Probing Novel Cancer Biology with High Throughput Screening
Probing Novel Cancer Biology with High Throughput Screening of Natural Products Jim Mc. Mahon Molecular Targets Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health
Mission and Goals • Collaborate with NCI investigators to develop and apply molecular target -specific assays. • Screen the NCI natural products repository and other sources of chemical diversity. • Identify and characterize target-specific compounds. • Provide active compounds to collaborating labs for further development. Target discovery/validation Assay development High-throughput screen Lead selection Lead optimization/candidate selection Preclinical testing 0 1 MTL 2 3 4 Years 5 6 Clinical trials 7 8 NATURE 2005
The MTL Plays a Unique Role in the NCI Basic Science Discoveries 14 patents issued 12 patents pending 13 current licensees 80 MTAs 100 papers published
NCI Natural Products Repository The NCI has one of the world’s largest, most diverse collections of natural product extracts (~200, 000 extracts).
NCI Screening Platforms Equipment • plate readers • liquid handlers • plate washers Primary screening • fluorescence – FI, FP, FRET, TRF • luminescence – LI, Dual Lumi, BRET • absorbance Secondary and follow-up screening • high content imaging • flow cytometry • quantitative real-time RT-PCR • in-cell western • biochemical assays
Application of Natural Products Extracts to HTS Challenges • interfering materials • • non-specific interactions with cells or proteins toxic compounds unknown/variable/low concentrations of components sample handling (e. g. viscosity, buffer compatibility, precipitation) Risks • plate failures • false positives/false negatives • poor reproducibility
Adaptation for HTS of Natural Products Extracts • Liquid handler settings • Assay optimization • Reagent selection • Washing plates • Defining optimal concentrations • Focus on classes of extracts • Parallel cytotoxicity assays • Comparisons to other assay results • Prefractionation of extracts
Recent MTL Screens with Natural Product Extracts • • • Mdm 2 E 3 ubiquitin ligase - electrochemiluminescence RNAse H – fluorescence polarization Estrogen Receptor – GFP translocation ABCG 2 transporter – cellular uptake of fluorophore Plk 1 – protein-protein interaction (ELISA-based) MALT 1 – fluorescence (AMC – homogenous format) Tdp 1 – fluorescence (eosin – surface bound) SUMOylation cascade – capillary separation Mutant p 38 kinase – ELISA based visualization AP 1 transcription factor– b-lactamase reporter EWS/Fli 1 response – dual luciferase reporter
Purification and Characterization of Active Components • Active components from active natural product extracts are purified by bioassay-guided fractionation. Separation schemes isolated both active compounds and analogs. • Chemical characterization employed extensive NMR analyses, mass spectrometry, and chemical derivatization. crude extracts resupply fractionation characterization
MTL Discoveries in Advanced Preclinical/Clinical Development Mithramycin Schweinfurthin analogue Englerin A Antiviral proteins
Summary • Natural products continue to be an important source of chemotypes resulting in approved drugs. • Screening crude natural product extracts in modern HTS screens requires assay modifications. • High hit rates with natural products extracts highlight the need for multiple secondary assays to prioritize active extracts. • The Molecular Targets Laboratory is uniquely positioned to engage in drug discovery from natural sources.
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