Screening approaches for the identification of Nrf2-Keap1 protein-protein interaction inhibitors targeting hot spot residues.

Protein-protein interactions (PPIs) play a crucial role in most biological processes and are important targets in the development of therapeutic agents. However, small molecule drug discovery that targets PPIs remains very challenging. Targeting hot spot residues is considered the best option for inhibiting such interactions, but there are few examples of how knowledge of hot spots can be used in high throughput screening to find hit compounds. A substrate adaptor protein for a ubiquitin ligase complex, Kelch-like ECH-associated protein 1 (Keap1), negatively modulates the expression of genes involved in cellular protection against oxidative stress. Here, we focused on three arginine hot spot residues in the Keap1 substrate binding pocket (Arg380, Arg415, and Arg483), and screened the carboxylic acid library owned by Japan Tobacco Inc. for compounds that interact with the arginine residues in differential scanning fluorescence assays. Furthermore, we identified several small molecule compounds that specifically bind to the Keap1 Kelch domain hot spots by comparing binding to alanine mutant proteins (R380A, R415A, and R483A) with binding to the wild-type protein using surface plasmon resonance (SPR) screening. These compounds inhibited the protein-protein interaction between the Keap1 Kelch domain and the nuclear factor erythroid 2-related factor 2 (Nrf2) peptide, and the ubiquitination of Nrf2 catalyzed by the Cul3/RINGBox 1 E3 ligase. In addition, the binding mode of one compound (Compound 4) was determined by X-ray crystallography after validation of binding by isothermal titration calorimetry, native mass spectrometry, and nuclear magnetic resonance. Compound 4 had favorable thermodynamic properties, and noncovalently bound to Keap1 with a stoichiometry of 1:1. Our results suggest that Compound 4 could potentially be developed into effective therapeutic or preventive agents for a variety of diseases and conditions such as oxidative stress response, inflammation, and carcinogenesis. We believe that the use of a set of complementary biophysical techniques including the SPR assay with single alanine mutant of hot spots provides opportunities to identify hit compounds for developing inhibitors of PPIs.

Optimization Efforts for Identification of Novel Highly Potent Keap1-Nrf2 Protein-Protein Interaction Inhibitors.

In research focused on protein-protein interaction (PPI) inhibitors, the optimization process to achieve both high inhibitory activity and favorable physicochemical properties remains challenging. Our previous study reported the discovery of novel and bioavailable Keap1-Nrf2 PPI inhibitor 8 which exhibited moderate in vivo activity in rats. In this work, we present our subsequent efforts to optimize this compound. Two distinct approaches were employed, targeting high energy water molecules and Ser602 as "hot spots" from the anchor with good aqueous solubility, metabolic stability, and membrane permeability. Through ligand efficiency (LE)-guided exploration, we identified two novel inhibitors 22 and 33 with good pharmacokinetics (PK) profiles and more potent in vivo activities, which appear to be promising chemical probes among the existing inhibitors.

Methylene acetal formation from 1,2- and 1,3-diols using an O,S-acetal, 1,3-dibromo-5,5-dimethylhydantoin, and BHT.

A mild and efficient method for formation of methylene acetals from 1,2- and 1,3-diols using methoxymethylphenylsulfide, 1,3-dibromo-5,5-dimethylhydantoin (DBDMH), and dibutylhydroxytoluene (BHT) is described. The use of BHT in this process suppresses side reactions and enables high-yielding formation of methylene acetals of various diols, including carbohydrate-type substrates.

Methyl and Fluorine Effects in Novel Orally Bioavailable Keap1-Nrf2 PPI Inhibitor.

Oxidative stress is one of the causes of progression of chronic kidney disease (CKD). Activation of the antioxidant protein regulator Nrf2 by inhibition of the Keap1-Nrf2 protein-protein interaction (PPI) is of interest as a potential treatment for CKD. We report the identification of the novel and weak PPI inhibitor 7 with good physical properties by a high throughput screening (HTS) campaign, followed by structural and computational analysis. The installation of only methyl and fluorine groups successfully provided the lead compound 25, which showed more than 400-fold stronger activity. Furthermore, these dramatic substituent effects can be explained by the analysis of using isothermal titration calorimetry (ITC). Thus, the resulting 25, which exhibited high oral absorption and durability, would be a CKD therapeutic agent because of the dose-dependent manner for up-regulation of the antioxidant protein heme oxigenase-1 (HO-1) in rat kidneys.

Effects of phosphorus substituents on reactions of α-alkoxyphosphonium salts with nucleophiles.

The effects of phosphorus substituents on the reactivity of α-alkoxyphosphonium salts with nucleophiles has been explored. Reactions of α-alkoxyphosphonium salts, prepared from various acetals and tris(o-tolyl)phosphine, with a variety of nucleophiles proceeded efficiently. These processes represent the first examples of high-yielding nucleophilic substitution reactions of α-alkoxyphosphonium salts. The reactivity of these salts was determined by a balance between steric and electronic factors, respectively, represented by cone angles θ and CO stretching frequencies ν (steric and electronic parameters, respectively). In addition, a novel reaction of α-alkoxyphosphonium salts derived from Ph(3)P with Grignard reagents was observed to take place in the presence of O(2) to afford alcohols in good yields. A radical mechanism is proposed for this process that has gained support from isotope-labeling and radical-inhibition experiments.

Direct conversion of acetals to esters with high regioselectivity via O,P-acetals.

A new direct conversion of O,O-acetals to esters via O,P-acetal intermediates was developed. The regioselective cleavage of unsymmetrical cyclic acetals occurred to give the more crowded esters as single isomers.

Method for the efficient synthesis of highly-substituted oxetan- and azetidin-, dihydrofuran- and pyrrolidin-3-ones and its application to the synthesis of (±)-pseudodeflectusin.

Highly substituted four- and five-membered heterocycles were prepared starting with O,P- and N,P-acetals by using a one-pot method involving base induced cyclization and a Horner-Wadsworth-Emmons (HWE) olefination reaction. Divergent synthesis of various heterocycles was achieved by using this method and transformations of the alkenyl group in the products of these processes were exemplified. Finally, a short and efficient synthesis of (±)-pseudodeflectusin based on the new methodology was achieved.

An unusual reaction of α-alkoxyphosphonium salts with Grignard reagents under an O2 atmosphere.

An unusual and novel reaction of α-alkoxyphosphonium salts, generated from O,O-acetals and Ph(3)P, with Grignard reagents under an O(2) atmosphere afforded alcohols in moderate to high yields. It was clarified by isotopic labelling experiments that the reaction proceeded via a novel radical pathway.

Remarkable effect of phosphine on the reactivity of O,P-acetal--efficient substitution reaction of O,P-acetal.

The structure and electronic nature of the phosphine have a significant influence on not only the formation, but also the subsequent transformation of O,P-acetals. The O,P-acetals generated from tris(o-tolyl)phosphine [(o-tol)(3)P] underwent efficient substitution reactions with various nucleophiles.