Assay Development and Screening Section, Curt Henrich (acting section head) Goals: The efforts of the MTP Assay Development and Screening Section generally focus on three main categories: 1) cell-based high-throughput screening (HTS), 2) assay support for MTP natural products chemistry efforts, and 3) biological characterization and mechanism of action (MOA) studies on hits from HTS. At any given time, 10-15 projects are active in the lab. All of these projects include significant collaborations with other sections of the MTP as well as with a range of NCI and academic scientists. The section develops, optimizes, validates, and applies HTS assays for identification of synthetic compound and natural product modulators of molecular and cellular targets and associated cellular phenotypes. Accomplishments: Collaborations with MTP and other NCI scientists for assay development and screening include assay development and HTS focused on 8 targets. Examples include growth inhibition assays (for example virus-dependent and -independent Merkel cell carcinoma), protein-protein interactions (e.g., binding of Raf to oncogenic Ras), substrate uptake (e.g., feasibility studies on androgen transporter activity assays), reporter gene assasy (e.g., preliminary assessment of dendritic cell activation assays) and biochemical assays (in collaboration with the MTP Protein Chemistry and Molecular Biology Section). The HTS assays are utilized in support of natural product chemistry efforts, i.e., purification and characterization of active compounds from natural product extracts identified from current or previously completed HTS campaigns. These include hundreds of chemistry projects focused on 10 cell-based and biochemical targets. In addition, preliminary pilot-scale evaluation of new partially purified (prefractionated) natural products extracts from the NCI Program for Natural Product Discovery has been initiated. Finally, the section collaborates extensively with NCI scientists to characterize active compounds that arise from HTS and natural products chemistry. These efforts include both standard cell biological and biochemical evaluation as well as application of a yeast chemical genetics platform for identification of potential molecular targets. Of particular note among the half dozen active projects are analysis of modulators of PAX3-FOXO1 in rhabdomysarcoma cells and effects of protein synthesis inhibitors on TRAIL response in renal cell carcinoma cells. Protein Chemistry and Molecular Biology Section, Barry OKeefe, Principal Investigator Goals: Investigation of specific protein-ligand interactions to assess their applicability to potential screening approaches. Development of cell-free high throughput screens robust enough to tolerate crude natural product extracts and sufficiently predictive to guide the identification and purification of bioactive compounds. Post-screen biochemical characterization of active compounds to determine specificity, mechanism of inhibition and specific binding interactions. Isolation and characterization of antiviral proteins from natural product extracts. Accomplishments: Developed biochemical screens for inhibitors of the E3 ubiquitin ligase Cbl-b, the kinase p38, and tyrosyl-DNA phosphodiestease 2. The PCMBS/OKeefe also biochemically evaluated hit compounds resulting from screens for inhibitors of Tdp1 and MALT1. Finally, the PCMBS/OKeefe characterized the activity of several new classes of anti-HIV proteins from natural product extracts. Chemical Diversity Development Section, John Beutler, Principal Investigator Goals: Provide chemical diversity to MTL HTS screening assays by accessing multiple sources of pure compounds and selected natural product extracts. Isolation and characterization of novel anti-cancer natural products from cell-based assays and their preclinical development. Accomplishments: Initiated collaborations with synthetic chemistry groups and natural product groups to obtainscreening samples, and established a library of 245,947 current samples for screening, including extracts, prefractionated samples, pure natural products and pure synthetics. Identified hits from diverse external sources and obtained resupply of hit material and analogues in multiple screening projects p38 and Tdp1. Developed a web based assay and sample management system which allows MTL staff to manage sample libraries, design assay plates and collect and analyze HTS data. Interfaced sample management system directly with sample storage and liquid handling infrastructure. Preclinical development of the englerins. Natural Products Chemistry Section, Kirk Gustafson, Principal Investigator Goals: Bioassay-directed isolation and structural elucidation of bioactive metabolites from extracts of marine invertebrate animals and algae, terrestrial plants, and microbes. Generation of an extract prefractionation library for more effective high-throughput screening outcomes. Adaptation and application of contemporary techniques for assigning the relative and absolute configuration of novel lead compounds. Provision of active natural products to support mechanism of action, target identification, and preclinical development studies. Accomplishments: Isolation and identification of natural products that inhibit a wide variety of molecular targets including transcription factors (AP-1 and HIF-2), tumor suppressors (Pdcd4, NF1), a multidrug resistance transporter (ABCG2), and a DNA repair enzyme (Tdp1). In addition, selective cytotoxic agents and anti-HIV natural products were identified using phenotypic screens.. Structural and stereochemical assignments of diverse natural product chemotypes were accomplished using a combination of chemical manipulations, NMR analyses, CD studies, and computational methods.
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