Identification of novel small molecule activators of nuclear factor‐κb with neuroprotective action via high‐throughput screening

Abstract

Neuronal noncytokine-dependent p50/p65 nuclear factor-κB (the primary NF-κB complex in the brain) activation has been shown to exert neuroprotective actions. Thus neuronal activation of NF-κB could represent a viable neuroprotective target. We have developed a cell-based assay able to detect NF-κB expression enhancement, and through its use we have identified small molecules able to up-regulate NF-κB expression and hence trigger its activation in neurons. We have successfully screened approximately 300,000 compounds and identified 1,647 active compounds. Cluster analysis of the structures within the hit population yielded 14 enriched chemical scaffolds. One high-potency and chemically attractive representative of each of these 14 scaffolds and four singleton structures were selected for follow-up. The experiments described here highlighted that seven compounds caused noncanonical long-lasting NF-κB activation in primary astrocytes. Molecular NF-κB docking experiments indicate that compounds could be modulating NF-κB-induced NF-κB expression via enhancement of NF-κB binding to its own promoter. Prototype compounds increased p65 expression in neurons and caused its nuclear translocation without affecting the inhibitor of NF-κB (I-κB). One of the prototypical compounds caused a large reduction of glutamate-induced neuronal death. In conclusion, we have provided evidence that we can use small molecules to activate p65 NF-κB expression in neurons in a cytokine receptor-independent manner, which results in both long-lasting p65 NF-κB translocation/activation and decreased glutamate neurotoxicity.

Discrete signal transduction pathway utilization by a neuropeptide (PACAP) and a cytokine (TNF-alpha) first messenger in chromaffin cells, inferred from coupled transcriptome-promoter analysis of regulated gene cohorts

“…This is in agreement with the well recognized of role of NFKB in the signal transduction of TNF-alpha in many other systems including brain [4,7,16,24,30,36,40]. In fact, there is a paucity of evidence in the literature for TNF-alpha-initiated signaling proceeding through any other pathway besides one involving NFKB.…”

Minimal NF-κB activity in neurons

“…Based on the extensive literature on neuronal NF-κB activation, we tested the most-studied candidates. Supported by published data (Tamatani et al, 1999, Albensi and Mattson, 2000, Marchetti et al, 2004, Manuvakhova et al, 2011, Shim et al, 2011), the strongest and most consistent activator of NF-κB in neurons was TNFα. We employed multiple assays to demonstrate the response dynamics, and we compared the magnitude of the neuronal response to that in mixed brain cells.…”

Firefly Luciferase in Chemical Biology: A Compendium of Inhibitors, Mechanistic Evaluation of Chemotypes, and Suggested Use As a Reporter

“…For example, examination of assays listed in PubChem indicated that FLuc inhibitors can comprise more than half the hits derived from FLuc-based assays (Auld et al, 2008b) and reports have noted higher enrichments, approaching 98% (Lyssiotis et al, 2009; Younis et al, 2010). Nonetheless, such chemotypes have been considered as lead series, in part based on suggestive secondary assay data, although the target underlying the activity has yet to be identified (Manuvakhova et al, 2011; Xiao et al, 2011). Potent FLuc inhibitors have also been characterized from natural product extracts (Cruz et al, 2011) and have confused the interpretation of virtual screening results (Pang et al, 2011).…”