DRUG DISCOVERY OF TLS INHIBITORS TO SELECTIVELY TARGET CANCER CELLS WITH HOMOLOGOUS RECOMBINATION REPAIR DEFICIENCIES

GARCÍA, IRIS ALEJANDRA; VILLAFAÑEZ F.; QUIROGA R; BOCCO JL; VILLAREAL M; SORIA G.

Salta

Congreso; Joint LV Annual SAIB Meeting and XIV PABMB Congress; 2019

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Translesion DNA Synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to ensure replication forks integrity and cell survival. Consequently, TLS inhibition emerges as a promising strategy for the therapeutic intervention of HR-deficient tumors by synthetic lethality (SL) induction. Given the current lack of selective TLS pharmacological inhibitors to evaluate this hypothesis, we developed different approaches to identify small molecules able to impair PCNA mono-ubiquitination, a key post-translational modification required for the efficient activation of TLS. Initially, we developed a miniaturized WB assay using complementary antibodies that simultaneously detect ubi-PCNA and total PCNA. Using this assay, we screened a library of 627 kinase inhibitors. We found that targeting the pro-survival kinase AKT leads to a strong impairment of PCNA ubiquitination. Remarkably, such inhibition triggered the induction of SL in BRCA-deficient cells submitted to replication stress. The follow-up strategy was to focus in the identification of PCNA ubiquitination inhibitors with more selective mechanisms of action. To tackle this challenge, we designed a virtual screening approach to identify direct blockers of PCNA-ubiquitination through molecular modeling from a 10K collection of structurally diverse small molecules. We found several putative compounds that block PCNA-ubiquitination in silico, which after experimental validation led to the identification of a small group of strong PCNA ubiquitination inhibitors. Collectively, this work shows for the first time that TLS inhibition can be achieved by the pharmacological impairment of PCNA ubiquitination and provides the proof-of-concept of TLS inhibition as a therapeutic strategy to selectively kill HR-deficient cells.