AID	Name	Type	Comment
602329	Identification of inhibitors of RAD54 Measured in Biochemical System Using Plate Reader - 2159-01_Inhibitor_SinglePoint_HTS_Activity	screening	Primary HTS
651673	Identification of Rad54 Inhibitors - Ruling Out Compounds That Bind DNA Measured in Biochemical System Using Plate Reader - 2159-04_Inhibitor_Dose_CherryPick_Activity	confirmatory
651651	Identification of Rad54 Inhibitors - Ruling Out Flurescence Quenchers Measured in Biochemical System Using Plate Reader - 2159-03_Inhibitor_Dose_CherryPick_Activity	confirmatory
651657	Identification of Inhibitors of RAD54 Measured in Biochemical System Using Plate Reader - 2159-01_Inhibitor_Dose_CherryPick_Activity	confirmatory
651692	Cytotoxicity assay Measured in Cell-Based System Using Plate Reader - 2159-05_Inhibitor_Dose_CherryPick_Activity	confirmatory

Primary Collaborators:
Alex Mazin,Drexel University College of Medicine,amazin@drexelmed.edu
Kyle Emmitte,Vanderbilt University,kyle.a.emmitte@Vanderbilt.Edu

Project Goal:

The goal of this proposal is to develop specific small-molecule inhibitors of human RAD54, one of the key homologous recombination (HR) proteins. In humans, the HR pathway is responsible for the repair of DNA double-strand breaks (DSB) and inter-strand cross-links (ICL). Since DSB- and ICL-inducing agents are commonly used in anticancer therapy we expect that inhibition of HR in cancer cells will increase the efficacy of this therapy.

Probe attributes:

Target Activity-IC50< 10 uM in the FqRET assay, IC50 <10 uM in Luc reporter and G3A content assays
Selectivity: Anti-target->5X- A3G assay
Biological Mode of Action (in vitro, cellular, in vivo)-In vitro: direct disruption of VIF self-assembly,Cellular: VIF-mediated antiviral effect through A3G
MOA (covalent/allosteric etc.)- Noncovalent preferred
Cellular Toxicity-non-toxic at 10X effective IC50
Functional Groups to be avoided (electrophiles, esters, etc.)-1. Chemically reactive groups, 2. Metabolically labile groups pH sensitive. or Hydrolytically instable
Chemical Solubility Criteria-Solubility > 20 uM in PBS

Biological Relevance:
In humans, the HR pathway is responsible for the repair of DNA double-strand breaks (DSB) and inter-strand cross-links (ICL). Since DSB- and ICL-inducing agents are commonly used in anticancer therapy we expect that inhibition of HR in cancer cells will increase the efficacy of this therapy.

Rad54 protein is evolutionarily conserved in all eukaryotes. In yeast, Rad54 mutants show the strongest DNA repair phenotype along with Rad51 and Rad52 mutants. In mammals, RAD54 knockouts display high sensitivity to ionizing radiation, and especially to ICL-inducing agents. In contrast, RAD51 knockouts are unviable and Rad52 knockouts lack any phenotype. Mutations in the RAD54 gene cause genome instability and lead to cancer. In vitro, Rad54 catalyzes branch migration of Holliday junctions, a basic step of homologous recombination, in which one DNA strand is progressively exchanged for another. However, while the biochemical properties of RAD54 are well characterized, its cellular functions remain poorly understood. Therefore, in addition to therapeutic applications, specific inhibitors would present an extremely valuable tool to study the RAD54 cellular functions in humans, where application of traditional genetic approaches is limited.

Grant Number: R03 DA033981-01A1