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1.  DUSP3 Phosphatase Deficiency or Inhibition Limit Platelet Activation and Arterial Thrombosis 
Circulation  2014;131(7):656-668.
Background
A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. Better understanding of the molecular mechanisms leading to platelet activation is of importance for the development of improved therapies. Recently, protein tyrosine phosphatases (PTPs) have emerged as critical regulators of platelet function.
Methods and Results
This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated through the collagen receptor glycoprotein VI (GPVI) and the C-type lectin-like receptor 2 (CLEC-2). DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism, compared to wild-type mice, and showed severely impaired thrombus formation upon ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of PLCγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen and CLEC-2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells.
Conclusions
DUSP3 plays a selective and essential role in collagen- and CLEC-2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a PTP, implicated in platelet signaling, has been targeted with a small-molecule drug.
doi:10.1161/CIRCULATIONAHA.114.010186
PMCID: PMC4334727  PMID: 25520375
platelets; signal transduction; thrombosis; collagen; inhibitors
2.  Design of High Throughput Screening Assays and Identification of a SUMO1-Specific Small Molecule Chemotype Targeting the SUMO-Interacting Motif-Binding Surface 
ACS combinatorial science  2015;17(4):239-246.
Protein-protein interactions are generally challenging to target by small molecules. To address the challenge, we have used a multi-disciplinary approach to identify small-molecule disruptors of protein-protein interactions that are mediated by SUMO (Small Ubiquitin-like MOdifier) proteins. SUMO modifications have emerged as a target with importance in treating cancer, neurodegenerative disorders, and viral infections. It has been shown that inhibiting SUMO-mediated protein-protein interactions can sensitize cancer cells to chemotherapy and radiation. We have developed highly sensitive assays using time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP) that were used for high-throughput screening (HTS) to identify inhibitors for SUMO-dependent protein-protein interactions. Using these assays, we have identified a non-peptidomimetic small molecule chemotype that binds to SUMO1 but not SUMO2 or 3. NMR chemical shift perturbation studies have shown that the compounds of this chemotype bind to the SUMO1 surface required for protein-protein interaction, despite the high sequence similarity of SUMO1 and SUMO2 and 3 at this surface.
doi:10.1021/co500181b
PMCID: PMC4609574  PMID: 25719760
Small ubiquitin-like modifier (SUMO); TR-FRET; fluorescence polarization; SUMO-interacting motif (SIM)
3.  Design, Synthesis and Evaluation of Benzoisothiazolones as Selective Inhibitors of PHOSPHO1 
We report the discovery and characterization of a series of benzisothiazolone inhibitors of PHOSPHO1, a newly identified soluble phosphatase implicated in skeletal mineralization and soft tissue ossification abnormalities. High-throughput screening (HTS) of a small molecule library led to the identification of benzoisothiazolones as potent and selective inhibitors of PHOSPHO1. Critical structural requirements for activity were determined, and the compounds were subsequently derivatized and measured for in vitro activity and ADME parameters including metabolic stability and permeability. On the basis of its overall profile the benzoisothiazolone analogue 2q was selected as MLPCN probe ML086
doi:10.1016/j.bmcl.2014.07.013
PMCID: PMC4170737  PMID: 25124115
Phosphatase; Vascular calcification; Probe compound; ML086; PHOSPHO1
4.  Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines 
Oncotarget  2015;6(21):18418-18428.
Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a “proof-of-principle” that enzymatic inhibition of QSOX1 may have clinical relevancy.
PMCID: PMC4621900  PMID: 26158899
QSOX1; LOPAC1280; ebselen; renal cancer; pancreatic cancer
5.  TRAIL-Based High Throughput Screening Reveals a Link between TRAIL-Mediated Apoptosis and Glutathione Reductase, a Key Component of Oxidative Stress Response 
PLoS ONE  2015;10(6):e0129566.
