Biol518 Lecture 2 HTS and Antibiotic Drug Discovery

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

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 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

Purified Potential Drug Targets FabB (A) 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., over- expression 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 Antisense cell No protein X X Antisense RNA Inducible promoter mRNA Normal cell Protein mRNA Plasmid DNA DNA

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

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

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 FabI Clone MurAClone TrpS Clone (Real et al., submitted)

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

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