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GENOMICS
FUNCTIONAL GENOMCS
PROTEOMICS
DRUG DISCOVERY
Trends Biotech 19: 288,2001
Role of microarrays in drug discovery for oncology
- “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”
Sanoudou Pharmacogenom J 12:185, 2012
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:
• TB treated by drugs (isoniazid) which block mycolic acid biosynthetic
pathway
… but resistant strains of M.t. are appearing
• Of ~ 4400 ORFs in 4.4 Mbp genome, large number for fatty acid
metabolism (lipid biosynthesis & degradation)
- complex membranes for defense? and enzymes to degrade
lipids 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?
- 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
red = INH-treated
green = untreated
Figure 1: 203 selected ORFs,
especially genes encoding
lipogenic or lipolytic enzymes
Figure 1a:
wild-type M.t. strain treated with INH
Figure 1b:
INH-resistant strain treated with INH
Figure 1c:
INH-resistant strain treated with
ethionamide
- also measured relative changes in mRNA abundance with INH
treatment over time
efpA – efflux transporter
- found induction of genes in
mycolic acid biosynthesis
pathway & several others
“Change in abundance of selected
transcripts in total RNA was estimated
by RT-PCR... Primers were designed to
amplify internal regions of efpA, fas,...”
Wilson et al. PNAS 96:12833, 1999
Value of this experimental strategy for drug development?
If identify genes for other proteins in same pathway
… or novel genes (eg with detoxifying effects)
- may be good targets for new drugs
If learn about physiological state of M.t.
- may predict action of novel compounds
If identify drug-responsive promoters
- may use in screens to test novel compounds
“The challenge of new drug discovery for tuberculosis”
“Bull’s-eye representation of
current clinical pipeline for
TB”
NDA = new drug application (for
regulatory approval)
Koul et al. Nature 469:483, 2011
PHARMACOGENOMICS
- application of genomic concepts & technologies to the study of
drug activity & metabolism
- genotyping of patients based on clinically-relevant genetic
polymorphisms (eg. SNPs linked to disease susceptibility…)
- study of how human genetic makeup influences drug response
(efficacy & toxicity of drugs)
- new drug development based on human genomic information
… customized drug programs for patients
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
Examples of drug responsiveness linked to genetic variation
Cytochrome P450 metabolizing enzymes (isozyme family)
- in liver, activate or inactivate different drugs
- known SNPs which affect enzyme activity
CYP2D6 gene (on chr 22)
eg. G-to-A mutation in exon 4 affects
splicing, so no protein
- cannot activate opioid analgesics
(eg codeine), so different form of pain
relief needed
CYP2C19 gene (on chr 10)
- 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
Ann. Rev. Genomics Hum. Genet. 2: 9-39, 2001
AmpliChip CYP450 test (Roche)
“The world's first pharmacogenomic
microarray designed for clinical applications.”
Powered by Affymetrix technology
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.
“... contains more that 15,000 different oligomers”
From Roche website about benefits
• Detects up to 33 CYP2D6 alleles and 3 CYP2C19 alleles
• Detects CYP2D6 gene duplication and deletions
• AmpliTaq GOLD polymerase carry-over protection prevents crosscontamination of samples
• Proprietary software enables the generation of customized reports containing
genotype and predicted phenotype information
Genotype-phenotype relationships of CYP2D6-polymorphisms
- drug uptake into cell
by receptor
- drug clearance from
circulation
- drug activity & metabolism
Meyer Nature Rev Genet 5:649, 2004
Population distribution of drug
metabolism phenotypes
Ann Rev Genom.Hum Genet. 2:9,2001
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
Use of isogenic human cancer cells for high-throughput
screening and drug discovery
DLD-1 - colon cancer cell line
with mutant K-Ras (oncogene G12V)
KO - knockout line with mutant K-ras deleted
Day -1
Day 0
“29,440 diverse chemical
compounds” in small molecule
(drug) libraries used
Days 1-6
Torrance Nature Biotech 19:940, 2001
Day 7 Export data and plot growth curves
Torrance Nature Biotech 19:940, 2001
Fluorescent micrographs
showing selective growth
inhibition
- then tested TPT & TPT-related compounds on various cancer cell lines
- found TPT effective for colon but not kidney cancer lines…
Torrance Nature Biotech 19:940, 2001
Categories of molecular targets widely used in human drug therapy:
Enzymes
G-protein coupled receptors
Ion channels
Nuclear hormone receptors
Trends Pharm Sci 22:23, 2001
Data mining for “novel” members of multi-gene families of interest
Transgenic mice models for pharmacogenomic research
Liggett, Nature Rev Genet 5:657, Sept. 2004
Bell, Nature 429:453, 2004
“Charting a course for genomic medicine from base pairs to bedside”
“Blue dots: single individual accomplishments;
green/yellow/red: sequentially higher densities of accomplishments”
Green, Nature 470:204, 2011
Nature November 29, 2012
“We observed that approximately 84% of the genome is
comprised of mobile elements or other repeat structures.”
“Landscape of the barley gene space”