A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.
doi:10.1371/journal.pone.0129566
PMCID: PMC4468210  PMID: 26075913
6.  Pathophysiological role of vascular smooth muscle alkaline phosphatase in medial artery calcification† 
Medial vascular calcification (MVC) is a pathological phenomenon common to a variety of conditions, including aging, chronic kidney disease, diabetes, obesity, and a variety of rare genetic diseases, that causes vascular stiffening and can lead to heart failure. These conditions share the common feature of tissue-nonspecific alkaline phosphatase (TNAP) upregulation in the vasculature. To evaluate the role of TNAP in MVC, we developed a mouse model that overexpresses human TNAP in vascular smooth muscle cells in an X-linked manner. Hemizygous overexpressor male mice (Tagln-Cre+/-; HprtALPL/Y, or TNAP-OE) show extensive vascular calcification, high blood pressure, cardiac hypertrophy and have a median age of death of 44 days, whereas the cardiovascular phenotype is much less pronounced and life expectancy is longer in heterozygous (Tagln-Cre+/-; HprtALPL/-) female TNAP-OE mice. Gene expression analysis showed upregulation of osteoblast and chondrocyte markers and decreased expression of vascular smooth muscle markers in the aortas of TNAP-OE mice. Through medicinal chemistry efforts, we developed inhibitors of TNAP with drug-like pharmacokinetic characteristics. TNAP-OE mice were treated with the prototypical TNAP inhibitor SBI-425 or vehicle to evaluate the feasibility of TNAP inhibition in vivo. Treatment with this inhibitor significantly reduced aortic calcification and cardiac hypertrophy, and extended lifespan over vehicle-treated controls, in the absence of secondary effects on the skeleton. This study shows that TNAP in the vasculature contributes to the pathology of MVC and that it is a druggable target. This article is protected by copyright. All rights reserved
doi:10.1002/jbmr.2420
PMCID: PMC4406354  PMID: 25428889
genetic animal models; preclinical studies; matrix mineralization; therapeutics
7.  pCAP-Based Peptide Substrates: The New Tool in the Box of Tyrosine Phosphatase Assays 
Methods (San Diego, Calif.)  2013;65(2):165-174.
Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.
doi:10.1016/j.ymeth.2013.07.022
PMCID: PMC3899110  PMID: 23886911
fluorogenic enzyme substrates; peptide synthesis; high-throughput screening; enzyme activity gel; multiply phosphorylated peptides
8.  Triple Hybrids of Steroids, Spiroketals, and Oligopeptides as New Biomolecular Chimeras 
Organic letters  2009;11(1):65-68.
An oxidative enol ether rearrangement methodology was the key methodology in the construction of steroid-spiroketal-RGD peptides. Biological studies demonstrated potent integrin CD11b/CD18 antagonistic effects.
doi:10.1021/ol802247m
PMCID: PMC4257705  PMID: 19067551
9.  TR-FRET-Based High-Throughput Screening Assay for Identification of UBC13 Inhibitors 
Journal of biomolecular screening  2011;17(2):163-176.
UBC13 is a non-canonical Ubiquitin Conjugating Enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of Lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair, and thus a potential radiosensitizer and chemosensitizer target for oncology. We developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. We defined conditions for optimized performance of the TR-FRET assay in both 384 and 1536-well formats. Chemical library screens (total 456,865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically > 0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.
doi:10.1177/1087057111423417
PMCID: PMC4172584  PMID: 22034497
10.  High-Throughput Screening of Tissue-Nonspecific Alkaline Phosphatase for Identification of Effectors with Diverse Modes of Action 
Nature protocols  2010;5(8):1431-1439.
