N-Biphenyl Pyrrolinones and Dibenzofurans as RNA-Binding Protein LIN28 Inhibitors Disrupting the LIN28-Let-7 Interaction.

The RNA-binding protein LIN28 is a regulator of miRNA let-7 biogenesis. Inhibitors of LIN28 are highly sought after given the central role that LIN28 plays in tumorigenesis and development of cancer stem cells as well as LIN28's association with poor clinical prognosis. Although LIN28 inhibitors of different scaffolds have been reported, the potential of most LIN28 inhibiting small molecules was not fully explored since very limited structure-activity relationship (SAR) studies have been performed. We previously identified trisubstituted pyrrolinones as a new class of LIN28 inhibitors disrupting the LIN28-let-7 interaction. Here, we performed extensive SAR by evaluating 95 small molecules and identified new trisubstituted pyrrolinones featuring either an N-biphenyl or N-dibenzofuran substituent, overthrowing the existing conclusion that a salicylic acid moiety is indispensable for activity. Exchange of the negatively charged salicylic acid moiety in LIN28 inhibitors with a heterocyclic substituent is beneficial for membrane permeability, leading to increased activity in a cellular assay, and will potentially reduce toxicity.

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.

Designing Selective Drug-like Molecular Glues for the Glucocorticoid Receptor/14-3-3 Protein-Protein Interaction.

The ubiquitously expressed glucocorticoid receptor (GR) is a nuclear receptor that controls a broad range of biological processes and is activated by steroidal glucocorticoids such as hydrocortisone or dexamethasone. Glucocorticoids are used to treat a wide variety of conditions, from inflammation to cancer but suffer from a range of side effects that motivate the search for safer GR modulators. GR is also regulated outside the steroid-binding site through protein-protein interactions (PPIs) with 14-3-3 adapter proteins. Manipulation of these PPIs will provide insights into noncanonical GR signaling as well as a new level of control over GR activity. We report the first molecular glues that selectively stabilize the 14-3-3/GR PPI using the related nuclear receptor estrogen receptor α (ERα) as a selectivity target to drive design. These 14-3-3/GR PPI stabilizers can be used to dissect noncanonical GR signaling and enable the development of novel atypical GR modulators.

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.

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.