Single-strand selective monofunctional uracil DNA glycosylase (SMUG1) is believed to be the primary DNA repair enzyme responsible for excising uracil from highly mutagenic genomic U:G lesions. Mutations or deficiencies in SMUG1 have been linked with increased cancer risks, and SMUG1 activity has been shown to mediate the efficacies of existing cancer therapies. Despite such significance, there is no existing strategy for modulating SMUG1 activity. To better understand the biological function of SMUG1 and potentially target it as a cancer prevention strategy, we endeavored to develop small molecule activators of the enzyme. Screening of a small library using a universal base excision reporter (UBER) assay recently developed in our laboratory led to several hit compounds with a conserved core structure. Iterative rounds of structure-activity relationship studies and structural optimizations eventually produced two quinazoline-based SMUG1 activators. These two lead compounds achieved maximum 90-fold enzyme activation <i>in vitro</i>, with significant activity remaining at sub-micromolar concentrations. Furthermore, they were shown to be specific toward SMUG1 activation over other DNA repair enzymes and kinase inhibition, and no loss of substrate specificity was observed. Finally, cellular experiments showed minimal toxicity in four human cell lines at 10 µM and confirmed the ability of the activators to enhance uracil excision in HeLa cells. These results suggest that the two activator compounds are promising tools for probing SMUG1 function.

Discovery of small molecule activators of single-strand selective monofunctional uracil DNA glycosylase

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