Inhibition of mitochondrial LonP1 protease by allosteric blockade of ATP binding and hydrolysis via CDDO and its derivatives

Jae Lee,Tadashi Honda,Kamal Singh,Ashutosh K Pandey,Sundararajan Venkatesh,Jayapalraja Thilagavathi,Carolyn K Suzuki

doi:10.1016/j.jbc.2022.101719

Abstract

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.

Highlights

Mitochondrial LonP1 is a cell-stress response protease that selectively eliminates misassembled or damaged proteins [1,2,3], and degrades certain rate-limiting proteins regulating mitochondrial metabolism and energetics [4,5,6,7]
We demonstrate that CDDO derivatives (Fig. 1A) inhibited the ATP-dependent protease activity of LonP1 as shown by degradation of fluorescently-labeled casein (FITC-casein) (Fig. 1 D, E and G), and the ATPase activity as shown by endpoint assays (Fig. 1F) and continuous enzyme-coupled assays (Fig. 2 and Fig. S3)
A pentacyclic triterpenoid enoxolone, lacking an electron-withdrawing group, did not inhibit the LonP1 ATPase (Fig. 1F). These results show that the electron-withdrawing moiety of CDDO derivatives is crucial for inhibiting LonP1

Summary

INTRODUCTION

Mitochondrial LonP1 is a cell-stress response protease that selectively eliminates misassembled or damaged proteins [1,2,3], and degrades certain rate-limiting proteins regulating mitochondrial metabolism and energetics [4,5,6,7]. Our results show that CDDO derivatives bind at two non-overlapping sites within a hydrophobic pocket contiguous with a channel lined by polar residues forming salt bridges, which is adjacent to the ATP/ADP-binding site The geometry of this binding site was scrutinized by engineered amino acid substitutions within the pockets that led to increased inhibition by CDDO, and by a naturally occurring pathogenic missense LonP1 mutation that blocked inhibition. We showed that CDDO does not inhibit the ATPase activity of purified 26S proteasome, and non-cytotoxic concentrations of CDDO derivatives in cultured cells blocked LonP1 but not the 26S proteasome Taken together, these findings demonstrate that CDDO derivatives inhibit LonP1 by a new mechanism and reveal the topological properties of an unidentified compound binding site, which can be exploited for developing chemical probes and chemotherapeutic agents to target this essential cell stress response regulator

RESULTS

DISCUSSION

EXPERIMENTAL PROCEDURES