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 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

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 cross-contamination 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 Fluorescent micrographs showing selective growth inhibition Torrance Nature Biotech 19:940, 2001

- 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”