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1.  Inhibition of Hematopoietic Protein Tyrosine Phosphatase Augments and Prolongs ERK1/2 and p38 Activation 
ACS Chemical Biology  2011;7(2):367-377.
The hematopoietic protein tyrosine phosphatase (HePTP) is implicated in the development of blood cancers through its ability to negatively regulate the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Small-molecule modulators of HePTP activity may become valuable in treating hematopoietic malignancies such as T cell acute lymphoblastic leukemia (T-ALL) and acute myelogenous leukemia (AML). Moreover, such compounds will further elucidate the regulation of MAPKs in hematopoietic cells. Although transient activation of MAPKs is crucial for growth and proliferation, prolonged activation of these important signaling molecules induces differentiation, cell cycle arrest, cell senescence, and apoptosis. Specific HePTP inhibitors may promote the latter and thereby may halt the growth of cancer cells. Here, we report the development of a small molecule that augments ERK1/2 and p38 activation in human T cells, specifically by inhibiting HePTP. Structure-activity relationship analysis, in silico docking studies, and mutagenesis experiments reveal how the inhibitor achieves selectivity for HePTP over related phosphatases by interacting with unique amino acid residues in the periphery of the highly conserved catalytic pocket. Importantly, we utilize this compound to show that pharmacological inhibition of HePTP not only augments, but also prolongs activation of ERK1/2 and, especially, p38. Moreover, we present similar effects in leukocytes from mice intraperitoneally injected with the inhibitor at doses as low as 3 mg/kg. Our results warrant future studies with this probe compound that may establish HePTP as a new drug target for acute leukemic conditions.
doi:10.1021/cb2004274
PMCID: PMC3288537  PMID: 22070201
2.  Identification of Inhibitors of NOD1-Induced Nuclear Factor-κB Activation 
ACS Medicinal Chemistry Letters  2011;2(10):780-785.
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 1c, ML130) as a potent and selective inhibitor of NOD1-induced NF-κB activation.
doi:10.1021/ml200158b
PMCID: PMC3193285  PMID: 22003428
NOD1; NF-κB activation; 2-aminobenzimidazole; hit-to-probe; ML130; MLPCN
3.  Potent, Selective, and Orally Available Benzoisothiazolone Phosphomannose Isomerase Inhibitors as Probes for Congenital Disorder of Glycosylation Ia 
Journal of medicinal chemistry  2011;54(10):3661-3668.
We report the discovery and validation of a series of benzoisothiazolones as potent inhibitors of phosphomannose isomerase (PMI), an enzyme which converts mannose-6-phosphate (Man-6-P) into fructose-6-phosphate (Fru-6-P), and more importantly, competes with phosphomannomutase 2 (PMM2) for Man-6-P, diverting this substrate from critical protein glycosylation events. In Congenital Disorder of Glycosylation type Ia, PMM2 activity is compromised, thus PMI inhibition is a potential strategy for the development of therapeutics. High-throughput screening (HTS) and subsequent chemical optimization led to the identification of a novel class of benzoisothiazolones as potent PMI inhibitors having little or no PMM2 inhibition. Two complimentary synthetic routes were developed enabling the critical structural requirements for activity to be determined, and the compounds were subsequently profiled in biochemical and cellular assays to assess efficacy. The most promising compounds were also profiled for bioavailability parameters including metabolic stability, plasma stability, and permeability. The pharmacokinetic profile of a representative of this series was also assessed, demonstrating the potential of this series for in vivo efficacy when dosed orally in disease models.
doi:10.1021/jm101401a
PMCID: PMC3437750  PMID: 21539312
4.  Discovery and characterization of 2-aminobenzimidazole derivatives as selective NOD1 inhibitors 
Chemistry & biology  2011;18(7):825-832.
