Development of Noncovalent Small-Molecule Keap1-Nrf2 Inhibitors by Fragment-Based Drug Discovery.

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 (Ki = 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.

Deconstructing Noncovalent Kelch-like ECH-Associated Protein 1 (Keap1) Inhibitors into Fragments to Reconstruct New Potent Compounds.

Targeting the protein-protein interaction (PPI) between nuclear factor erythroid 2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) is a potential therapeutic strategy to control diseases involving oxidative stress. Here, six classes of known small-molecule Keap1-Nrf2 PPI inhibitors were dissected into 77 fragments in a fragment-based deconstruction reconstruction (FBDR) study and tested in four orthogonal assays. This gave 17 fragment hits of which six were shown by X-ray crystallography to bind in the Keap1 Kelch binding pocket. Two hits were merged into compound 8 with a 220-380-fold stronger affinity (Ki = 16 µM) relative to the parent fragments. Systematic optimization resulted in several novel analogues with Ki values of 0.04-0.5 µM, binding modes determined by X-ray crystallography, and enhanced microsomal stability. This demonstrates how FBDR can be used to find new fragment hits, elucidate important ligand-protein interactions, and identify new potent inhibitors of the Keap1-Nrf2 PPI.

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

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 central nervous system (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, thermal shift assay, and surface plasmon resonance-and further evaluate the compounds in an NQO1 induction cell assay and in counter tests for nonspecific activities. Surprisingly, half of the compounds were inactive or deviated substantially from reported activities, while we confirm the cross-assay activities for others. Through this study, we have identified the most promising Keap1-Nrf2 inhibitors that can serve as pharmacological probes or starting points for developing CNS-active Keap1 inhibitors.

A homeostatic model of IkappaB metabolism to control constitutive NF-kappaB activity.

Cellular signal transduction pathways are usually studied following administration of an external stimulus. However, disease-associated aberrant activity of the pathway is often due to misregulation of the equilibrium state. The transcription factor NF-kappaB is typically described as being held inactive in the cytoplasm by binding its inhibitor, IkappaB, until an external stimulus triggers IkappaB degradation through an IkappaB kinase-dependent degradation pathway. Combining genetic, biochemical, and computational tools, we investigate steady-state regulation of the NF-kappaB signaling module and its impact on stimulus responsiveness. We present newly measured in vivo degradation rate constants for NF-kappaB-bound and -unbound IkappaB proteins that are critical for accurate computational predictions of steady-state IkappaB protein levels and basal NF-kappaB activity. Simulations reveal a homeostatic NF-kappaB signaling module in which differential degradation rates of free and bound pools of IkappaB represent a novel cross-regulation mechanism that imparts functional robustness to the signaling module.

Non-covalent Small-Molecule Kelch-like ECH-Associated Protein 1-Nuclear Factor Erythroid 2-Related Factor 2 (Keap1-Nrf2) Inhibitors and Their Potential for Targeting Central Nervous System Diseases.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) has a protective effect against oxidative stress and plays a major role in inflammation and central nervous system (CNS) diseases. Inhibition of the protein-protein interaction (PPI) between Nrf2 and its repressor protein, Kelch-like ECH-associated protein 1 (Keap1), leads to translocation of Nrf2 from the cytosol to the nucleus and expression of detoxifying antioxidant enzymes. To date, several non-covalent small-molecule Keap1-Nrf2 inhibitors have been identified; however, many of them contain carboxylic acids and are rather large in size, which likely prevents or decreases CNS permeability. This Perspective describes current small-molecule Keap1-Nrf2 inhibitors with experimental evidence for the ability to inhibit the Keap1-Nrf2 interaction by binding to Keap1 in a non-covalent manner. Binding data, biostructural studies, and biological activity are summarized for the inhibitors, and their potential as CNS tool compounds is discussed by analyzing physicochemical properties, including CNS multiparameter optimization (MPO) scoring algorithms. Finally, several strategies for identifying CNS-targeting Keap1 inhibitors are described.

Do Correlates of Pain-Related Stoicism and Cautiousness Differ in Younger and Older People With Advanced Cancer?

Age differences are not evident in pain-related stoicism and cautiousness in people with cancer pain. Little is known about the factors associated with these pain-related attitudes or age-related patterns in these associations. The present cross-sectional study investigated the biopsychosocial correlates of the attitudes in younger and older patients with advanced cancer. Pain-related stoicism (fortitude, concealment, superiority) and cautiousness (self-doubt, reluctance) were assessed using the Pain Attitudes Questionnaire-Revised (PAQ-R). Participants, 155 younger (younger than 60 years old) and 114 older (60 years old or older) patients with advanced cancer completed the PAQ-R and measures of sociodemographic and medical characteristics, pain intensity, cognitive-affective pain-related responses, physical functioning, psychological distress and well-being, and psychosocial functioning. Backwards regression analyses identified correlates for each PAQ-R factor separately for younger and older patients. Activity engagement was a frequent correlate, but its relationship with concealment was the only association common to both age groups. Younger and older patients exhibited different avoidance-related constructs suggesting relational challenges in the former group (avoidant attachment) and intrapersonal fear in the latter (cognitive avoidance). Medical correlates also showed age differences: younger patients showed symptom-focused correlates, whereas older patients showed aging-related correlates. Findings support a biopsychosocial framework of cancer-pain adaptation incorporating a lifespan-developmental perspective. PERSPECTIVE: To our knowledge, this article is the first to identify biopsychosocial correlates of stoic and cautious attitudes toward cancer pain in younger and older patients with advanced cancer. Findings highlight possible age-related motivations for greater pain-related stoicism or cautiousness and can potentially inform interventions addressing challenges in cancer-pain adaptation in advanced cancer.

Psychometric Evaluation of the Pain Attitudes Questionnaire-Revised for People With Advanced Cancer.

Pain-related stoicism and cautiousness are theorized to be more prevalent in older than younger patients and to lead to greater pain under-reporting and consequently inadequate pain management in older patients. The Pain Attitudes Questionnaire-Revised (PAQ-R), which measures 5 pain-related stoicism (fortitude, concealment, superiority) and cautiousness (self-doubt, reluctance) factors in chronic pain, can help test this hypothesis in advanced cancer but requires validation. We conducted a psychometric evaluation of the PAQ-R in 155 younger (younger than 60 years) and 114 older (aged 60 years and older) patients with advanced cancer. Participants showed disagreement with self-doubt items and floor effects with the subscale. Confirmatory factor analyses revealed good fit of the PAQ-R's 5 factors to younger and older groups' data but collinearity between fortitude and concealment. Multisample confirmatory factor analyses supported partial scalar invariance between age groups. Few hypothesized age-related differences were observed. Younger patients reported higher superiority scores than older patients. Whereas older patients showed greater fortitude and superiority with lower average pain intensity, younger patients showed greater concealment or fortitude with greater worst and average pain intensity. Furthermore, whereas older patients displayed greater superiority with lower interference in relations with others, younger patients displayed greater concealment and superiority with greater interference in walking ability and greater concealment and self-doubt with more interference in relations with others. Cross-validation of the PAQ-R's factor structure and identification of pathways to the factors and effect on pain-related outcomes using multivariate approaches are warranted. PERSPECTIVE: This article presents the psychometric properties of a measure of 2 particular pain-related attitudes. The measure can help clarify whether these attitudes adversely influence pain reporting in older patients with advanced cancer as hypothesized and, in turn, explain the inadequate pain management frequently reported with this clinical group.