Biol518

1. Biol518 Lecture 2 HTS and Antibiotic Drug Discovery

2. Modern Drug Discovery Program Selection Target Selection/ Validation Assay Development HTS Lead Optimization Drug Candidate Selection Clinical Trials Drug Approval Follow-up Monitoring

3. HTS Workflow

4. 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

5. Yeast halo assay

6. Reverse Chemical Genomics • Now we know many essential genes (whose products are essential), we can simply clone the genes and over-express 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

7. Purified Potential Drug Targets FabB (A) Def (B) FabD (C)

8. Traditional Paradigm with a twist • Target-specific sensitized cell-based assays (antisense expression) • Cell growth inhibition followed by rapid target identifications (e.g., over-expression of essential genes)

9. 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

11. Plasmid DNA Protein Antisense RNA mRNA mRNA DNA DNA Antisense cell Normal cell X Conditional Antisense Inhibitionof Protein Synthesis Inducible promoter No protein

12. Millions of random DNA fragments Essential gene blocked by antisense Non essential gene blocked by antisense No cell growth Essential Protein mRNA DNA Shotgun Antisense Expression Determines Essentiality of Genes Pathogen genome

13. Ultra-Rapid Functional Genomics Identify >100 essential gene drug targets per month Antisense (+ inducer) No antisense (- inducer)

15. Selective Sensitization

16. GyrA Clone – antibiotic profile

17. FabF Clone – antibiotic profile

18. IleS Clone – antibiotic profile

21. Microbiological profiles

22. Molecular Interaction

23. 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

24. Over-expression of Essential genes

25. Triclosan Dose Response (Xu et al., 2006 BBRC)

26. Inhibitor-Target Specificity MurAClone TrpS Clone FabI Clone (Real et al., submitted)

27. Target Identification Using Mixed Clone Assay A C B (Real et al., submitted)

28. Target Identification Using Individual Array (Real et al., submitted) indolmycin phosphomycin triclosan