A protocol for the identification of effectors of tissue-nonspecific alkaline phosphatase (TNAP) is described. It is based on highly sensitive method for detecting TNAP activity. A dioxetane-based substrate after dephosphorylation by TNAP undergoes a series of chemical transformations resulting in light production. The light intensity serves as a quantitative measure of the velocity of the TNAP catalysed reaction in the steady state. The protocol includes guidelines for the optimization of the assay and execution of the high-throughput screening in multiwell plates. The assay is sensitive to the influence of diverse effectors of TNAP as long as the assay optimization steps are repeated for each new batch of the enzyme; full optimization is accomplished in under two days. Depending on the available equipment 10,000-100,000 compounds could be screened in 8-hour period. This protocol provides thousands-fold more sensitive and tenfold faster way of screening TNAP, when compared with a conventional colorimetric assay with p-nitrophenyl phosphate.
doi:10.1038/nprot.2010.86
PMCID: PMC3908691  PMID: 20671726
alkaline phosphatase; chemiluminescent assay; enzyme assay; functional assay; high-throughput screening
11.  Discovery of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221) as a functional antagonist of the apelin (APJ) receptor 
The recently discovered apelin/APJ system has emerged as a critical mediator of cardiovascular homeostasis and is associated with the pathogenesis of cardiovascular disease. A role for apelin/APJ in energy metabolism and gastrointestinal function has also recently emerged. We disclose the discovery and characterization of 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APJ functional antagonist in cell-based assays that is >37-fold selective over the closely related angiotensin II type 1 (AT1) receptor. ML221 was derived from an HTS of the ∼330,600 compound MLSMR collection. This antagonist showed no significant binding activity against 29 other GPCRs, except to the κ-opioid and benzodiazepinone receptors (<50/<70%I at 10 μM). The synthetic methodology, development of structure-activity relationship (SAR), and initial in vitro pharmacologic characterization are also presented.
doi:10.1016/j.bmcl.2012.08.105
PMCID: PMC3729231  PMID: 23010269
GPCRs; HTS; APJ; Apelin; Cardiovascular disease; Antagonists; SAR
12.  Discovery of a Plasmodium falciparum glucose-6-phosphate dehydrogenase 6- phosphogluconolactonase inhibitor (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2-(2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide (ML276) that reduces parasite growth in vitro 
Journal of medicinal chemistry  2012;55(16):7262-7272.
A high throughput screen of the NIH’s MLSMR collection of ~340,000 compounds was undertaken to identify compounds that inhibit Plasmodium falciparum glucose-6-phosphate dehydrogenase (PfG6PD). PfG6PD is essential for proliferating and propagating P. falciparum and differs structurally and mechanistically from the human ortholog. 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 fastgrowing cells. In P. falciparum the bifunctional enzyme glucose-6-phosphate dehydrogenase-6- phosphogluconolactonase (PfGluPho) 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 PfGluPho. The lead compound identified from this effort, (R,Z)-N-((1-ethylpyrrolidin-2-yl)methyl)-2- (2-fluorobenzylidene)-3-oxo-3,4-dihydro-2H-benzo[b][1,4]thiazine-6-carboxamide, 11, (ML276), is a submicromolar inhibitor of PfG6PD (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 PfG6PD in vivo are in progress.
doi:10.1021/jm300833h
PMCID: PMC3530835  PMID: 22813531
6-phosphogluconolactonase; glucose-6-phosphate; glucose-6-phosphate dehydrogenase; highthroughput screening; malaria; pentose phosphate pathway; Plasmodium; redox metabolism; benzothiazinone
13.  Discovery of Small Molecule Kappa Opioid Receptor Agonist and Antagonist Chemotypes through a HTS and Hit Refinement Strategy 
ACS chemical neuroscience  2012;3(3):221-236.
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 (Ki < 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.
doi:10.1021/cn200128x
PMCID: PMC3378255  PMID: 22737280
kappa opioid receptor agonist; kappa opioid receptor antagonist; high-throughput screening
14.  Discovery of Small Molecule Kappa Opioid Receptor Agonist and Antagonist Chemotypes through a HTS and Hit Refinement Strategy 
ACS Chemical Neuroscience  2012;3(3):221-236.
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 (Ki < 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.
doi:10.1021/cn200128x
PMCID: PMC3378255  PMID: 22737280
Kappa opioid receptor agonist; kappa opioid receptor antagonist; high-throughput screening
15.  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
16.  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
17.  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
18.  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
19.  Assay Format as a Critical Success Factor for Identification of Novel Inhibitor Chemotypes of Tissue-Nonspecific Alkaline Phosphatase from High-Throughput Screening 
Molecules (Basel, Switzerland)  2010;15(5):3010-3037.
