Subject(s)
Drug Evaluation/methods , Genome, Human , Pharmacogenetics , Polymorphism, Single Nucleotide/genetics , Algorithms , Databases as Topic , Gene Frequency , Genetic Linkage/genetics , Genetic Variation/genetics , Haplotypes/genetics , Humans , Phenotype , Physical Chromosome Mapping , Protein BindingABSTRACT
The explosion of information generated by large-scale functional genomics technologies has resulted in an exponential increase in the number of potential genes and proteins available for pharmaceutical and diagnostic research development. In order to tap this potential, the primary challenge is to develop a strategy to effectively integrate and extract meaning from the human genomic sequence information that has been generated since the start of the Human Genome Project. This article deals with the strategies being applied by academics and by the biotechnology sector to sort and triage this information with the ultimate goal of identifying future therapeutic targets for cancer and other diseases.
Subject(s)
Carcinogens/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , Neoplasms/drug therapy , Neoplasms/genetics , Pharmacogenetics/methods , Animals , Drug Design , Genetic Therapy/methods , Genome, Human , Humans , Information Storage and Retrieval/methods , Medical Oncology , Oligonucleotide Array Sequence Analysis/methods , OncogenesABSTRACT
Expression pharmacogenomics includes differential gene expression (DGE) profiling of drug responses in model systems to generate a set of differentially modulated drug-responsive genes which can serve as a surrogate measure for drug action. In this manner, expression pharmacogenomics bridges the fields of genomics and medicinal chemistry. Additionally, modulated genes can be organized into metabolic and signaling pathways that highlight the mechanism of drug activity in a selected tissue. Here, we describe the application of expression pharmacogenomics to characterize a drug response in the clinically relevant in vivo model, the Sprague-Dawley rat. Following oral dosing of rats with GW9578, a novel synthetic peroxisome proliferator activated receptor alpha (PPAR alpha) ligand indicated for lipid disorders, we applied GeneCalling, a differential mRNA transcript profiling technique, to rat liver cDNA. Following GW9578 treatment, 2.4% of the rat liver genes were differentially expressed. We confirmed the sequence identity of 50 distinctly modulated genes. DGE was observed among genes representative of at least six discrete metabolic pathways. Furthermore, we observed up-regulation of 20 genes involved in mitochondrial, peroxisomal and microsomal fatty acid oxidation, consistent with molecular biological and clinical data indicating PPAR alpha ligand principal efficacy to be through increasing fatty acid metabolism. Those pathways regulated in our study that are potentially contributory to target effect, non-target adverse effects, or of unknown consequence include xenobiotic detoxification and steroid modification. Finally, comprehensive drug response profiling can lead to the serendipitous discovery of novel disease indications. In this case, these results suggest a potential novel indication for GW9578 in the treatment of X-linked adrenoleukodystrophy. We have shown, therefore, that the organization of DGE results into metabolic and signaling pathways can elucidate mechanisms of pharmacologically desired (i.e., efficacious) and, where appropriate, undesired (i.e., potentially deleterious) effects.
Subject(s)
Butyrates/pharmacology , Hypolipidemic Agents/pharmacology , Liver/drug effects , Nuclear Proteins/genetics , Phenylurea Compounds/pharmacology , Receptors, Cytoplasmic and Nuclear/genetics , Transcription Factors/genetics , Administration, Oral , Algorithms , Animals , Gene Expression Profiling/methods , Ligands , Liver/metabolism , Male , Nuclear Proteins/metabolism , Peroxisomes/drug effects , Peroxisomes/metabolism , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolismABSTRACT
Expression pharmacogenomics applies genome/proteome scale differential expression technologies to both in vivo and in vitro models of drug response to identify candidate markers correlative with and predictive of drug toxicity and efficacy. It is anticipated to streamline drug development by triaging towards lead compounds and clinical candidates that maximize efficacy while minimizing safety risks. As the majority of expression pharmacogenomics will be performed on preclinical therapeutic candidates, compatibility with favored preclinical animal model systems will be essential. This review will address expression pharmacogenomics in the context of those animal model systems commonly used for pharmacokinetic, pharmacodynamic and toxicologic analyses. Specific discussions will cover: (A) relative robustness of genomic and proteomic technology platforms used to generate drug response data in critical model systems; (B) animal handling, treatment and other experimental design optimizations; (C) data analysis strategies for extracting and validating candidate pharmacogenomic markers; and (D) overarching limitations in applying expression pharmacogenomics to animal model systems.
Subject(s)
Gene Expression Profiling , Models, Animal , Pharmacogenetics , Animals , HumansABSTRACT
Short-chain fatty acids (SCFAs) butyrate, propionate, and acetate produced during fiber fermentation promote colonic differentiation and can reverse or suppress neoplastic progression. We sought to identify candidate genes responsible for SCFA activity on colonocytes and to compare the relative activities of independent SCFAs. cDNA was generated from polyA+ mRNA isolated from control Caco-2 cells and cells treated with equimolar butyrate, propionate, and acetate. GeneCalling, a restriction-based differential RNA expression platform linked to a DNA sequence database lookup, was applied. A total of 30,000 individual genetic sequences were analyzed for differential expression among the three SCFAs. Differentially expressed peaks corresponding to cancer-related genes were isolated, sequenced, and cross-referenced to the GenBank human database. Gene identities were independently confirmed by oligonucleotide poisoning. More than 1000 gene fragments were identified as being substantially modulated in expression by butyrate. Butyrate tended to have the most pronounced effects and acetate the least. Five fragments selected for further study were fully sequenced and proved 100% homologous with human sequences for clusterin, amyloid precursor-like protein 2, and caudal homeobox 2 protein, not previously known to be modulated by SCFAs. In each case, a similar order of potency for the three SCFAs studied was observed. The common SCFAs appear to exert different effects. This study suggests the diversity of the SCFA response at the molecular level and facilitates identifying genes important in the biologic activity of dietary fiber.
Subject(s)
Colon/cytology , Enterocytes/metabolism , Fatty Acids, Volatile/genetics , Gene Expression , Molecular Chaperones , Alzheimer Disease , Amyloid beta-Protein Precursor , Butyrates , Caco-2 Cells , Clusterin , Dietary Fiber , Genes, Homeobox , Glycoproteins/genetics , Humans , Nerve Tissue ProteinsABSTRACT
We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.