A Comparative Assessment Study of Known Small-Molecule Keap1−Nrf2 Protein–Protein Interaction Inhibitors: Chemical Synthesis, Binding Properties, and Cellular Activity

Abstract

Inhibiting the protein-protein interaction (PPI) between the transcription factor Nrf2 and its repressor protein Keap1 has emerged as a promising strategy to target oxidative stress in diseases, including CNS disorders. Numerous non-covalent small-molecule Keap1-Nrf2 PPI inhibitors have been reported to date, but many feature suboptimal physicochemical properties for permeating the blood-brain barrier, while others contain problematic structural moieties. Here, we present the first side-by-side assessment of all reported Keap1-Nrf2 PPI inhibitor classes using fluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR)-and further evaluate the compounds in an NQO1 induction cell assay and in counter tests for non-boronate ester building block 41 as the crucial carbon skeleton-building step. Synthesis of the enol triflate using the one-step NaHMDS-mediated enolization/PhNTf2-induced trapping procedure reported in the patent application 39 was not efficient in our hands, giving low yield (43% vs. 88% reported in literature 39) and significant byproduct formation. We found that using a freshly-made LiHMDS as an alternative base gave a cleaner reaction and excellent yield (quantitative). The converging SM reaction step between 38 and 41 gave several wellknown by-products, including the boronic acid, aryl boronate homo-coupling product and protodeboronation product, but could still afford the desired cross-coupling product 42 in good yield (69% vs. 33% reported in literature 39). Catalytic hydrogenation to deprotect the carboxylic acid and reduce the alkene double bond diastereoselectively furnished only the cis-cyclohexane 43 in accordance with literature; 39 this was revealed by nuclear Overhauser effect (NOE) NMR (Supporting Information Figure S1). This facial selectivity can be explained by a steric directing effect of the carboxybenzyl group. The cyclohexane carboxylic acid of 43 was finally coupled with 2-butylpyrrolidine and the pyrazole carboxylic acid deprotected to give 10 as a mixture of four stereoisomers. Attempted separation of the two diastereoisomers by preparative HPLC was unsuccessful. In the patent application, purification by HPLC is reported to give two different fractions, each containing all four stereoisomers in slightly different proportions, of which one was directly tested as a mixture. 39 Having this literature result as a reference point, we did not proceed with further purification of 10.

Bardoxolone conjugation enables targeted protein degradation of BRD4

“…In summary, the first bifunctional protein degrader (PROTAC) based on the known KEAP1 ligand bardoxolone (CDDO) is reported. 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] .…”

Structural basis of the anti-ageing effects of polyphenolics: mitigation of oxidative stress

“…Compounds of this class can increase NRF2 activity in vivo [79]. Furthermore, while some structures do not contain, nor have the propensity to form, α, β-unsaturated carbonyls, ketones themselves (for example in flavanones) are electrophilic and could potentially react with KEAP1 or other sensor-type cysteine proteins forming the respective hemithioacetals; additionally, compounds may also act upon the NRF2/KEAP1 axis in a non-covalent manner [80][81][82][83][84][85].…”

Targeting the Redox Landscape in Cancer Therapy

“…Due to insufficient specificity of these covalent modifiers and low bioavailability and cell permeability of the employed peptides, current efforts focus on alternative classes of compounds. Recent studies show successful induction of Nrf2 through targeting of the Nrf2-Keap1 protein-protein interaction with non-covalently interacting small molecules [180]. One example is the synthetic oleanane triterpenoid compound RTA 405, which showed antitumor activity in cell culture [76,77,181].…”