Figure 1.

The triterpenoids CDDO and its derivatives inhibit the protease and ATPase activities of LonP1. Structures of (A) electrophilic CDDO derivatives; (B) TP-82, a nonelectrophilic CDDO analog that lacks the C-2 electron-withdrawing group; (C) enoxolone, another pentacyclic triterpenoid. D, CDDO and CDDO-Me inhibit LonP1-mediated degradation of FITC-casein, whereas TP-82 does not. LonP1 (1.0 μM, monomer) was preincubated (30 min, 30 °C) with or without inhibitor or DMSO vehicle control (≤1%). Reactions were initiated by adding FITC-casein (0.1 mg/ml) and ATP (4 mM) followed by incubation at 37 °C for the indicated times. Representative of N ≥ 3 independent experiments. E, effect of preincubation time on LonP1 inhibition by CDDO derivatives at 30 °C. Reactions were initiated by adding FITC-casein (0.1 mg/ml) and ATP (4 mM) followed by incubation at 37 °C for 30 min. F, CDDO derivatives inhibit the ATPase activity of LonP1. A dose–response curve is shown for each compound tested. LonP1 (400 nM, monomer) was preincubated with compound (60 min, 25 °C), after which ATP (1 mM final) was added, and reactions were incubated (60 min, 25 °C), quenched and luminescence measured using the ADP-Glo endpoint assay. The error bars indicate the SD of replicate experiments (N = 4). G–I, kinetics of LonP1 inhibition by CDDO derivatives. Reactions were carried out as in D and E and relative fluorescence units (RFU) were measured using a plate reader. H and I, reversibility of CDDO-Me inhibition of LonP1 before and after dialysis. ATP-dependent degradation of FITC-casein was performed as in D and E. H, before dialysis, CDDO-Me inhibition of LonP1 was determined. An aliquot (50 μl) of the reaction mixture (500 μl) containing LonP1 incubated with CDDO-Me (10 μM) or the DMSO control was removed and the kinetics of FITC-casein degradation was assayed. I, after dialysis, the remainder of the reaction mixture in (H) was transferred to a Slide-A Lyzer cassette and dialyzed overnight at 4 °C with Buffer K (50 mM Hepes KOH, pH 8.0, 150 mM NaCl, 10 mM MgOAc₂, 20% glycerol). After dialysis, the kinetics of FITC-casein degradation was assayed as in G. CDDO, 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid.