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. Definitions: Essential genes – genes absolutely required for growth and survival. Essential proteins – proteins encoded by essential genes that are required for growth and survival. Non essential genes – genes whose destruction does not lead to significant growth defects in a cell.

10. Why study essential genes? Essential genes are important for cellular function and physiology; to study them will reveal details about microbial physiology Practical application: essential genes encode essential proteins which are excellent drug targets to develop new antibiotics

11. Strategies for Essential ID Saturated transposon mutagenesis Systematic gene knock-out (or inability to knock-out) Antisense expression controlling gene expression

12. Number of essential genes determined for various bacterial species.

13. Transposition Transposons – DNA elements that can hop (transpose) from one place in DNA to another Transposons are known to exist in all organisms on earth Movement by a transposon is called transposition, catalyzed by enzymes called transposases Transposons usually encode their own transposases

14. Transposition Many transposons are essentially cut out of one DNA and inserted into another Other transposons are copied and then inserted elsewhere Donor DNA and target DNA

16. Structure of Bacterial Transposons All contain repeats at their ends, usually inverted repeats (IR) Presence of short direct repeats in the target DNA that bracket the transposon The sites of insertion are different among target DNAs

18. Gene Knock-out Gene replacement (knock-out). The purpose is to remove (knock-out) most of one gene and see what happens to the phenotype of the organism. Suicide vector is used.

20. Gene Knock-In Gene replacement (knock-In): The purpose is to disrupt the structure of a gene by inserting a resistance marker gene and see what happens to the phenotype of the organism. Suicide vector is used. Also known as plasmid insertion mutagenesis.

21. Gene Knock-In A suicide plasmid containing an antibiotic resistance gene (AbR) is constructed to contain a small region of sequence homology (denoted by the solid box) to orfX (denoted by the wide arrow). When the plasmid is introduced into wild-type cells (W+), a single cross-over recombination event between these two regions of homology leads to insertion of plasmid sequences and disruption of the orfX reading frame. The resulting mutant is antibiotic resistant and defective for orfX (orfX−).

24. 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

26. Conditional Antisense Inhibition of Protein Synthesis Antisense cell No protein X X Antisense RNA Inducible promoter mRNA Normal cell Protein mRNA Plasmid DNA DNA

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

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

30. Selective Sensitization

31. GyrA Clone – antibiotic profile Xu et al, 2010

32. FabF Clone – antibiotic profile Xu et al, 2010

33. IleS Clone – antibiotic profile Xu et al, 2010

35. 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

36. Over-expression of Essential genes

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

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

39. Target Identification Using Mixed Clone Assay A BC (Real et al., submitted)

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