Synthesis and Biological Evaluation of Novel Triazine Analogs as Rad6 Inhibitors.

Rad6 is an E2 ubiquitin-conjugating enzyme that plays critical roles in genome maintenance and proteostasis. Rad6 is frequently overexpressed in many cancers and promotes cancer development, progression, and chemotherapy resistance.

Purpose: Given its role in cancer development and progression, Rad6 is an underexplored therapeutic target. Previous research identified compound SMI#9 as a small molecule inhibitor of Rad6. Despite its potency, SMI#9 has limited efficacy in vivo due to its limiting water solubility and the presence of a labile ester group.

Methods: To address these limitations, we prepared a series of SMI#9 analogs in which the ester group was replaced with a secondary amine, and their effects on Rad6B-mediated ubiquitination of histone H2A were evaluated. In vivo interaction with Rad6 was assessed using cellular thermal shift assays. SMI#9 analog effects on cell survival and migration of triple negative and endocrine-resistant breast cancer, and melanoma cells were measured using MTT and Boyden chamber assays. Autophagy, mitochondrial function, and β-catenin localization were measured using CytoID, Mitotracker, and immunostaining, respectively. Cellular uptakes of analogs were determined by mass spectroscopy.

Results: Analogs #4 and #6 inhibited H2A ubiquitination, induced autophagy and mitochondrial dysfunction, downregulated intracellular β-catenin, and inhibited proliferation. #6 targets Rad6 in vivo. #4 and #6 are chemically related, and #4 undergoes in vivo conversion to #6.

Conclusions: #6 retains all the properties of SMI#9 but with lesser potency. However, its improved water solubility and metabolic stability allows for in vivo studies that were previously precluded due to the poor physicochemical properties of SMI#9.