The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PPi), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.
doi:10.3390/molecules15053010
PMCID: PMC3392958  PMID: 20657462
diethanolamine (DEA); absorption spectroscopy; luminescence; high throughput screening; CDP-Star®; Molecular Libraries; tissue-nonspecific alkaline phosphatase; alkaline phosphatase; chemical library; para-nitrophenylphosphate
20.  Inhibition of the Hematopoietic Protein Tyrosine Phosphatase by Phenoxyacetic Acids 
ACS Medicinal Chemistry Letters  2010;2(2):113-118.
Protein tyrosine phosphatases have only recently become the focus of attention in the search for novel drug targets despite the fact that they play vital roles in numerous cellular processes and are implicated in many human diseases. The hematopoietic protein tyrosine phosphatase (HePTP) is often found dysregulated in preleukemic myelodysplastic syndrome (MDS) as well as in acute myelogenous leukemia (AML). Physiological substrates of HePTP include the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Specific modulators of HePTP catalytic activity will be useful for elucidating mechanisms of MAPK regulation in hematopietic cells and may also provide treatments for hematopoietic malignancies such as AML. Here, we report the discovery of phenoxyacetic acids as inhibitors of HePTP. Structure−activity relationship analysis and in silico docking studies reveal the molecular basis of HePTP inhibition by these compounds. We also show that these compounds are able to penetrate cell membranes and inhibit HePTP in human T lymphocytes.
doi:10.1021/ml100103p
PMCID: PMC3077561  PMID: 21503265
HePTP; PTPN7; inhibitors; p38; myelodysplastic syndrome; myelogenous leukemia
21.  Inhibition of the Hematopoietic Protein Tyrosine Phosphatase by Phenoxyacetic Acids 
ACS medicinal chemistry letters  2011;2(2):113-118.
Protein tyrosine phosphatases (PTPs) have only recently become the focus of attention in the search for novel drug targets despite the fact that they play vital roles in numerous cellular processes and are implicated in many human diseases. The hematopoietic protein tyrosine phosphatase (HePTP) is often found dysregulated in preleukemic myelodysplastic syndrome (MDS), as well as in acute myelogenous leukemia (AML). Physiological substrates of HePTP include the mitogen-activated protein kinases (MAPKs) ERK1/2 and p38. Specific modulators of HePTP catalytic activity will be useful for elucidating mechanisms of MAPK regulation in hematopietic cells, and may also provide treatments for hematopoietic malignancies such as AML. Here we report the discovery of phenoxyacetic acids as inhibitors of HePTP. Structure-activity relationship (SAR) analysis and in silico docking studies reveal the molecular basis of HePTP inhibition by these compounds. We also show that these compounds are able to penetrate cell membranes and inhibit HePTP in human T lymphocytes.
doi:10.1021/ml100103p
PMCID: PMC3077561  PMID: 21503265
HePTP; PTPN7; inhibitors; p38; myelodysplastic syndrome; myelogenous leukemia
22.  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
23.  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
24.  Design and Synthesis of Selective Inhibitors of Placental Alkaline Phosphatase 
Placental Alkaline Phosphatase (PLAP) is a tissue-restricted isozyme of the Alkaline Phosphatase (AP) superfamily. PLAP is an oncodevelopmental enzyme expressed during pregnancy and in a variety of human cancers, but its biological function remains unknown. We report here a series of catechol compounds with great affinity for the PLAP isozyme and significant selectivity over other members of the AP superfamily. These selective PLAP inhibitors will provide small molecule probes for the study of the pathophysiological role of PLAP.
doi:10.1016/j.bmc.2009.12.012
PMCID: PMC2818863  PMID: 20031422
Enzyme inhibitor; Placental Alkaline Phosphatase; cancer; oncodevelopmental
25.  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

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