The
mitochondrial protein LonP1 is an
ATP-dependent protease that mitigates cell stress and calibrates mitochondrial metabolism and energetics. Biallelic mutations in the LONP1 gene are known to cause a broad spectrum of diseases, and LonP1 dysregulation is also implicated in
cancer and age-related disorders. Despite the importance of LonP1 in health and disease, specific inhibitors of this
protease are unknown. Here, we demonstrate that 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic
acid (
CDDO) and its -methyl and -
imidazole derivatives reversibly inhibit LonP1 by a noncompetitive mechanism, blocking
ATP-hydrolysis and thus proteolysis. By contrast, we found that
CDDO-
anhydride inhibits the LonP1
ATPase competitively. Docking of
CDDO derivatives in the cryo-EM structure of LonP1 shows these compounds bind a hydrophobic pocket adjacent to the
ATP-binding site. The binding site of
CDDO derivatives was validated by amino acid substitutions that increased LonP1 inhibition and also by a pathogenic mutation that causes cerebral, ocular, dental, auricular and skeletal (
CODAS) syndrome, which ablated inhibition.
CDDO failed to inhibit the
ATPase activity of the purified
26S proteasome, which like LonP1 belongs to the
AAA+ superfamily of
ATPases Associated with diverse cellular Activities, suggesting that
CDDO shows selectivity within this family of
ATPases. Furthermore, we show that noncytotoxic concentrations of
CDDO derivatives in cultured cells inhibited LonP1, but not the
26S proteasome. Taken together, these findings provide insights for future development of LonP1-specific inhibitors with chemotherapeutic potential.