Trends Biotech 19: 288,2001

1
GENOMICS
FUNCTIONAL GENOMCS
PROTEOMICS
DRUG DISCOVERY
Trends Biotech 19 288,2001
2
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 12185, 2012
3
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
Press release 19 November 2012
The last drug with a new mechanism of action
approved for TB was rifampicin (discovered in
1963) Koul et al. Nature 2011 review
4
EXPLORING DRUG-INDUCED CHANGES IN GENE EXPRESSION
in Mycobacterium tuberculosis (Wilson et al.
PNAS 9612833, 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?
5

• 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
6
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
7

• 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 9612833, 1999
8
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
9
The challenge of new drug discovery for
tuberculosis
Bulls-eye representation of current clinical
pipeline for TB
NDA new drug application (for regulatory
approval)
Koul et al. Nature 469483, 2011
10
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
11
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 gt20 Asians
Ann. Rev. Genomics Hum. Genet. 2 9-39, 2001
12
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 cross-contamination of samples

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

13
Genotype-phenotype relationships of
CYP2D6-polymorphisms
- drug uptake into cell by receptor
- drug clearance from circulation
- drug activity metabolism
Meyer Nature Rev Genet 5649, 2004
14
Population distribution of drug metabolism
phenotypes
Ann Rev Genom.Hum Genet. 29,2001
15
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 NAT24
alleles
- mutations in NAT2 gene account for the majority
of the slow acetylator genotypes in the human
population (NAT25A, NAT25B, and NAT26A).
Ramachandran Pharmacogenom Personalized Med 289,
2012
16
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 19940, 2001
17
Day 7 Export data and plot growth curves
Fluorescent micrographs showing selective
growth inhibition
Torrance Nature Biotech 19940, 2001
18
- then tested TPT TPT-related compounds on
various cancer cell lines
- found TPT effective for colon but not kidney
cancer lines
Torrance Nature Biotech 19940, 2001
19
Categories of molecular targets widely used in
human drug therapy
Enzymes
G-protein coupled receptors
Ion channels
Nuclear hormone receptors
Trends Pharm Sci 2223, 2001
Data mining for novel members of multi-gene
families of interest
20
Transgenic mice models for pharmacogenomic
research
Liggett, Nature Rev Genet 5657, Sept. 2004
21
Bell, Nature 429453, 2004
22
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 470204, 2011
23
Nature November 29, 2012
We observed that approximately 84 of the genome
is comprised of mobile elements or other repeat
structures.
24
Landscape of the barley gene space