Trends Biotech 19: 288,2001

GENOMICS

FUNCTIONAL GENOMCS

PROTEOMICS

DRUG DISCOVERY

Sanoudou Pharmacogenom J 12:185, 2012

- “to decipher molecular mechanisms of drug action, identify novel therapeutic targets and suitable agents to target them with, & discover molecular markers/signatures that predict response to therapy or optimal drug dose for each patient”

Role of microarrays in drug discovery for oncology

DRUG DISCOVERY STRATEGY

How select gene X as potential target for anti-microbial drug?

1. Gene present in all target species

2. Gene absent from host (or very different form)

3. Gene has essential function

4. Other useful features- info about gene expression/function

- known compounds which inhibit activity

- not associated with known resistance mechanisms

“The last drug with a new mechanism of action approved for TB was rifampicin (discovered in 1963)” Koul et al. Nature 2011 review

Press release: 19 November 2012

EXPLORING DRUG-INDUCED CHANGES IN GENE EXPRESSION in Mycobacterium tuberculosis (Wilson et al. PNAS 96:12833, 1999)

Background info:

• Of ~ 4400 ORFs in 4.4 Mbp genome, large number for fatty acid metabolism (lipid biosynthesis & degradation)

- complex membranes for defense? and enzymes to degradelipids in host cell membranes?

• Large families of novel, glycine-rich protein genes

comprise ~ 10% of genome, 100 – 3500 aa in length[Gly-Gly-Ala] repeats…

- role as variable cell surface antigens “change their coat” to evade host immune response?

• TB treated by drugs (isoniazid) which block mycolic acid biosynthetic pathway … but resistant strains of M.t. are appearing

- M.t. cells treated with or without anti-TB drug INH (isoniazid inhibits mycolic acid synthesis in lipid membrane)

- RNA isolated at different time points,fluorescently labeled, hybridized with microarray of ORFs

Figure 1: 203 selected ORFs,especially genes encoding lipogenic or lipolytic enzymes

red = INH-treatedgreen = untreated

Figure 1b:INH-resistant strain treated with INH

Figure 1c:INH-resistant strain treated with ethionamide

Figure 1a:wild-type M.t. strain treated with INH

- also measured relative changes in mRNA abundance with INH treatment over time

- found induction of genes in mycolic acid biosynthesis pathway & several others

efpA – efflux transporter

Wilson et al. PNAS 96:12833, 1999

“Change in abundance of selected transcripts in total RNA was estimated by RT-PCR... Primers were designed to amplify internal regions of efpA, fas,...”

Value of this experimental strategy for drug development?

If identify genes for other proteins in same pathway… or novel genes (eg with detoxifying effects)

If learn about physiological state of M.t.

If identify drug-responsive promoters

- may be good targets for new drugs

- may predict action of novel compounds

- may use in screens to test novel compounds

“The challenge of new drug discovery for tuberculosis”

Koul et al. Nature 469:483, 2011

“Bull’s-eye representation of current clinical pipeline for TB”

NDA = new drug application (for regulatory approval)

PHARMACOGENOMICS

- study of how human genetic makeup influences drug response

- new drug development based on human genomic information

- genotyping of patients based on clinically-relevant genetic polymorphisms (eg. SNPs linked to disease susceptibility…)

… customized drug programs for patients

(efficacy & toxicity of drugs)

- application of genomic concepts & technologies to the study of drug activity & metabolism

Among patients with the same diagnosis:

There may be individuals who do not respond to a particular drug... and others who show a toxic response...

because of differences in genetic make-up

Cytochrome P450 metabolizing enzymes (isozyme family)- in liver, activate or inactivate different drugs- known SNPs which affect enzyme activity

Examples of drug responsiveness linked to genetic variation

CYP2D6 gene (on chr 22)

eg. G-to-A mutation in exon 4 affects splicing, so no protein

- SNPs correlated with “poor metabolizers” of mephenytoin used to treat epilepsy

eg. SNP in exon 5 affects splicing, present in ~2% Caucasians, but >20% Asians

CYP2C19 gene (on chr 10)

- cannot activate opioid analgesics (eg codeine), so different form of pain relief needed

Ann. Rev. Genomics Hum. Genet. 2: 9-39, 2001

• Detects up to 33 CYP2D6 alleles and 3 CYP2C19 alleles

From Roche website about benefits

• Detects CYP2D6 gene duplication and deletions

• AmpliTaq GOLD polymerase carry-over protection prevents cross-contamination of samples

• Proprietary software enables the generation of customized reports containing genotype and predicted phenotype information

AmpliChip CYP450 test (Roche)

“The world's first pharmacogenomic microarray designed for clinical applications.”

“... contains more that 15,000 different oligomers”

Comprehensive detection of gene variations for the CYP2D6 and CYP2C19 genes, which play a major role in the metabolism of an estimated 25 percent of all prescription drugs.

Powered by Affymetrix technology

Meyer Nature Rev Genet 5:649, 2004

- drug activity & metabolism

- drug clearance from circulation

Genotype-phenotype relationships of CYP2D6-polymorphisms

- drug uptake into cell by receptor

Ann Rev Genom.Hum Genet. 2:9,2001

Population distribution of drug metabolism phenotypes

Pharmacogenomics and TB treatment - isoniazid (INH) is metabolized by acetylation catalyzed by N-acetyl transferase (NAT2)

- genetic variation among individuals in rate of acetylation is correlated with number of NAT2*4 alleles

- mutations in NAT2 gene account for the majority of the “slow acetylator” genotypes in the human population (NAT2*5A, NAT2*5B, and NAT2*6A).

Ramachandran Pharmacogenom Personalized Med 2:89, 2012

Day -1

Torrance Nature Biotech 19:940, 2001

Day 0

Days 1-6

Use of isogenic human cancer cells for high-throughput screening and drug discovery

DLD-1 - colon cancer cell linewith mutant K-Ras (oncogene G12V)

KO - knockout line with mutant K-ras deleted

“29,440 diverse chemical compounds” in small molecule (drug) libraries used

Torrance Nature Biotech 19:940, 2001Fluorescent micrographsshowing selective growthinhibition

Day 7 Export data and plot growth curves

- then tested TPT & TPT-related compounds on various cancer cell lines

Torrance Nature Biotech 19:940, 2001

- found TPT effective for colon but not kidney cancer lines…

Categories of molecular targets widely used in human drug therapy:

Enzymes

G-protein coupled receptors

Ion channels

Nuclear hormone receptors

Data mining for “novel” members of multi-gene families of interest

Trends Pharm Sci 22:23, 2001

Liggett, Nature Rev Genet 5:657, Sept. 2004

Transgenic mice models for pharmacogenomic research

Bell, Nature 429:453, 2004

“Charting a course for genomic medicine from base pairs to bedside”

Green, Nature 470:204, 2011“Blue dots: single individual accomplishments; green/yellow/red: sequentially higher densities of accomplishments”

“We observed that approximately 84% of the genome is comprised of mobile elements or other repeat structures.”

Nature November 29, 2012

“Landscape of the barley gene space”