Natural products inhibitors targeting homology-directed DNA repair for cancer therapy - ABSTRACT Genomic instability is a hallmark of cancer. Genome destabilizing defects in DNA repair enable tumorigenesis and cancer progression pushing cancer cells into dependence on a specific subset of DNA repair and genome maintenance factors. Two such targets of the proposed program are DNA repair proteins RAD51 and RAD52. The flexibility and adaptability of DNA repair networks allows cancer cells to employ them to enable rapid evolution, progressive accumulation of genetic variation, genomic instability, the acquisition of invasiveness, drug and radiation resistance and disease progression. Cells deficient in homologous recombination (due, for example to defects in tumor suppressors BRCA1 and BRCA2) require RAD52 for survival, while overexpression of RAD51 in cancers promotes radiation and chemotherapy resistance. Both proteins rely on their ssDNA binding to carry out their cellular functions. Inhibition of DNA repair by interfering with RAD52 or RAD51 binding, or entrapment of the toxic DNA repair intermediates with the help of small molecules has significant therapeutic potential. Identifying disruptors of ssDNA binding specific to each protein represents a tough medicinal chemistry challenge, but is a prerequisite for tailored low toxicity therapies. Natural products have an unrivaled place in early stage drug discovery and occupy an unparalleled chemical space for protein binding, which will be employed to elucidate the structural and chemical aspects of specific inhibition of our target proteins. Our published and preliminary studies established robust biophysical (FRET-based) assays to monitor the ssDNA binding by RAD51, RAD52 and Replication Protein A (RPA), a major human ssDNA binding protein whose inhibition is likely to be toxic. Proof-of-principle screens confirmed that we can readily identify small molecules and natural products specifically inhibiting each of the three proteins. We have also developed a computational workflow that accurately predicts interactions between small molecules and the DNA binding groove of RAD52. Here, we propose to carry out a screening campaign to identify a range of natural product scaffolds that can be developed into potent, specific and non-toxic disruptors of the RAD52-ssDNA interaction and to build a framework for discovery of RAD51 natural products inhibitors. We will screen a unique library of natural products, an NCI Program for Natural Product Discovery (NPNPD) Prefractionated Library consisting of 320,000 fractions, which were produced by separating the unique crude extracts derived from plant, marine, and microbial organisms. Our high throughput screening and compound validation (Aim1) followed by in silico analysis (Aim2) will yield sets of natural products scaffolds that will inform us on the regions and structural features within the target site of RAD52, that can be targeted to achieve specificity and high affinity, and on the chemical signature of the potential specific hits. Structure-activity relationships and rules for specificity learned from these campaigns can be then utilized in scaffold hopping approaches to new synthetic RAD52 inhibitors.
