SYNTHESIS AND EVALUATION OF NOVOBIOCIN ANALOGUES AS POTENTIAL INHIBITORS OF POLYMERASE THETA POLΘ IN DNA DAMAGE AND REPAIR

Abstract

Killing tumors while leaving normal cells unharmed is the goal of precision cancer therapy, a challenging feat that is enabled by targeting tumor-specific vulnerabilities [1]. Targeting Polθ potentiates PARP inhibitors (PARPi) in specifically killing homology-directed repair (HDR)-deficient tumor cells and re-sensitize PARPi resistant tumor cells [1]. The induced essentiality between Polθ and HDR-deficiency underscores the value of Polθ as a therapeutic target in the context of tumors with HDR genetic mutations [1]. The helicase-like domain (HLD) is a super family 2 (SF2)-type domain with DNA-dependent ATPase activity [1]. We previously discovered that the antibiotic NVB acts as an inhibitor of N-terminal HLD of Polθ [1]. Consequently, a Phase I clinical trial of Novobiocin (NVB) is currently underway in patients with tumors that harbor aberrant DNA repair genes [1]. To inform enigmatic theta-mediated end joining (TMEJ) and support ongoing preclinical and clinical studies, we therefore investigated the mechanism of action of NVB-mediated inhibition of Polθ ATPase activity [1]. By combining hydrogen deuterium exchange-mass spectrometry (HX-MS), biochemical assays, microscale thermophoresis (MST), cellular assays and computational modelling, we determined that NVB is a non-ATP competitive inhibitor that binds to an allosteric site near the ssDNA binding channel in the ATPase core [1]. This is contrary to the current view of NVB as an ATP competitive inhibitor of DNA gyrase [1]. This NVB binding mode blocks ssDNA binding and inhibits ssDNA-mediated stimulation of Polθ ATPase activity [1]. Importantly, we find that NVB blocks Polθ binding to ssDNA both in vitro and in cells [1]. Using novobiocic acid (the aglycone of NVB), we investigated the orientation of NVB binding and established the contribution of the sugar group in enhancing the potency of NVB [1]. Our study identified the NVB binding pocket and provides a path for optimization and investigation of the importance of ssDNA binding for Polθ biological functions at double-strand breaks (DSBs) and stalled replication forks [1].