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1.
SLAS Discov ; 25(2): 215-222, 2020 Feb.
Article in English | MEDLINE | ID: mdl-31849250

ABSTRACT

The real-time quantification of target engagement (TE) by small-molecule ligands in living cells remains technically challenging. Systematic quantification of such interactions in a high-throughput setting holds promise for identification of target-specific, potent small molecules within a pathophysiological and biologically relevant cellular context. The salt-inducible kinases (SIKs) belong to a subfamily of the AMP-activated protein kinase (AMPK) family and are composed of three isoforms in humans (SIK1, SIK2, and SIK3). They modulate the production of pro- and anti-inflammatory cytokines in immune cells. Although pan-SIK inhibitors are sufficient to reverse SIK-dependent inflammatory responses, the apparent toxicity associated with SIK3 inhibition suggests that isoform-specific inhibition is required to realize therapeutic benefit with acceptable safety margins. Here, we used the NanoBRET TE intracellular kinase assay, a sensitive energy transfer technique, to directly measure molecular proximity and quantify TE in HEK293T cells overexpressing SIK2 or SIK3. Our 384-well high-throughput screening of 530 compounds demonstrates that the NanoBRET TE intracellular kinase assay was sensitive and robust enough to reveal differential engagement of candidate compounds with the two SIK isoforms and further highlights the feasibility of high-throughput implementation of NanoBRET TE intracellular kinase assays for target-driven small-molecule screening.


Subject(s)
Phosphotransferases/isolation & purification , Protein Isoforms/genetics , Protein Kinases/genetics , Protein Serine-Threonine Kinases/genetics , AMP-Activated Protein Kinase Kinases , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Fluorescence Resonance Energy Transfer , Gene Expression Regulation, Enzymologic/drug effects , HEK293 Cells , High-Throughput Screening Assays/methods , Humans , Phosphotransferases/genetics , Protein Isoforms/antagonists & inhibitors , Protein Kinases/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors
2.
Clin Cancer Res ; 19(15): 4282-9, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23741071

ABSTRACT

PURPOSE: This planned exploratory analysis assessed the predictive nature of baseline circulating factors of the insulin-like growth factor (IGF) axis on the treatment effect of ganitumab (monoclonal antibody inhibitor of IGF-1 receptor) plus gemcitabine in a randomized phase II study in metastatic pancreatic adenocarcinoma. EXPERIMENTAL DESIGN: Baseline levels of IGFs/IGF binding proteins (IGFBP) were analyzed in serum or plasma. Mutations and gene expression were analyzed in archival samples. Treatment effects between biomarker subgroups were compared for overall survival (OS). Associations of tumor markers with OS were evaluated. RESULTS: For patients with evaluable samples, ganitumab was associated with improved OS versus placebo (HR, 0.49; 95% CI: 0.28-0.87). The treatment effect on improved OS was strong in the patient subset with higher levels of IGF-1, IGF-2, or IGFBP-3, or lower levels of IGFBP-2, but not so on the other corresponding subset. Median OS of ganitumab versus placebo in patients with higher levels of IGF-1, IGF-2, and IGFBP-3 was 16 versus 6.8 months (HR, 0.25; 95% CI: 0.09-0.67), 16 versus 5.9 months (HR, 0.24; 95% CI: 0.09-0.68), and 16 versus 6.8 months (HR, 0.28; 95% CI: 0.11-0.73), and in patients with lower IGFBP-2 levels was 12.7 versus 6.6 months (HR, 0.19; 95% CI: 0.07-0.55). Interaction between treatment and IGFs/IGFBPs in multivariate analyses suggested predictive potential for IGF-2 (P = 0.002) and IGFBP-2 (P = 0.02). KRAS mutation status and PTEN expression were not associated with OS. CONCLUSIONS: Baseline circulating factors of the IGF axis may predict OS benefit from ganitumab plus gemcitabine in metastatic pancreatic adenocarcinoma.


