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1.  Identification and Characterization of Novel Tissue-Nonspecific Alkaline Phosphatase Inhibitors with Diverse Modes of Action 
Journal of Biomolecular Screening  2009;14(7):824-837.
Tissue-nonspecific alkaline phosphatase (TNAP) plays a major role in maintaining a ratio of phosphate to inorganic pyrophosphate (Pi/PPi) in biological fluids that is conducive to controlled skeletal mineralization while preventing inappropriate ectopic calcification. Medial calcification associated with Enpp1 or Ank deficiency or with end–stage renal disease is associated with an increase in TNAP activity in arteries that leads to reduced levels of PPi and increased vascular calcification. Here, we describe in detail a high-throughput screening (HTS) campaign to identify inhibitors of TNAP, performed within the Molecular Library Screening Center Network (MLSCN). A homogeneous luminescent TNAP assay was developed and optimized for identification of compounds with diverse mechanism of action (MOA). The MLSCN compound collection, containing 64,394 molecules at the time of screening, was tested in the assay. Several novel inhibitory scaffold classes were identified and demonstrated to have diverse selectivity and mode of inhibition (MOI) profiles. Representatives of the novel scaffolds exhibited nanomolar potency surpassing the inhibitors known to date.
This paper sets a successful example in which pharmacologically active compounds, with outstanding selectivity in a panel of more than 200 assays, are identified from high throughput screening. Integral to the success of the project were a well-designed compound collection, an industrial-level screening facility and a deep knowledge of target biology that were brought together through the NIH-sponsored Roadmap Initiative.
PMCID: PMC3403534  PMID: 19556612
NIH Roadmap Initiatives; MLSCN; TNAP inhibitors; diverse MOA; compound selectivity
2.  Chemical Biology Strategy Reveals Pathway-Selective Inhibitor of NF-κB Activation Induced by Protein Kinase C 
ACS chemical biology  2010;5(3):287-299.
Dysregulation of NF-κB activity contributes to many autoimmune and inflammatory diseases. At least nine pathways for NF-κB activation have been identified, most of which converge on the IκB kinases (IKKs). Although IKKs represent logical targets for potential drug discovery, chemical inhibitors of IKKs suppress all known NF-κB activation pathways, and thus lack the selectivity required for safe use. A unique NF-κB activation pathway is initiated by protein kinase C (PKC) that is stimulated by antigen receptors and many growth factor receptors. Using a cell-based high throughput screening (HTS) assay and chemical biology strategy, we identified a 2-aminobenzimidazole compound, CID-2858522, which selectively inhibits the NF-κB pathway induced by PKC, operating downstream of PKC but upstream of IKKβ, without inhibiting other NF-κB activation pathways. In human B cells stimulated through surface immunoglobulin, CID-2858522 inhibited NF-κB DNA-binding activity and expression of endogenous NF-κB-dependent target gene, TRAF1. Altogether, as a selective chemical inhibitor of the NF-κB pathway induced by PKC, CID-2858522 serves as a powerful research tool, and may reveal new paths towards therapeutically useful NF-κB inhibitors.
PMCID: PMC2842467  PMID: 20141195

Results 1-2 (2)