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Biol518 Lecture 2 HTS and Antibiotic Drug Discovery

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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. - PowerPoint PPT Presentation

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Biol518

Lecture 2HTS and Antibiotic Drug

Discovery

Modern Drug DiscoveryP

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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

A CBkDa230

130

95

17

1 21 2

72

56

36

28

1 2

11

kDA230

56

28

130 9572

36

17

11

130

17

11

72

28

36

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 cellAntisense cell

Noprotein

XXAntisense RNAAntisense RNA

Inducible promoterInducible promoter

mRNAmRNA

Normal cellNormal cell

Protein

mRNAmRNA

Plasmid DNA

DNADNA

Shotgun Antisense Expression Determines Essentiality of Genes

Shotgun Antisense Expression Determines Essentiality of Genes

Non essential geneblocked by antisenseNon essential geneblocked by antisense

Essential geneblocked by antisenseEssential geneblocked by antisense

Millions of random DNA fragmentsMillions of random DNA fragments

No cell growthNo cell growthmRNAmRNA

Essential Protein

DNA

Pathogen genome

Ultra-Rapid Functional GenomicsUltra-Rapid Functional Genomics

Identify >100 essential gene drug targets per monthIdentify >100 essential gene drug targets per monthAntisense

(+ 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 SpecificityInhibitor-Target Specificity

FabI Clone

MurAClone

TrpS Clone

(Real et al., submitted)

Target Identification Using Mixed Target Identification Using Mixed Clone AssayClone Assay

A BC

(Real et al., submitted)

Target Identification Using Target Identification Using Individual ArrayIndividual Array

ArgS AsnS AspS CysS Efp FabA

FbaA FabD FabG FabI FabZ FtsE

FtsI FtsX FtsY FtsZ GyrA GlnS

GlyS HisS LolD LolE MrdB MurA

MurG NrdB NadE PheS PheT PlsC

PrfA PrfB Ppa RplE RplJ RpsL

RpoD TrpS SerS Rho MurI MurD

MurF PolA TrmA ThrS TmK ZipA

A B

C D

indolmycin

phosphomycin triclosan

(Real et al., submitted)