Proapoptotic effect of WS-299 induced by NOXA accumulation and NRF2-counterbalanced oxidative stress damage through targeting RBX1-UBE2M interaction in gastric cancer

The abnormal activation of Cullin RING E3 Ligases (CRLs) is closely associated with the occurrence and development of various cancers. Targeting the neddylation pathway represents an effective approach for cancer treatment. In this work, we reported that WS-299, structurally featuring a coumarin moiety attached to the triazolopyrimidine, exhibited excellent anti-proliferative activity in MGC-803 and HGC-27 cells. WS-299 exerted potent anticancer effects by inhibiting clone formation, EdU incorporation and inducing cell cycle arrest. WS-299 inhibited CUL3/5 neddylation and caused an obvious accumulation of Nrf2 and NOXA, substrates of CRL3 and CRL5, respectively. Biochemical studies showed that WS-299 inhibited CUL3 neddylation by inhibiting RBX1-UBE2M interaction. The anti-proliferative effect of WS-299 was mainly induced by NOXA-mediated apoptosis. Of note, Nrf2 attenuated WS-299-induced reactive oxygen species (ROS) levels. Furthermore, Nrf2 accumulation also had an antagonistic effect on NOXA-induced apoptosis. Therefore, WS-299 and siNrf2 synergistically increased ROS levels, apoptotic cells and suppressed tumor growth in vivo. Taken together, our research clarified the anti-cancer mechanisms of WS-299 through targeting RBX1-UBE2M protein–protein interaction and inhibiting the neddylation modification of CUL3 and CUL5. More importantly, our studies also demonstrated that combination of WS-299 with Nrf2 inhibitors could be an effective strategy for treating gastric cancers.