The Hip-Hop Chemical Genomics Lab-Research Overview
The primary interests of our labís research are to screen known and novel anti-proliferatives to:
- understand precise mechanisms of drug action by identifying the major drug target as well off-target effects including those potentially advantageous (for repurposing of approved drugs) and/or detrimental (resulting in unwanted side-effects)
- identify novel, "undruggable" targets to expand the fraction of the genome that can be exploited for chemotherapeutic intervention
- identify novel, specific chemical probes for use as rapid, reversible molecular biology tools to interrogate essential biological function and elucidate pathway architecture
To accomplish these aims we will use our well-validated, automated and high-throughput chemogenomic assay, HaploInsufficiency Profiling (HIP), based on our laboratoryís observation of "drug-induced haploinsufficiency". Drug-induced haploinsufficiency is the observed growth sensitivity in the presence of a drug of a diploid yeast strain heterozygous for the gene encoding the drug target (Nat Genet 21, March 1999). In our rapid and cost-effective HIP assay, a complete collection of heterozygous deletion strains are pooled, grown in the presence of compound and sampled as a function of time. Molecular bar-codes incorporated into each strain allow parallel analysis and relative strain abundance to be quantitatively assessed either by hybridization to oligonucleotide arrays, or more recently, by Next Generation Sequencing Technologies. The result is a list of genes ranked in order of their importance for growth and survival, a quantitative metric termed "fitness". Strains most sensitive to drug often carry deletions in genes that encode the drug target. The HIP assay is currently the only assay that allows the in vivo identification of all drug or small molecule targets in the cell, thereby identifying any polypharmacology effects of drug.
Once the primary mechanism has been identified and confirmed in secondary genetic and/or biochemical assays, further pathway specific genes that act to buffer the drug target pathway can be uncovered using our HOP (Homozygous deletion Profiling) assay. This assay identifies the drug effects on the nonessential fraction of the genome and reveals the genes important for buffering the drug target pathway. These genes typically comprise other pathway components and/or genes involved in multi-drug resistance (e.g. drug transport, detoxification and metabolism). Currently we are engaged in a large-scale discovery effort to identify novel specific chemical probes that can be employed to further understand the effects of inhibiting essential biological pathways and that will ultimately assist in deconvolution of these pathways through epistatic analysis.
While yeast cannot completely reflect the complexities of a mammalian cell, the high degree of homology shared with human (~70% of all essential yeast genes have a significant human homolog) provides hypotheses for the mechanism of action of any compound of interest. Using siRNA methodologies these hypotheses can then be readily tested in mammalian cells to phenocopy the effect of drug. Indeed, the availability of a molecularly bar-coded shRNA library representing ~15,000 human genes (TRC consortium http://www.broadinstitute.org/rnai/trc) has inspired us to leverage the HIP assay into mammalian cells in collaboration with Dr. Jason Moffat at the Donnelly Centre.