To accomplish these goals we will employ a validated chemogenomic assay, HaploInsufficiency Profiling (HIP), based on our laboratory’s observation that lowering the dosage of a single gene from two copies to one copy in diploid yeast results in a heterozygote that is sensitized to any compound that acts on the product of this gene. In our assay, a complete collection of heterozygous deletion strains is 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 fitness to be quantitatively assessed by hybridization to oligonucleotide arrays. Strains most sensitive to drug often carry deletions in genes that interact directly with the test compounds and inhibit cell proliferation. In this way, all ~6,000 yeast proteins are screened simultaneously. This approach is distinguished from other genomic approaches because 1) it is a cellular in vivo assay and 2) it allows ranking of genes most important for survival to compound that can be rapidly confirmed biologically. The results of this assay typically identify the drug target. Moreover, because such probes act in a reversible fashion, they can be employed to further understand the effects of inhibiting essential biological pathways. Once the primary mechanism has been uncovered, further pathway specific synthetic genetic effects of a particular compound on a pathway can be uncovered using HOP (Homozygous deletion Profiling) screening. By assessing the effects of these chemical probes on the nonessential fraction of the genome, we will uncover genes that buffer inhibition of the target pathway and that will ultimately assist in deconvolution of involved pathways. Over the past 3 years we have:

• scaled and miniaturized the assay
• built the informatics and custom robotics infrastructure to support this technology
• rigorously validated the technology such that it can now be applied to a large-scale systems discovery effort