Biol518 Lecture 2 HTS and Antibiotic Drug Discovery.

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

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130 9572

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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)

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.

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

Strategies for Essential ID

Saturated transposon mutagenesis Systematic gene knock-out (or

inability to knock-out) Antisense expression controlling

gene expression

Number of essential genes determined for various bacterial species.

Species No. of Essential Genes Methods Used ReferencesBacillus subtilis 271 Gene disruption 43

Mycoplasma genitalium 265-350 Transposon Mutagenesis 39Streptococcus pneumoniae 113 Gene disruption 72

Haemophilus influenzae 478 Transposon Mutagenesis 1Escherichia coli 620 Transposon Mutagenesis 28

E. coli (PEC data base) 250 Various methods 52Staphylococcus aureus 150 Antisense expression 41Staphylococcus aureus 168 Antisense expression 22Typical bacterial species 206 Theoretical analyses 32

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

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

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

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.

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.

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−).

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

Xu et al, 2010

FabF Clone – antibiotic profile

Xu et al, 2010

IleS Clone – antibiotic profile

Xu et al, 2010

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)