How Difficult Is It to Discover New Antibacterials?

1. How Difficult Is It to Discover New Novel Antibacterials? How Difficult Is It to Discover New Antibacterials? Lynn L. Silver, Ph.D. LL Silver Consulting, LLC

2. Antibacterials at FDA 2000-2011

3. 2005 2000 1995 1990 1985 1980 1975 1970 1965 1960 1955 1950 1945 1940 1935 1930 Fidaxomicin Discovery Timeline 2010 Retapamulin Last novelagent to reach the clinic was discovered in 1987 Linezolid Daptomycin Synercid Bactroban daptomycin Norfloxacin monobactams Imipenem lipiarmycin oxazolidinones cephamycin carbapenem fosfomycin mupirocin lincomycin fusidic acid metronidazole novobiocin nalidixic acid cycloserine trimethoprim isoniazid rifamycin erythromycin Although development and modification of old classes has proceeded – no newly discovered novel classes have made it to the clinic in 24 years vancomycin cephalosporin streptogramins pleuromutilin bacitracin chlortetracycline chloramphenicol polymyxin streptomycin sulfonamide penicillin

4. 2005 2000 1995 Whole cell phenotypic screens Microbial physiology, biochemistry and genetics used to ID antibiotic targets and essential genes 1990 Genomics IDs novel conserved targets Enzyme and binding assays 1985 1980 1975 1970 1965 1960 1955 Empirical “kill the bug” screens 1950 1945 1940 1935 Discovery Strategies 2010 The Golden Age Screening for and design of novelantibacterials was vigorously pursued by Big Pharma until recently

5. Consider… • If Big Pharma (and biotechs) have been largely unsuccessful in finding novel antibacterials to develop… • Will that be reversed by • Increasing financial incentives? • Revising regulatory policy? • What has prevented novel discovery? • The need to address scientific obstacles

6. Gene-to-Drug Approach Genomics Novel antibacterial targets High Throughput Screening Inhibit the enzyme Small molecule ‘Hits’ Small molecule ‘Hits’ Compounds can’t enter Inhibit bacterial growth Small molecule ‘Leads’ Small molecule ‘Leads’ Inhibit bacterial growth by inhibiting the enzyme Compounds kill by other means ez ab Candidates Candidates ab ez Preclinical testing Same as for other drugs Druglike properties Almost all have high resistance potential Low resistance potential Clinical Trials Drug

7. The Obstacles to Antibacterial Discovery • Improve chemical sources • Remove toxic, detergent, reactive compounds from libraries • Define physicochemical characteristics specifying bacterial entry & efflux • Revive natural product screening • Pursue targets with low resistance potential

8. Gram negative Periplasm Cytoplasm CM OM -lactams Glycopeptides Cycloserine Fosfomycin Rifampin Aminoglycosides Tetracyclines Chloramphenicol Macrolides Lincosamides Oxazolidinones Fusidic Acid Mupirocin Novobiocin Fluoroquinolones Sulfas Trimethoprim Metronidazole Daptomycin Polymyxin The bacterial entry problem CM P. Aeruginosa is more problematic due to strong efflux and reduced permeability P. aeruginosa gram positive Cytoplasm Impermeability and efflux of G- render many agents inactive Almost all “gram positive” drugs are active (biochemically) on the analogous gram negative targets – but the drugs are not antibacterial vs gram negatives

9. Antibacterials Useful in SystemicMonotherapy Targets with low resistance potential • Examine successful antibacterials enzymes All have multiple targets or targets encoded by multiple genes No high-level resistance by single-step mutation ANTIBACTERIAL TARGET -lactamsmultiple penicillin binding proteins [PBPs] synthesis of cell wall peptidoglycan GlycopeptidesD-ala-D-ala of peptidoglycan substrate TetracyclinerRNA of 30s ribosome subunit AminoglycosidesrRNA of 30s ribosome subunit MacrolidesrRNA of 50s ribosome subunit LincosamidesrRNA of 50s ribosome subunit ChloramphenicolrRNA of 50s ribosome subunit OxazolidinonesrRNA of 50s ribosome subunit Fluoroquinolonesbacterial topoisomerases (gyrase and topo IV) MetronidazoleDNA Daptomycinmembranes

10. Single Enzyme Targets of Antibiotics in Clinical Use USE Multi-drugTB therapy Multi-drug TB therapy Multi-drug TB therapy Combo w/ Sulfas Combo w/ Trimethoprim Multi-drug therapy Topical therapy UTI All are subject to single-step high level resistance ANTIBIOTIC TARGET rifampicin RNA polymerase isoniazidInhA streptomycin 30s ribosome/rpsL trimethoprim DHFR (FolA) sulfamethoxazole PABA synthase (FolP) novobiocin DNA gyrase B subunit mupirocin Ile tRNA-synthetase fosfomycin MurA

11. Based on existing antibacterial drugs… • Successful monotherapeutic antibacterials • Not subject to single-site mutation to high level resistancebecause they are multi-targeted • Current drugs inhibiting single enzymes • Generally used in combination because they are subject to single mutation to significant resistance THUS: "Multitargets" are preferable to single enzyme targets for systemic monotherapy BUT: The search for single enzyme inhibitors has been the mainstay of novel discovery for at least 20 years …

12. If single enzyme targets give rise to resistance in the laboratory… • Determine if the in vitro (laboratory) resistance is likely to translate to resistance in the clinic • Standardize the use of models for evolution of resistance under therapeutic conditions • To validate targets, test target/lead pairs in these models • Pursue multitargets

13. A way forward • Targets • For single-enzyme inhibitors: Robust modeling of resistance • Pursue multi-targets • Chemicals • Deduce rules for bacterial entry and efflux, especially in G- • Clean up libraries and incorporate rules for entry • Revive Natural Products • With better chemicals, return to empirical discovery • Collaboration between academe and industry • Computation for multitargeting • Modeling of resistance • Chemistry for cell entry and efflux avoidance

15. gram negative gram positive only other drugs + Antibacterials Are Chemically Unlike other Drugs cLogD7.4 = GREASINESS MW = SIZE Mammalian targets ≠ antibacterial targets Many antibacterials must enter bacterial cells

16. Cytoplasm-targeted antibacterials Gram positive only Cytoplasmic Gram negative cytoplasmic entry by diffusion cLogP = Greasiness Gram negative cytoplasmic carrier-mediated transport MW = SIZE

17. An approach to new multitargets: Sorting targets by their ligands • Compound and fragment profiling binding/docking to bacterial proteins Can be done computationally Candidate multitargets

18. PP-C55 GlcNAc MurNAc What is Antibacterial Multitargeting? ciprofloxacin GyraseTopo IV gentamicin tetracycline chloramphenicol linezolid erythromycin Lipid II vancomycin daptomycin Targeting the products of multiple genes – or the product of their function – such that single mutations cannot lead to high level resistance Two or more essential gene products with similar active sites: DNA Gyrase & Topisomerase IV Products of identical genes : rRNA Essential structures produced by a pathway where structural changes cannot be made by single mutations: Membranes These and other known multiargets have been pursued More may be uncovered by computation based on structural studies of bacterial proteins and the small molecule “ligands” that bind to them