How Difficult Is It to Discover New Novel

Discovery Strategies The Golden Age 2010 Screening for and design of novelantibacterials was vigorously pursued by Big Pharma until recently<br>

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<br>

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

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 The Obstacles to Antibacterial Discovery<br>

-lactams
Glycopeptides
Cycloserine
Fosfomycin

Rifampin

Aminoglycosides
Tetracyclines
Chloramphenicol
Macrolides
Lincosamides
Oxazolidinones
Fusidic Acid
Mupirocin

Novobiocin
Fluoroquinolones
Sulfas
Trimethoprim
Metronidazole

Daptomycin
Polymyxin gram positive CM Cytoplasm P. aeruginosa Almost all “gram positive”
drugs are active (biochemically)
on the analogous gram negative targets –
but the drugs are not antibacterial vs gram negatives Impermeability
and efflux of G-
render many
agents inactive P. Aeruginosa is more problematic due to strong efflux and reduced permeability The bacterial entry problem<br>

Antibacterials Useful in Systemic Monotherapy ANTIBACTERIAL TARGET
-lactams multiple penicillin binding proteins [PBPs]  synthesis of cell wall peptidoglycan
Glycopeptides D-ala-D-ala of peptidoglycan substrate
Tetracycline rRNA of 30s ribosome subunit
Aminoglycosides rRNA of 30s ribosome subunit
Macrolides rRNA of 50s ribosome subunit
Lincosamides rRNA of 50s ribosome subunit
Chloramphenicol rRNA of 50s ribosome subunit
Oxazolidinones rRNA of 50s ribosome subunit
Fluoroquinolones bacterial topoisomerases (gyrase and topo IV)
Metronidazole DNA
Daptomycin membranes No high-level resistance by single-step mutation All have multiple targets or targets encoded by multiple genes Targets with low resistance potential Examine successful antibacterials<br>

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

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 …<br>

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<br>

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<br>

Antibacterials Are Chemically Unlike other Drugs Mammalian targets ≠ antibacterial targets
Many antibacterials must enter bacterial cells MW = SIZE cLogD7.4 = GREASINESS<br>

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

Compound and fragment profiling




binding/docking to bacterial proteins An approach to new multitargets:
Sorting targets by their ligands Candidate multitargets Can be done computationally<br>

What is Antibacterial Multitargeting? Lipid II ciprofloxacin daptomycin vancomycin gentamicin
tetracycline
chloramphenicol
linezolid
erythromycin 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<br>