Fig 2: BT317 acts as a dual LonP1 and chymotrypsin-like proteasome inhibitor.(A) BT317 inhibition of LonP1 protease activity was assessed using a FITC-Casein substrate. (B) 10μM BT317 or 100nM BTZ were evaluated for proteasome inhibition at 1, 4, 6, 8 hours. NAC was used to ablate proteasome inhibition. (C) The IDH1 wildtype U-251 and IDH1 mutant U-87 MG lines were treated with 10μM BT317 and show increased Aco2 and TFAM levels. Data are presented as mean ± SEM of atleast 3 replicates. Statistical significance was determined by t-test. * P <0.05, **P <0.01, ***P <0.001; n.s., not significant.

Fig 3: Dual LonP1 and Chymotrypsin-like Proteasome Inhibition has Greater Synergy in IDH1 Mutant Astrocytoma and Enhances Autophagy and ROS Production.(A) In combination with Carfilzomib, a selective chymotrypsin-like proteasome inhibitor, CDDO-ME, a known LonP1 inhibitor, demonstrates strong synergy at 200nM in IDH1 mutant lines, whereas (B) the IDH1 wildtype lines only show limited synergy at 400–500nM. (C) Lc3B levels and (D) ROS levels were assessed after the combination of 5nM CFZ and 200nM CDDO-ME at 1, 12 and 24 hour time points in the DB70 and DB76 lines. (E) Similarly ROS levels were analyzed in the 83MES line. Statistical significance was determined by t-test. * P <0.05, **P <0.01, ***P <0.001; n.s., not significant.