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

GARCÍA, ALEJANDRA I.; VILLAFAÑEZ, FLORENCIA; QUIROGA, RODRIGO; BOCCO, JOSÉ L.; VILLARREAL, MARCOS; SORIA, GASTÓN

Salta

Workshop; PABMB-SAIB 2019; 2019

Translesion DNA Synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to ensure replication forks integrity and cellsurvival. Consequently, TLS inhibition emerges as a promising strategy for the therapeutic intervention of HR-deficient tumors by syntheticlethality (SL) induction. Given the current lack of selective TLS pharmacological inhibitors to evaluate this hypothesis, we developed differentapproaches to identify small molecules able to impair PCNA mono-ubiquitination, a key post-translational modification required for the efficientactivation of TLS. Initially, we developed a miniaturized WB assay using complementary antibodies that simultaneously detect ubi-PCNA andtotal PCNA. Using this assay, we screened a library of 627 kinase inhibitors. We found that targeting the pro-survival kinase AKT leads to a strongimpairment of PCNA ubiquitination. Remarkably, such inhibition triggered the induction of SL in BRCA-deficient cells submitted to replicationstress. The follow-up strategy was to focus on the identification of PCNA ubiquitination inhibitors with more selective mechanisms of action. Totackle this challenge, we designed a virtual screening approach to identify direct blockers of PCNA-ubiquitination through molecular modelingfrom 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 workshows for the first time that TLS inhibition can be achieved by the pharmacological impairment of PCNA ubiquitination and provides the proofof-concept of TLS inhibition as a therapeutic strategy to selectively kill HR-deficient cells.