of novel drugs for the treatment of AIDS. ? ? ? ?
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