Identification of a Small Molecule Inhibitor of RAD52 by Structure-Based Selection

Abstract

It has been reported that inhibition of RAD52 either by specific shRNA or a small peptide aptamer induced synthetic lethality in tumor cell lines carrying BRCA1 and BRCA2 inactivating mutations. Molecular docking was used to screen two chemical libraries: 1) 1,217 FDA approved drugs, and 2) 139,735 drug-like compounds to identify candidates for interacting with DNA binding domain of human RAD52. Thirty six lead candidate compounds were identified that were predicted to interfere with RAD52 –DNA binding. Further biological testing confirmed that 9 of 36 candidate compounds were able to inhibit the binding of RAD52 to single-stranded DNA in vitro. Based on molecular binding combined with functional assays, we propose a model in which the active compounds bind to a critical “hotspot” in RAD52 DNA binding domain 1. In addition, one of the 9 active compounds, adenosine 5’-monophosphate (A5MP), and also its mimic 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) 5’ phosphate (ZMP) inhibited RAD52 activity in vivo and exerted synthetic lethality against BRCA1 and BRCA2–mutated carcinomas. These data suggest that active, inhibitory RAD52 binding compounds could be further refined for efficacy and safety to develop drugs inducing synthetic lethality in tumors displaying deficiencies in BRCA1/2-mediated homologous recombination.

Homologous recombination in budding yeast expressing the human RAD52 gene reveals a Rad51-independent mechanism of conservative double-strand break repair

“…Several studies using small molecule or peptide aptamer inhibitors of HsRAD52 describe an inhibition of the growth of human tumor cells possessing attenuated canonical HR pathway function, demonstrating the utility of this strategy (73–75). High throughput screening with the ‘humanized’ yeast strains described here could be used to identify chemical compounds that inhibit the function of HsRAD52, while detailed studies of HsRAD52 function at the molecular level could reveal the specific effects of known inhibitors.…”

Small-Molecule Inhibitors Targeting DNA Repair and DNA Repair Deficiency in Research and Cancer Therapy

“…Such a binding mode offers at least two targetable features, the integrity of the ring-shaped RAD52 oligomer and a narrow (though somewhat featureless) ssDNA binding groove. Several groups including ours have discovered small-molecule inhibitors of these two activities (Chandramouly et al, 2015; Hengel et al, 2016; Huang et al, 2016; Sullivan et al, 2016). These various studies screened different libraries, yielding hits with distinctly disparate chemical space from one another (Table 2).…”

“…Using a different fluorescence-based screen, Huang et al, identified two compounds, D-103 and D-G23 that inhibit the ssDNA annealing activity of RAD52, suppresses growth of BRCA-deficient cells, and at high concentrations inhibits SSA in human cells (Huang et al, 2016). A molecular docking of two compound libraries (total of ~141,000 compounds) into the site within the RAD52 DNA-binding domain that contains the ssDNA binding residues was used to identify several additional disrupters of the RAD52-ssDNA interaction (Sullivan et al, 2016). Among these, 5-Aminoimidazole-4-carboxamide ribonucleotide ( AICAR ) 5’ phosphate ( ZMP ) specifically inhibited the RAD52-ssDNA interactions.…”

Aberrant DNA Double-strand Break Repair Threads in Breast Carcinoma: Orchestrating Genomic Insult Survival

“…Tumors carrying BRCA1- and BRCA2-inactivating mutations have been reported to become lethal through RAD52 inhibition either by a specific shRNA or a small peptide aptamer 41. To achieve better outcomes in breast carcinoma, DNA repair protein inhibition strategies are highly necessary.…”