PROJECT SUMMARY – MOLECULAR SCREENING AND PROTEIN EXPRESSION The Molecular Screening and Protein Expression Shared Resource (MSPESR) provides Cancer Center (CC) members with state-of-the-art high-throughput screening capabilities of shRNA and small molecule libraries to identify genes and tool inhibitors of candidate therapeutic targets. Identifying drug-like, small molecules that regulate the activity of therapeutic targets holds promise in defining new treatment paradigms, especially for recalcitrant tumor types, where current clinical practice is suboptimal. In addition, expert technical assistance is provided in recombinant DNA plasmid engineering, protein expression in bacteria and baculovirus-infected insect cells, purification of recombinant proteins, and production of high-titer retroviruses (e.g. lentiviruses) for delivery of shRNA and cDNAs to mammalian cells. CC investigators require high quality recombinant proteins for characterization of enzymatic activities, crystallization for structural analysis, characterization of structure- function relationships of protein-protein, protein-nucleic acid, and protein-small molecule interactions; and immunization of rabbits/mice to generate custom antibodies. Through an inter-institutional agreement with the Helen F. Graham Cancer Center (HFGCC), the MSPESR offers CC members access to CRISPR/Cas9 gene editing services. The MSPESR fosters collaboration by providing expertise in biochemical and cell-based assay development. Such assays enable researchers to identify small molecule compounds which can then be used as tools to further study the target protein functions and signaling pathways in cells, or alternatively they may represent hit or lead compounds and form the basis for 'hit-to-lead" optimization to identify a lead series for drug discovery initiatives. Currently, the MSPESR offers: 1) biochemical-, cell-, and high-content based assays amenable to high-throughput screening in 384-well microtiter plates; 2) libraries of small molecules and shRNA; 3) high-throughput screening of small molecule libraries; 4) analysis of biological and chemistry datasets; 5) characterization of potency and selectivity of newly identified compounds in secondary, orthogonal assays. These services are provided through a centralized laboratory equipped with robotics, libraries of drug- like molecules arrayed in high-density microplate formats, and computational infrastructure for efficient analysis, interpretation, and management of biological and chemistry datasets. The MSPESR is operated by an experienced Managing Director and dedicated laboratory staff, cross-trained in all services offered. This allows for timely project management, quality assurance, and dissemination/integration of data critical for translation of basic biological observations into potential therapeutic strategies. Through its activity over the last budget cycle, the MSPESR has enabled dissection of complex signaling pathways of tumor onset and progression, validation of anticancer agent(s), and proof of concept results that were ultimately incorporated into early phase clinical trials. As an engine for multidisciplinary research collaboration, the MSPESR has contributed to critical publications and grant funding across all three CC Programs.