Assay Development and Screening Section, James McMahon, Principal Investigator Goals: Assay development efforts focused on the configuration, optimization and validation of high-throughput assays to screen pure compound libraries and natural product extracts for modulators of specific molecular and cellular targets and associated cellular phenotypes. Preliminary characterization of screen hits for better prioritization in downstream follow-up. Accomplishments: Nine assays developed, 11 high-throughput screening (HTS) campaigns completed and 10 assays performed in support of natural products chemistry. Preliminary biological characterization work done on several hits from completed HTS campaigns. One lead compound obtained from HTS is currently in clinical trial while several others are under preclinical development. 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 MDM2, tyrosyl-DNA phosphodiestease 1, the interaction of the polo-box domain of polo-like kinase 1 and its phosphorylated peptide ligand, and for the novel paracaspase mucosa-associated lymphoid tissue transformation protein 1. The PCMBS/OKeefe also biochemically evaluated hit compounds resulting from screens for inhibitors MDM2, the anti-HIV target RNase H and tyrosyl-DNA phosphodiestease 1. 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 obtain screening samples, and established a library of 261,223 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 including AP-1, Pdcd4, and Plk-1. 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 both the englerins and schweinfurthins. 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). New compounds were discovered that synergistically enhance tumor cell killing by the TRAIL death receptor ligand. In addition, selective cytotoxic agents and anti-HIV natural products were identified using phenotypic screens. An extract prefractionation library with >100,000 samples was generated and shown to be a value-added screening resource. Structural and stereochemical assignments of diverse natural product chemotypes were accomplished using a combination of chemical manipulations, NMR analyses, CD studies, and computational methods.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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