Discovery and Characterization of Chemical Inhibitors of UBC13.

Abstract 2950

Poly-ubiquitination of signaling proteins via lysine 63 (K63)-linked chains is recognized as a critical post-translational modification involved in activation of NF-kB and stress kinases in the context of signaling by Tumor Necrosis Factor Receptors (TNFRs), Toll-like receptors (TLRs), and antigen receptors. UBC13 is a K63-specific ubiquitin conjugating enzyme that partners with TNFR-associated factors (TRAFs) to mediate K63-linked ubiquitination. Gene ablation studies have shown UBC13 is required for NF-kB signaling induced by a variety of stimuli in specific types of immune cells, making it a potential target for certain cancers, autoimmune and inflammatory diseases. UBC13 operates together with obligatory cofactors, either UEV1A in the cytosol or MMS2 in the nucleus. The nuclear function of UBC13 is evolutionarily conserved, where it plays a critical role in double strand DNA repair, making UBC13 a potential chemo- and radio-sensitizer target for oncology.

To identify chemical inhibitors of UBC13, we developed a HTS assay measuring UBC13-UEV1A enzymatic activity by TR-FRET, screening altogether ∼450,000 diverse compounds. Hit compounds were characterized using a rigorous testing funnel consisting of (a) informatics filtering against a database of > 100 HTS campaigns conducted with the same libraries, to eliminate promiscuous compounds; (b) counter-screens against E1, another cysteine-dependent enzyme (caspase-3), and against an irrelevant target formatted as a TR-FRET assay; and (c) ordering compounds from fresh powders and demonstrating reproducible concentration-dependent inhibition of UBC13. The surviving hits were then analyzed by cell-based assays for suppression of TRAF6 ubiquitination but not Mdm2-mediated ubiquitination of p53, resulting in 14 promising hits that included two chemical series. While suppressing TRAF6 ubiquitination (UBC13-dependent) in cells, these compounds did not interfere with either SUMOylation (UBC9-dependent) or NEDDylation (UBC12-dependent) of cellular proteins. UBC13 inhibitors also suppressed NF-kB activity (measured using stably integrated NF-kB-driven luciferase reporter gene) induced by PKC activators (Carma/Bcl-10/MALT pathway) and DNA damaging agent (Doxorubicin) but not by TNF-a. Investigations of the bioactivity of these UBC13 inhibitory compounds and their analogs will be described for a variety of hematolymphoid malignancies. (Supported by NIH R03-MH085677, NIH U54–005033, and by a fellowship grant from International Myeloma Foundation).