Reverse Chemical Genetics: Comprehensive Fitness Profiling Reveals the Spectrum of Drug Target Interactions

Abstract

The emergence and prevalence of drug resistance demands streamlined strategies to identify drug resistant variants in a fast, systematic and cost-effective way. Methods commonly used to understand and predict drug resistance rely on limited clinical studies from patients who are refractory to drugs or on laborious evolution experiments with poor coverage of the gene variants. Here, we report an integrative functional variomics methodology combining deep sequencing and a Bayesian statistical model to provide a comprehensive list of drug resistance alleles from complex variant populations. Dihydrofolate reductase, the target of methotrexate chemotherapy drug, was used as a model to identify functional mutant alleles correlated with methotrexate resistance. This systematic approach identified previously reported resistance mutations, as well as novel point mutations that were validated in vivo. Use of this systematic strategy as a routine diagnostics tool widens the scope of successful drug research and development.

Overcoming cancer therapeutic bottleneck by drug repurposing

“…[502][503][504] Approaches based on chemical genetics are also dependent on intermolecular forces, and can help explain the relationship between binding and drug efficacy. 505,506 An example of the success of this technique comes from Karaman et al They evaluated several kinase inhibitors against pathologically significant human protein kinases using a competition binding assay in vitro. Interaction maps for 38 kinase inhibitors across a panel of 317 kinases representing >50% of the predicted human protein kinome generated a heat map of 3175 binding interactions.…”

Single-cell Analysis for Drug Development Using Convex Lens-Induced Confinement Imaging

“…For the growth comparison experiment, we used the dfr1/DFR1 heterozygous deletion strain, derived from BY4743 [20]. This strain is deleted for a single copy of the enzyme dihydrofolate reductase, which is the established target of methotrexate [21].…”

“…In yeast, the dihydrofolate reductase enzyme is encoded by DFR1 and is required for tetrahydrofolate biosynthesis. This enzyme is functionally conserved between yeast and human cells; that is, the viability and antifolate resistance of a yeast dfr1 mutant can be rescued by expression of the human enzyme DHFR [20]. Inhibition by metho-trexate results in fitness defects of yeast cells, which is likely due to the adverse effects of the drug on nucleic acid and amino acid biosynthesis [21].…”

How Surrogate and Chemical Genetics in Model Organisms Can Suggest Therapies for Human Genetic Diseases

“…In yeast, similar approaches to SGA have been taken with respect to chemical genetic analysis ( Fairn and McMaster 2005 ; Fairn et al 2007 ; Hillenmeyer et al 2008 ; Smith et al 2010 ; Ho et al 2011 ; Cong et al 2012 ; Enserink 2012 ; Wohlbold et al 2012 ; Cuesta-Marbán et al 2013 ; Czyz et al 2013 ; Lehner 2013 ; Golla et al 2016 ; Silberberg et al 2016 ; Wong et al 2016 ). In chemical genetics, how a drug/compound affects the growth of the entire yeast knockout collection is assessed ( Figure 2, A and B ), often referred to as either drug-induced haploinsufficiency profiling (HIP) or homozygous profiling (HOP) ( Giaever et al 1999 ; Lee et al 2014 ).…”