NLR 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 pathways, 2-aminobenzimidazole compounds were identified that selectively inhibit NOD1. Mechanistic studies of a prototypical compound, Nodinitib-1 (ML130; CID-1088438), suggest 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.
doi:10.1016/j.chembiol.2011.06.009
PMCID: PMC3152441  PMID: 21802003
NOD1; NOD2; NLR; NF-κB; HTS
5.  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.
doi:10.1177/1087057109338517
PMCID: PMC3403534  PMID: 19556612
NIH Roadmap Initiatives; MLSCN; TNAP inhibitors; diverse MOA; compound selectivity
6.  Inhibition of Bfl-1 with N-Aryl Maleimides 
High throughput screening of 66,000 compounds using competitive binding of peptides comprising the BH3 domain to anti-apoptotic Bfl-1 led to the identification of fourteen validated “hits” as inhibitors of Bfl-1. N-Aryl maleimide 1 was among the validated “hits”. A chemical library encompassing over 280 analogs of 1 was prepared following a two-step synthesis. Structure-activity studies for inhibition of Bfl-1 by analogs of N-aryl maleimide 1 revealed a preference for electron-withdrawing substituents in the N-aryl ring and hydrophilic amines appended to the maleimide core. Inhibitors of Bfl-1 are potential development candidates for anti-cancer therapeutics.
doi:10.1016/j.bmcl.2010.09.046
PMCID: PMC2987701  PMID: 20933419
Bfl-1 inhibitors; N-aryl maleimides; high-throughput screening; anti-cancer agents
7.  High-Throughput Screening based Identification of Small Molecule Antagonists of Integrin CD11b/CD18 Ligand Binding 
Binding of leukocyte specific integrin CD11b/CD18 to its physiologic ligands is important for the development of normal immune response in vivo. Integrin CD11b/CD18 is also a key cellular effector of various inflammatory and autoimmune diseases. However, small molecules selectively inhibiting the function of integrin CD11b/CD18 are currently lacking. We used a newly described cell-based high throughput screening assay to identify a number of highly potent antagonists of integrin CD11b/CD18 from chemical libraries containing >100,000 unique compounds. Computational analyses suggest that the identified compounds cluster into several different chemical classes. A number of the newly identified compounds blocked adhesion of wild-type mouse neutrophils to CD11b/CD18 ligand fibrinogen. Mapping the most active compounds against chemical fingerprints of known antagonists of related integrin CD11a/CD18 shows little structural similarity, suggesting that the newly identified compounds are novel and unique.
doi:10.1016/j.bbrc.2010.02.151
PMCID: PMC3065194  PMID: 20188705
Integrin; CD11b/CD18; inhibitor; screening; HTS assay; adhesion assay
8.  Discovery and Validation of a Series of Aryl Sulfonamides as Selective Inhibitors of Tissue-Nonspecific Alkaline Phosphatase (TNAP) 
Journal of medicinal chemistry  2009;52(21):6919-6925.
We report the characterization and optimization of drug-like small molecule inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme critical for the regulation of extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) of a small molecule library led to the identification of arylsulfonamides as potent and selective inhibitors of TNAP. Critical structural requirements for activity were determined, and the compounds were subsequently profiled for in vitro activity and bioavailability parameters including metabolic stability and permeability. The plasma levels following subcutaneous administration of a member of the lead series in rat was determined, demonstrating the potential of these TNAP inhibitors as systemically active therapeutic agents to target various diseases involving soft tissue calcification. A representative member of the series was also characterized in mechanistic and kinetic studies.
doi:10.1021/jm900383s
PMCID: PMC2783186  PMID: 19821572
9.  Design and synthesis of pyrazole derivatives as potent and selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP) 
Tissue-nonspecific alkaline phosphatase (TNAP) plays a central role in regulating extracellular matrix calcification during bone formation and growth. High throughput screening (HTS) for small molecule TNAP inhibitors led to the identification of hits in the sub-micromolar potency range. We report the design, synthesis and in vitro evaluation of a series of pyrazole derivatives of a screening hit which are potent TNAP inhibitors exhibiting IC50 values as low as 5 nM. A representative of the series was characterized in kinetic studies and determined to have a mode of inhibition not previously observed for TNAP inhibitors.
doi:10.1016/j.bmcl.2008.10.107
PMCID: PMC2752324  PMID: 19038545
10.  Identification of Inhibitors of NOD1-Induced Nuclear Factor-κB Activation 
ACS Medicinal Chemistry Letters  2011;2(10):780-785.
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 1c, ML130) as a potent and selective inhibitor of NOD1-induced NF-κB activation.
doi:10.1021/ml200158b
PMCID: PMC3193285  PMID: 22003428
NOD1; NF-κB activation; 2-aminobenzimidazole; hit-to-probe; ML130; MLPCN

Results 1-11 (11)