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Biol518 Lecture 2 HTS and Antibiotic Drug Discovery Follow-up Monitoring Drug Approval Clinical Trials Drug Candidate Selection Lead Optimization HTS Assay Development Target Selection/ Validation Program Selection Modern Drug Discovery HTS Workflow Traditional Approach: cell growth inhibition Discovery of most antibiotics and antifungal drugs was accomplished by looking for cell growth inhibition by natural compounds Once potent compounds are identified, their targets are discovered through extensive biochemical and physiological research This is also a chemical genomics approach Yeast halo assay Reverse Chemical Genomics Now we know many essential genes (whose products are essential), we can simply clone the genes and overexpress and purify proteins Using purified proteins (enzymes), we can search for compounds inhibiting enzyme activity Test compounds on cells to see if cell growth is inhibited Purified Potential Drug Targets 1 A 2 kDa 230 1 2 B 130 95 72 56 36 28 1 2 C 130 95 72 56 130 36 28 36 28 17 72 17 11 17 11 11 FabB (A) kDA 230 Def (B) FabD (C) Traditional Paradigm with a twist Target-specific sensitized cell-based assays (antisense expression) Cell growth inhibition followed by rapid target identifications (e.g., overexpression of essential genes) Antisense RNA Antisense RNA expression. Random cloning and expression of short pieces of genomic DNA on a plasmid in an microorganism to elucidate the function of the genes Conditional Antisense Inhibition of Protein Synthesis Plasmid DNA Protein mRNA Inducible promoter Antisense RNA X mRNA DNA Normal cell No protein DNA Antisense cell Shotgun Antisense Expression Determines Essentiality of Genes Pathogen genome Millions of random DNA fragments Non essential gene blocked by antisense Essential Protein mRNA DNA Essential gene blocked by antisense No cell growth Ultra-Rapid Functional Genomics Antisense (+ inducer) No antisense (- inducer) Identify >100 essential gene drug targets per month Selective Sensitization GyrA Clone – antibiotic profile FabF Clone – antibiotic profile IleS Clone – antibiotic profile Microbiological profiles Molecular Interaction Over-expression of Essential genes Concept: over-expression of a target protein in a cell renders the cell resistant to an inhibitor specifically targeting the protein target Strategy: create a large collection of cell clones each over-expressing one essential protein Expose cell array to inhibitory concentration of a compound -> cell growth conferred by a specific clone Over-expression of Essential genes Triclosan Dose Response (Xu et al., 2006 BBRC) Inhibitor-Target Specificity MurAClone TrpS Clone FabI Clone (Real et al., submitted) Target Identification Using Mixed Clone Assay A C B (Real et al., submitted) Target Identification Using Individual Array (Real et al., submitted) B A ArgS AsnS AspS CysS Efp FabA FbaA FabD FabG FabI FabZ FtsE FtsI FtsX FtsY FtsZ GyrA GlnS GlyS HisS LolD LolE MrdB MurA MurG NrdB NadE PheS PheT PlsC PrfA PrfB Ppa RplE RplJ RpsL RpoD TrpS SerS Rho MurI MurD MurF PolA TrmA ThrS TmK ZipA indolmycin D C phosphomycin triclosan