Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

Abstract

The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing.DOI: http://dx.doi.org/10.7554/eLife.14740.001

Integrating Experimental and In Silico HTS in the Discovery of Inhibitors of Protein–Nucleic Acid Interactions

“…This compound, NP-004255 , is a macrocyle ester, consisting of three trihydroxylated phenolic moieties; it is called corilagin , which belongs to a family of secondary plant metabolites called ellagitannins . Importantly, the hit was validated by both NMR WaterLOGSY and the FRET-based method described above, as fully described in Hengel et al ., 2016 (Hengel et al, 2016), as shown in Figure 9. NP-004255 was shown to bind to RAD52, and compete for ssDNA binding.…”

“…Amazingly, a single natural product was selected for detailed biophysical study, and found to be in the high nM to low μM range (depending on the assay employed). A rational strategy for targeting the RAD52 PNI is, ostensibly, a nightmarish challenge for structure based design, in that only an unliganded crystal structure of RAD52 was available (PDB 1KN0), which suggests an exotic circular continuous ssDNA binding epitope (Hengel et al, 2016, Kagawa et al, 2002), with a patchwork of alternating hydrophobic and hydrophilic regions and complex water networks in the biding groove. Given the uncertainty of PDB 1KN0 representing the liganded conformation of the protein in solution, the lack of any known ligands that bind in this groove and the complex role of water, we sought to build an integrated workflow for PNI inhibitor discovery which capitalized on in vitro HTS initially, followed by computational studies built on a rigorous statistical framework for understanding why certain chemotypes disrupt the RAD52 PNI.…”

“…Additionally, interstitial water molecules were determined and retained to be used by the VINA scoring function. Our workflow (Hengel et al, 2016), featured a user-specified parameter for defining what constituted an interstitial water. This allowed us to parse all the docking poses from the docking workflow outlined above.…”

“…In these studies, we found that interstitial water structure was a major factor contributing to ligand pose and the quality of the docking scores. In Hengel et al ., we referred to this automated process as Simulated Annealing Energy Minimization (SEAM) (Hengel et al, 2016). …”

“…Indeed, true prospective structure based discovery of specific low μM to high nM hits using strictly in silico screening, especially against challenging or novel targets, is quite rare (Head, 2010). Our philosophy for the campaign described in Hengel et al(Hengel et al, 2016), was to use ROC curve analysis in such a way as to be able to focus on screening of interesting and relatively precious natural products. Natural products clearly have an unrivaled history in drug discovery, and often represent the primary hits against targets.…”