Inhibition of Protein Kinase C-Driven Nuclear Factor-κB Activation: Synthesis, Structure−Activity Relationship, and Pharmacological Profiling of Pathway Specific Benzimidazole Probe Molecules

A unique series of biologically active chemical probes that selectively inhibit NF-κB activation induced by protein kinase C (PKC) pathway activators have been identified through a cell-based phenotypic reporter gene assay. These 2-aminobenzimidazoles represent initial chemical tools to be used in gaining further understanding on the cellular mechanisms driven by B and T cell antigen receptors. Starting from the founding member of this chemical series <b>1a</b> (notated in PubChem as CID-2858522), we report the chemical synthesis, SAR studies, and pharmacological profiling of this pathway-selective inhibitor of NF-κB activation

Discovery of ML358, a Selective Small Molecule Inhibitor of the SKN‑1 Pathway Involved in Drug Detoxification and Resistance in Nematodes

Nematodes parasitize ∼1/3 of humans worldwide, and effective treatment via administration of anthelmintics is threatened by growing resistance to current therapies. The nematode transcription factor SKN-1 is essential for development of embryos and upregulates the expression of genes that result in modification, conjugation, and export of xenobiotics, which can promote resistance. Distinct differences in regulation and DNA binding relative to mammalian Nrf2 make SKN-1 a promising and selective target for the development of anthelmintics with a novel mode of action that targets stress resistance and drug detoxification. We report <b>17</b> (<b>ML358</b>), a first in class small molecule inhibitor of the SKN-1 pathway. Compound <b>17</b> resulted from a vanillamine-derived hit identified by high throughput screening that was advanced through analog synthesis and structure–activity studies. Compound <b>17</b> is a potent (IC₅₀ = 0.24 μM, <i>E</i>_max = 100%) and selective inhibitor of the SKN-1 pathway and sensitizes the model nematode <i>C. elegans</i> to oxidants and anthelmintics. Compound <b>17</b> is inactive against Nrf2, the homologous mammalian detoxification pathway, and is not toxic to <i>C. elegans</i> (LC₅₀ > 64 μM) and Fa2N-4 immortalized human hepatocytes (LC₅₀ > 5.0 μM). In addition, <b>17</b> exhibits good solubility, permeability, and chemical and metabolic stability in human and mouse liver microsomes. Therefore, <b>17</b> is a valuable probe to study regulation and function of SKN-1 <i>in vivo</i>. By selective targeting of the SKN-1 pathway, <b>17</b> could potentially lead to drug candidates that may be used as adjuvants to increase the efficacy and useful life of current anthelmintics

Identification of Inhibitors of NOD1-Induced Nuclear Factor-κB Activation

NOD1 (nucleotide-binding oligomerization domain 1) protein is a member of the NLR (NACHT and leucine rich repeat domain containing proteins) protein family, which plays a key role in innate immunity as a sensor of specific microbial components derived from bacterial peptidoglycans and induction of inflammatory responses. Mutations in NOD proteins have been associated with various inflammatory diseases that affect NF-κB (nuclear factor κB) activity, a major signaling pathway involved in apoptosis, inflammation, and immune response. A luciferase-based reporter gene assay was utilized in a high-throughput screening program conducted under the NIH-sponsored Molecular Libraries Probe Production Center Network program to identify the active scaffolds. Herein, we report the chemical synthesis, structure–activity relationship studies, downstream counterscreens, secondary assay data, and pharmacological profiling of the 2-aminobenzimidazole lead (compound <b>1c</b>, ML130) as a potent and selective inhibitor of NOD1-induced NF-κB activation

Discovery of a Plasmodium falciparum Glucose-6-phosphate Dehydrogenase 6‑phosphogluconolactonase Inhibitor (<i>R</i>,<i>Z</i>)‑<i>N</i>‑((1-Ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro‑2<i>H</i>‑benzo[<i>b</i>][1,4]thiazine-6-carboxamide (ML276) That Reduces Parasite Growth in Vitro

A high-throughput screen of the NIH’s MLSMR collection of ∼340000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (<i>Pf</i>G6PD). <i>Pf</i>G6PD is important for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human orthologue. The reaction catalyzed by glucose-6-phosphate dehydrogenase (G6PD) is the first, rate-limiting step in the pentose phosphate pathway (PPP), a key metabolic pathway sustaining anabolic needs in reductive equivalents and synthetic materials in fast-growing cells. In P. falciparum, the bifunctional enzyme glucose-6-phosphate dehydrogenase-6-phosphogluconolactonase (<i>Pf</i>GluPho) catalyzes the first two steps of the PPP. Because P. falciparum and infected host red blood cells rely on accelerated glucose flux, they depend on the G6PD activity of <i>Pf</i>GluPho. The lead compound identified from this effort, (<i>R</i>,<i>Z</i>)-<i>N</i>-((1-ethylpyrrolidin-2-yl)­methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2<i>H</i>-benzo­[<i>b</i>]­[1,4]­thiazine-6-carboxamide, <b>11</b> (ML276), is a submicromolar inhibitor of <i>Pf</i>G6PD (IC₅₀ = 889 nM). It is completely selective for the enzyme’s human isoform, displays micromolar potency (IC₅₀ = 2.6 μM) against P. falciparum in culture, and has good drug-like properties, including high solubility and moderate microsomal stability. Studies testing the potential advantage of inhibiting <i>Pf</i>G6PD in vivo are in progress

Discovery of Small Molecule Kappa Opioid Receptor Agonist and Antagonist Chemotypes through a HTS and Hit Refinement Strategy

Herein we present the outcome of a high throughput screening (HTS) campaign-based strategy for the rapid identification and optimization of selective and general chemotypes for both kappa (κ) opioid receptor (KOR) activation and inhibition. In this program, we have developed potent antagonists (IC₅₀ < 120 nM) or agonists of high binding affinity (<i>K</i>_i < 3 nM). In contrast to many important KOR ligands, the compounds presented here are highly modular, readily synthesized, and, in most cases, achiral. The four new chemotypes hold promise for further development into chemical tools for studying the KOR or as potential therapeutic lead candidates

Discovery of ML314, a Brain Penetrant Nonpeptidic β‑Arrestin Biased Agonist of the Neurotensin NTR1 Receptor

The neurotensin 1 receptor (NTR1) is an important therapeutic target for a range of disease states including addiction. A high-throughput screening campaign, followed by medicinal chemistry optimization, led to the discovery of a nonpeptidic β-arrestin biased agonist for NTR1. The lead compound, 2-cyclopropyl-6,7-dimethoxy-4-(4-(2-methoxyphenyl)-piperazin-1-yl)­quinazoline, <b>32</b> (ML314), exhibits full agonist behavior against NTR1 (EC₅₀ = 2.0 μM) in the primary assay and selectivity against NTR2. The effect of <b>32</b> is blocked by the NTR1 antagonist SR142948A in a dose-dependent manner. Unlike peptide-based NTR1 agonists, compound <b>32</b> has no significant response in a Ca²⁺ mobilization assay and is thus a biased agonist that activates the β-arrestin pathway rather than the traditional G_<i>q</i> coupled pathway. This bias has distinct biochemical and functional consequences that may lead to physiological advantages. Compound <b>32</b> displays good brain penetration in rodents, and studies examining its in vivo properties are underway