Discipline: Biological Sciences
Subcategory: Cancer Research
Swikriti Shrestha - Claflin University
Co-Author(s): Sarah R. Hengel, University of Iowa, IA; Laura Folly da Silva Constantino, University of Iowa, IA; M. Ashley Spies, University of Iowa, IA;Maria Spies, University of Iowa, IA
The DNA repair protein RAD52 plays important roles in genome maintenance. RAD52 plays an important role in break-induced DNA replication, supports recovery of stalled replication forks, and participates in homology-directed DNA repair by single-strand annealing. Inhibition or depletion of RAD52 synthetically lethal with biallelic defects in tumor suppressors BRCA1, BRCA2 and PALB2, commonly associated with breast cancer. Our previous work identified selective inhibitors that disrupt the ssDNA binding and annealing activity of RAD52. The follow-up computational study has identified three new inhibitors R35, R36, and R37. Using Foster Resonance Energy Transfer (FRET) based assays we assessed the ability of these compounds to inhibit the RAD52-ssDNA interaction. All three compounds inhibited the RAD52-ssDNA interaction with IC50 values in the low micro molar range, which were similar to our most potent inhibitor epigallocatechin identified by high throughput screening. All three compounds also inhibited the RAD52 interaction with its physiological substrate, which is ssDNA coated with the ssDNA binding protein RPA. Notably, these compounds also perturbed the RPA-ssDNA interaction. The three compounds represent a chemical space different from previously identified compounds and thus confirm the robustness of our computational approach. The fact that they also inhibit the RPA-ssDNA interaction may suggest that they act as generic ssDNA mimetics and therefore will require further biochemical and structural analysis, as well as modifications to improve specificity.
Not SubmittedFunder Acknowledgement(s): I would like to thank Dr Maria Spies and Dr Sarah Hengel,University of Iowa with whom I worked in this project. The summer program was supported by Carver college of Medicine, Office of Cultural affairs and Diveristy, University of Iowa and Claflin University.
Faculty Advisor: Derrick Swinton, deswinton@claflin.edu
Role: In this research project, I worked on characterizing novel in-silico inhibitors of RAD52 protein. I started with expressing RAD52 protein in bacterial cells and performed protein purification using nickel, heparin and resource column. Using the fractions of protein obtained from purification, I performed biophysical assays using Foster Resonance energy transfer to study the binding of RAD52 protein with single stranded DNA and the effects of different inhibitors on the protein-DNA interaction.