Discovery and Characterization of 2-Aminobenzimidazole Derivatives as Selective NOD1 Inhibitors

SummaryNLR family proteins play important roles in innate immune response. NOD1 (NLRC1) activates various signaling pathways including NF-κB in response to bacterial ligands. Hereditary polymorphisms in the NOD1 gene are associated with asthma, inflammatory bowel disease, and other disorders. Using a high throughput screening (HTS) assay measuring NOD1-induced NF-κB reporter gene activity, followed by multiple downstream counter screens that eliminated compounds impacting other NF-κB effectors, 2-aminobenzimidazole compounds were identified that selectively inhibit NOD1. Mechanistic studies of a prototypical compound, Nodinitib-1 (ML130; CID-1088438), suggest that these small molecules cause conformational changes of NOD1 in vitro and alter NOD1 subcellular targeting in cells. Altogether, this inaugural class of inhibitors provides chemical probes for interrogating mechanisms regulating NOD1 activity and tools for exploring the roles of NOD1 in various infectious and inflammatory diseases

PPAR-δ is repressed in Huntington's disease, is required for normal neuronal function and can be targeted therapeutically

Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a CAG-polyglutamine repeat expansion in the huntingtin (htt) gene. We found that peroxisome proliferator-activated receptor delta (PPARδ) interacts with htt and that mutant htt represses PPARδ-mediated transactivation. Increased PPARδ transactivation ameliorated mitochondrial dysfunction and improved cell survival of HD neurons. Expression of dominant-negative PPARδ in CNS was sufficient to induce motor dysfunction, neurodegeneration, mitochondrial abnormalities, and transcriptional alterations that recapitulated HD-like phenotypes. Expression of dominant-negative PPARδ specifically in the striatum of medium spiny neurons in mice yielded HD-like motor phenotypes, accompanied by striatal neuron loss. In mouse models of HD, pharmacologic activation of PPAR δ, using the agonist KD3010, improved motor function, reduced neurodegeneration, and increased survival. PPAR δ activation also reduced htt-induced neurotoxicity in vitro and in medium spiny-like neurons generated from human HD stem cells, indicating that PPAR δ activation may be beneficial in individuals with HD and related disorders

Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy

Adolescent Reproductive Knowledge, Attitudes, and Beliefs and Future Fatherhood

BACKGROUND: With a growing focus on the importance of men’s reproductive health--including preconception health--the ways in which young men’s knowledge, attitudes, and beliefs (KAB) predict their reproductive paths are understudied. OBJECTIVE: To determine if reproductive KAB predicts fatherhood status, timing and residency (living with child or not). METHODS: Reproductive KAB and fatherhood outcomes were analyzed from the National Longitudinal Study of Adolescent Health, a 20-year, nationally representative study of individuals from adolescence into adulthood. Four measures of reproductive KAB were assessed during adolescence in Waves I and II. A generalized linear latent and mixed model predicted future fatherhood status (non-father, resident/nonresident father, adolescent father) and timing while controlling for other socio-demographic variables. RESULTS: Of the 10,253 males, 3425 were fathers (686 non-resident/2739 resident) by wave IV. Higher risky sexual behavior scores significantly increased the odds of becoming nonresident father (OR=1.30, p < 0.0001), resident father (OR=1.07, p = 0.007), and adolescent father (OR=1.71, p < 0.0001); higher pregnancy attitudes scores significantly increased the odds of becoming a nonresident father (OR=1.20, p < 0.0001) and resident father (O =1.11, p < 0.0001); higher birth control self-efficacy scores significantly decreased the odds of becoming a nonresident father (O =0.72, p < 0.0001), and adolescent father (OR=0.56, p = 0.01). CONCLUSION: Young men’s KAB in adolescence predicts their future fatherhood and residency status. Strategies that address adolescent males' reproductive KAB are needed in the prevention of unintended reproductive consequences such as early and nonresident fatherhood

Benzodiazepinone Derivatives Protect against Endoplasmic Reticulum Stress-Mediated Cell Death in Human Neuronal Cell Lines

Endoplasmic reticulum (ER) stress causes neuronal dysfunction followed by cell death and is recognized as a feature of many neurodegenerative diseases. Using a phenotypic screen, we recently identified benzodiazepinone derivatives that reduce ER stress-mediated apoptosis in a rat neuronal progenitor cell line (CSM14.1). Herein we describe how structure–activity relationship (SAR) studies around these screening hits led to compounds that display robust cytoprotective activity against thapsigargin-induced ER stress in SH-SY5Y and H4 human neuronal cell lines. We demonstrate that the most potent of these derivatives, compound <b>4hh</b>, inhibits the activation of p38 MAP kinase (p38) and c-Jun N-terminal kinase (JNK), protein kinases that are downstream signal effectors of the unfolded protein response (UPR). Compound <b>4hh</b> specifically protects against thapsigargin-induced cell death and displays no protection against other insults known to induce cellular stress or activate p38. However, compound <b>4hh</b> provides moderate inhibition of p38 activity stimulated by compounds that disrupt calcium homeostasis. Our data indicate that probe compound <b>4hh</b> is a valuable small molecule tool that can be used to investigate the effects of ER stress on human neurons. This approach may provide the basis for the future development of therapeutics for the treatment of neurodegenerative diseases

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