Design and Synthesis of Irreversible Analogues of Bardoxolone Methyl for the Identification of Pharmacologically Relevant Targets and Interaction Sites

Abstract

Semisynthetic triterpenoids such as bardoxolone methyl (methyl-2-cyano 3,12-dioxooleano-1,9-dien-28-oate; CDDO-Me) (4) are potent inducers of antioxidant and anti-inflammatory signaling pathways, including those regulated by the transcription factor Nrf2. However, the reversible nature of the interaction between triterpenoids and thiols has hindered attempts to identify pharmacologically relevant targets and characterize the sites of interaction. Here, we report a shortened synthesis and SAR profiling of 4, enabling the design of analogues that react irreversibly with model thiols, as well as the model protein glutathione S-transferase P1, in vitro. We show that one of these analogues, CDDO-epoxide (13), is comparable to 4 in terms of cytotoxicity and potency toward Nrf2 in rat hepatoma cells and stably modifies specific cysteine residues (namely, Cys-257, -273, -288, -434, -489, and -613) within Keap1, the major repressor of Nrf2, both in vitro and in living cells. Supported by molecular modeling, these data demonstrate the value of 13 for identifying site(s) of interaction with pharmacologically relevant targets and informing the continuing development of triterpenoids as novel drug candidates.

Bardoxolone conjugation enables targeted protein degradation of BRD4

“…While robust, proteasome-dependent degradation of BRD4 was observed, a number of mechanistic questions remain: firstly, CDDO is not known to bind the Kelch domain of the KEAP1/Cul3 complex, the key area for recognition of Nrf2 and where degrader 44 , and multiple small molecule inhibitors presumably bind [45][46][47][48] . While a crystal structure of CDDO bound to Cys-151 of the BTB domain of KEAP1 has been solved, various reports have also posited that multiple cysteines are targeted by this class of compounds during the Nrf2 activation process in cells [49][50][51][52][53] . This raises the question how (or if) a CDDO-based PROTAC can mechanistically induce neosubstrate degradation in a KEAP1-dependent manner.…”

“…Secondly, while bardoxolone is thought to activate Nrf2 through the targeting of reactive cysteines on KEAP1, it also interacts with additional pharmacological targets, including IKKb which modulates NF-kB signaling 54 , mTOR 55 , and others 55 , 56 . It should be noted that the direct detection of all proteome-wide targets of CDDO by pulldown studies has proven challenging given the highly reversible nature of its cysteine interactions 30 , 50 , 53 , 54 – 56 . Thus, we cannot rule out at this moment that the degradation observed here may be due to one (or more) other cullin-family E3 ligases that are targeted by CDDO–JQ1.…”

Keap1-Nrf2 signaling activation by Bardoxolone-methyl ameliorates high glucose-induced oxidative injury in human umbilical vein endothelial cells

“…BARD can induce Nrf2 signaling cascade activation by releasing Nrf2 from Keap1 [ 21 , 22 ]. A co-immunoprecipitation (Co-IP) assay was carried out in cultured HUVECs.…”

“…BARD activates Nrf2 signaling cascade through disrupting Nrf2-Keap1 association and inhibiting Nrf2 protein degradation [ 21 – 25 ]. We therefore proposed that Nrf2 activation by Keap1 silencing should mimic BARD-induced cytoprotection in HG-stimulated HUVECs.…”

“…Bardoxolone-methyl (BARD) is a synthetic triterpenoid [ 21 – 25 ]. BARD has displayed antioxidant, anti-inflammatory, anti-proliferative and anti-fibrotic activities under experimental and clinical settings [ 21 – 25 ]. BARD is an extremely efficient activator of Nrf2.…”