Saudi Cultural Missions Theses & Dissertations
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Item Restricted SYNTHESIS AND EVALUATION OF NOVOBIOCIN ANALOGUES AS POTENTIAL INHIBITORS OF POLYMERASE THETA POLΘ IN DNA DAMAGE AND REPAIR(University of Arkansas at Little Rock, 2025) Albuhluli, Mohammed; Anindya, Ghosh; Darin, JonesKilling 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].20 0