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

Synthetic
triterpenoids including CDDO, its methyl ester (CDDO-Me,
bardoxolone methyl), and its imidazolide (CDDO-Im) enhance Nrf2-mediated
antioxidant 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 eight different
cysteines, and acyl adducts with lysine and several tyrosine residues.
Modeling studies suggest that the Tyr 85 adduct stabilizes the Keap1-Cul3
complex, thereby enhancing the potency of CDDO-Im.