Drug discovery of TLS inhibitors to selectively target cancer cells with homologous recombination repair deficiencies.

QUIROGA, RODRIGO*; SORIA, GASTÓN; GARCÍA, IRIS ALEJANDRA*; BOCCO, JOSÉ LUIS; VILLAFAÑEZ, FLORENCIA*; VILLARREAL, MARCOS

Salta

Congreso; LV Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular; 2019

SAIB PABMB

Translesion DNA Synthesis (TLS) and homologous recombination (HR) cooperate during S-phase to ensure replicationforks integrity and cell survival. Consequently, TLS inhibition emerges as a promising strategy for the therapeuticintervention of HR-deficient tumors by synthetic lethality (SL) induction. Given the current lack of selective TLSpharmacological inhibitors to evaluate this hypothesis, we developed different approaches to identify small molecules able toimpair 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 andtotal PCNA. Using this assay, we screened a library of 627 kinase inhibitors. We found that targeting the pro-survival kinaseAKT leads to a strong impairment of PCNA ubiquitination. Remarkably, such inhibition triggered the induction of SL inBRCA-deficient cells submitted to replication stress. The follow-up strategy was to focus in the identification of PCNAubiquitination inhibitors with more selective mechanisms of action. To tackle this challenge, we designed a virtual screeningapproach to identify direct blockers of PCNA-ubiquitination through molecular modeling from a 10K collection ofstructurally diverse small molecules. We found several putative compounds that block PCNA-ubiquitination in silico, whichafter 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 ofPCNA ubiquitination and provides the proof-of-concept of TLS inhibition as a therapeutic strategy to selectively kill HR-deficient cells.