Therapeutic disruption of RAD52–ssDNA complexation via novel drug-like inhibitors

Abstract

RAD52 protein is a coveted target for anticancer drug discovery. Similar to poly-ADP-ribose polymerase (PARP) inhibitors, pharmacological inhibition of RAD52 is synthetically lethal with defects in genome caretakers BRCA1 and BRCA2 (∼25% of breast and ovarian cancers). Emerging structure activity relationships for RAD52 are complex, making it challenging to transform previously identified disruptors of the RAD52–ssDNA interaction into drug-like leads using traditional medicinal chemistry approaches. Using pharmacophoric informatics on the RAD52 complexation by epigallocatechin (EGC), and the Enamine in silico REAL database, we identified six distinct chemical scaffolds that occupy the same physical space on RAD52 as EGC. All six were RAD52 inhibitors (IC50 ∼23–1200 μM) with two of the compounds (Z56 and Z99) selectively killing BRCA-mutant cells and inhibiting cellular activities of RAD52 at micromolar inhibitor concentrations. While Z56 had no effect on the ssDNA-binding protein RPA and was toxic to BRCA-mutant cells only, Z99 inhibited both proteins and displayed toxicity towards BRCA-complemented cells. Optimization of the Z99 scaffold resulted in a set of more powerful and selective inhibitors (IC50 ∼1.3–8 μM), which were only toxic to BRCA-mutant cells. RAD52 complexation by Z56, Z99 and its more specific derivatives provide a roadmap for next generation of cancer therapeutics.

A double-ring of human RAD52 remodels replication forks restricting fork reversal

“…Fig.1I and Supplemental Fig. S2B&D show that the RAD52 is recruited to the parental ssDNA in hydroxyurea (HU) treated cells, confirming our previous observations 5,25 . In contrast, both the RAD52 IBD and the RAD52 OBD mutants showed defects in association with parental ssDNA, the RAD52 OBD being more severely affected.…”

“…In contrast, both the RAD52 IBD and the RAD52 OBD mutants showed defects in association with parental ssDNA, the RAD52 OBD being more severely affected. As expected, RAD52-depleted cells showed increased levels of nascent ssDNA upon replication fork arrest 5,25 . Adding-back wild type RAD52 restored the normal levels of exposed ssDNA.…”

“…Increasing concentration of RAD52 populate peaks corresponding to two and to a lesser degree three undecamers of RAD52 per fork ( Fig.2C&D , Supplemental Table S2 ). Double rings bound to the fork DNA were also observed in the RAD52 IBD mutant, but were much reduced in the RAD52 OBD mutant despite its capacity to bind DNA 25 ( Supplemental Fig. S3 , Supplemental Table S2 ).…”

“…To quantify the stoichiometry of the RAD52-fork interaction we employed mass photometry, a light scattering based single-molecule technique 39,40 . In the absence of DNA, RAD52 exists in solution as a mixture of undecamers, decamers and monomers 25 ( Fig.2A , Supplemental Table S2 ). The undecameric rings of RAD52 are readily observed in the 2D class of the cryo-EM analysis described below ( Fig.…”

“…This gatekeeper role of RAD52 is distinct from its residual activity in homologous recombinaiton 11,12 , as well as RAD52 functions in potentially mutagenic single-strand annealing (SSA) 13 , antagonizing DNA polymerase  and theta-mediated end joining 14 , recruitment of MRE11 nuclease to reversed forks 15 , or mitotic DNA synthesis (MiDAS) 16,17 , but is related to fork cleavage by RAD52/MUS81 axis in response to prolonged replication stress 18 . The RAD52 gatekeeper function is temporally upstream of the reversed fork protection by recombination machinery and may therefore represent a basis for the synthetically lethal interaction between RAD52 loss or pharmacological inhibition and BRCAness 7,11,[19][20][21][22][23][24][25] . In the absence of RAD52, or upon RAD52 inhibition, the reversed forks are restored, but with large ssDNA gaps, i.e.…”