NMR STUDIES REVEAL UNEXPECTED BINDING SITE FOR APE1 REDOX INHIBITOR

AP endo 1 (APE1) is a multi‐functional enzyme involved in DNA repair and transcriptional regulation. APE1 uses a redox function to modulate gene expression by regulating the redox state of transcription factors including p53, NF‐KB, among others. APE1 is upregulated in many types of cancer and its redox activity plays significant role in tumor development and migration. Accordingly, much research has focused on development of effective small molecule inhibitors of APE1 redox activity. A well‐studied example is E3330, which was found to directly associate with APE1 and inhibit its redox activity, but not its AP endo activity. Due to this selective functional inhibition, it was suggested that E3330 binds near Cys65, a buried residue that is proposed to be required for redox activity. Using NMR techniques and docking studies, we have elucidated the binding site of E3330. Surprisingly, E3330 binds in the active site, near the pocket occupied by the flipped abasic nucleotide of a bound DNA substrate. To further explore redox inhibition by small molecules, we synthesized another previously identified APE1 redox inhibitor, 16F, and determined that binding site. It binds a site distinct from the repair active site and the putative redox domain (C65). The disparate binding sites of these two redox inhibitors, and the solvent inaccessibility of Cys65, suggests conformational dynamics may be involved in APE1 redox activity. Funding NIH T32 GM066706 , “Graduate Training at the Chemistry Biology Interface”