CDDO-imidazolide Targets Multiple Amino Acid Residues on the Nrf2 Adaptor, Keap1

Abstract

Synthetic triterpenoids including CDDO, its methyl ester (CDDO-Me, bardoxolone methyl), and its imidazolide (CDDO-Im) enhance Nrf2-mediated anti-oxidant and anti-inflammatory activity in many diseases by reacting with thiols on the adaptor protein, Keap1. Unlike monofunctional CDDO-Me, the bifunctional analog, CDDO-Im, has a second reactive site (imidazolide) and can covalently bind to amino acids other than cysteine on target proteins such as glutathione S-transferase pi (GSTP), serum albumin, or Keap1. Here we show for the first time that bifunctional CDDO-Im (in contrast to CDDO-Me), as low as 50 nM, can covalently transacylate arginine and serine residues in GSTP and cross link them to adjacent cysteine residues. Moreover, we show that CDDO-Im binds covalently to Keap1, by forming permanent Michael adducts with 8 different cysteines, and acyl adducts with lysine and several tyrosine residues. Modeling studies suggest the Tyr 85 adduct stabilizes the Keap1-Cul3 complex, thereby enhancing the potency of CDDO-Im.