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.

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Reactive oxygen species (ROS) are produced predominantly by the mitochondrial electron transport chain and by NADPH oxidases in peroxisomes and in the endoplasmic reticulum. The antioxidative defense counters overproduction of ROS with detoxifying enzymes and molecular scavengers, for instance, superoxide dismutase and glutathione, in order to restore redox homeostasis. Mutations in the redox landscape can induce carcinogenesis, whereas increased ROS production can perpetuate cancer development. Moreover, cancer cells can increase production of antioxidants, leading to resistance against chemo- or radiotherapy. Research has been developing pharmaceuticals to target the redox landscape in cancer. For instance, inhibition of key players in the redox landscape aims to modulate ROS production in order to prevent tumor development or to sensitize cancer cells in radiotherapy. Besides the redox landscape of a single cell, alternative strategies take aim at the multi-cellular level. Extracellular vesicles, such as exosomes, are crucial for the development of the hypoxic tumor microenvironment, and hence are explored as target and as drug delivery systems in cancer therapy. This review summarizes the current pharmaceutical and experimental interventions of the cancer redox landscape.

Targeting the protein–protein interaction (PPI) between the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and its repressor, Kelch-like ECH-associated protein 1 (Keap1), constitutes a promising strategy for treating diseases involving oxidative stress and inflammation. Here, a fragment-based drug discovery (FBDD) campaign resulted in novel, high-affinity (K i = 280 nM), and cell-active noncovalent small-molecule Keap1-Nrf2 PPI inhibitors. We screened 2500 fragments using orthogonal assaysfluorescence polarization (FP), thermal shift assay (TSA), and surface plasmon resonance (SPR)and validated the hits by saturation transfer difference (STD) NMR, leading to 28 high-priority hits. Thirteen co-structures showed fragments binding mainly in the P4 and P5 subpockets of Keap1’s Kelch domain, and three fluorenone-based fragments featuring a novel binding mode were optimized by structure-based drug discovery. We thereby disclose several fragment hits, including their binding modes, and show how FBDD can be performed to find new small-molecule Keap1-Nrf2 PPI inhibitors.

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Drug design of protein kinase inhibitors is now greatly enabled by thousands of publicly available X-ray structures, extensive ligand binding data, and optimized scaffolds coming off patent. The extensive data begin to enable design against a spectrum of targets (polypharmacology); however, the data also reveal heterogeneities of structure, subtleties of chemical interactions, and apparent inconsistencies between diverse data types. As a result, incorporation of all relevant data requires expert choices to combine computational and informatics methods, along with human insight. Here we consider polypharmacological targeting of protein kinases ALK, MET, and EGFR (and its drug resistant mutant T790M) in non small cell lung cancer as an example. Both EGFR and ALK represent sources of primary oncogenic lesions, while drug resistance arises from MET amplification and EGFR mutation. A drug which inhibits these targets will expand relevant patient populations and forestall drug resistance. Crizotinib co-targets ALK and MET. Analysis of the crystal structures reveals few shared interaction types, highlighting proton-arene and key CH–O hydrogen bonding interactions. These are not typically encoded into molecular mechanics force fields. Cheminformatics analyses of binding data show EGFR to be dissimilar to ALK and MET, but its structure shows how it may be co-targeted with the addition of a covalent trap. This suggests a strategy for the design of a focussed chemical library based on a pan-kinome scaffold. Tests of model compounds show these to be compatible with the goal of ALK, MET, and EGFR polypharmacology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13321-017-0229-8) contains supplementary material, which is available to authorized users.

Virtual screening methods are now widely used in early stages of drug discovery, aiming to rank potential inhibitors. However, any practical ligand set (of active or inactive compounds) chosen for deriving new virtual screening approaches cannot fully represent all relevant chemical space for potential new compounds. In this study, we have taken a retrospective approach to evaluate virtual screening methods for the leukemia target kinase ABL1 and its drug-resistant mutant ABL1-T315I. ‘Dual active’ inhibitors against both targets were grouped together with inactive ligands chosen from different decoy sets and tested with virtual screening approaches with and without explicit use of target structures (docking). We show how various scoring functions and choice of inactive ligand sets influence overall and early enrichment of the libraries. Although ligand-based methods, for example principal component analyses of chemical properties, can distinguish some decoy sets from active compounds, the addition of target structural information via docking improves enrichment, and explicit consideration of multiple target conformations (i.e. types I and II) achieves best enrichment of active versus inactive ligands, even without assuming knowledge of the binding mode. We believe that this study can be extended to other therapeutically important kinases in prospective virtual screening studies.