Subject(s)
Adenocarcinoma/drug therapy , Antibodies, Monoclonal/administration & dosage , Deoxycytidine/analogs & derivatives , Pancreatic Neoplasms/drug therapy , Receptor, IGF Type 1/immunology , Adenocarcinoma/blood , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Antibodies, Monoclonal, Humanized , Biomarkers, Tumor/blood , Deoxycytidine/administration & dosage , Humans , Insulin-Like Growth Factor Binding Protein 2/blood , Insulin-Like Growth Factor Binding Protein 3/blood , Insulin-Like Growth Factor I/metabolism , Neoplasm Metastasis/drug therapy , Neoplasm Metastasis/genetics , Neoplasm Metastasis/pathology , PTEN Phosphohydrolase/genetics , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Receptor, IGF Type 1/genetics , Survival Analysis , ras Proteins/genetics , Gemcitabine
3.
J Med Chem ; 51(20): 6280-92, 2008 Oct 23.
Article in English | MEDLINE | ID: mdl-18817364

ABSTRACT

The p38 mitogen-activated protein kinase (MAPK) is a central signaling molecule in many proinflammatory pathways, regulating the cellular response to a multitude of external stimuli including heat, ultraviolet radiation, osmotic shock, and a variety of cytokines especially interleukin-1beta and tumor necrosis factor alpha. Thus, inhibitors of this enzyme are postulated to have significant therapeutic potential for the treatment of rheumatoid arthritis, inflammatory bowel disease, osteoporosis, and many other diseases where aberrant cytokine signaling is the driver of disease. Herein, we describe a novel class of 3-amino-7-phthalazinylbenzoisoxazole-based inhibitors. With relatively low molecular weight, these compounds are highly potent in enzyme and cell-based assays, with minimal protein shift in 50% human whole blood. Compound 3c was efficacious (ED 50 = 0.05 mg/kg) in the rat collagen induced arthritis (CIA) model.


Subject(s)
Amines/chemistry , Benzene/chemistry , Isoxazoles/administration & dosage , Isoxazoles/pharmacology , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Phthalazines/chemistry , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/pharmacology , Administration, Oral , Animals , Arthritis/chemically induced , Arthritis/drug therapy , Arthritis/enzymology , Crystallography, X-Ray , Disease Models, Animal , Humans , Isoxazoles/chemistry , Isoxazoles/therapeutic use , Mitogen-Activated Protein Kinase 14/chemistry , Mitogen-Activated Protein Kinase 14/metabolism , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Rats , Structure-Activity Relationship
4.
Bioorg Med Chem Lett ; 18(18): 5115-7, 2008 Sep 15.
Article in English | MEDLINE | ID: mdl-18723346

ABSTRACT

Deregulation of the receptor tyrosine kinase c-Kit is associated with an increasing number of human diseases, including certain cancers and mast cell diseases. Interference of c-Kit signaling with multi-kinase inhibitors has been shown clinically to successfully treat gastrointestinal stromal tumors and mastocytosis. Targeted therapy of c-Kit activity may provide therapeutic advantages against off-target effects for non-oncology applications. A new structural class of c-Kit inhibitors is described, including in vitro c-Kit potency, kinase selectivity, and the observed binding mode.


Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Isoxazoles/chemical synthesis , Isoxazoles/pharmacology , Proto-Oncogene Proteins c-kit/drug effects , Proto-Oncogene Proteins c-kit/metabolism , Amides/chemistry , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Isoxazoles/chemistry , Molecular Conformation , Molecular Structure , Receptor Protein-Tyrosine Kinases/metabolism , Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/drug effects
6.
J Med Chem ; 51(11): 3065-8, 2008 Jun 12.
Article in English | MEDLINE | ID: mdl-18447379

ABSTRACT

Inhibition of c-Kit has the potential to treat mast cell associated fibrotic diseases. We report the discovery of several aminoquinazoline pyridones that are potent inhibitors of c-Kit with greater than 200-fold selectivity against KDR, p38, Lck, and Src. In vivo efficacy of pyridone 16 by dose-dependent inhibition of histamine release was demonstrated in a rodent pharmacodynamic model of mast cell activation.


Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins c-kit/metabolism , Pyridones/chemical synthesis , Quinazolines/chemical synthesis , Administration, Oral , Animals , Crystallography, X-Ray , Histamine Release/drug effects , Mast Cells/drug effects , Mast Cells/metabolism , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Pyridones/pharmacokinetics , Pyridones/pharmacology , Quinazolines/pharmacokinetics , Quinazolines/pharmacology , Rats , Structure-Activity Relationship
7.
J Med Chem ; 51(6): 1681-94, 2008 Mar 27.
Article in English | MEDLINE | ID: mdl-18321037

ABSTRACT

The lymphocyte-specific kinase (Lck), a member of the Src family of cytoplasmic tyrosine kinases, is expressed in T cells and natural killer (NK) cells. Genetic evidence, including knockout mice and human mutations, demonstrates that Lck kinase activity is critical for normal T cell development, activation, and signaling. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. With the aid of X-ray structure-based analysis, aminopyrimidine amides 2 and 3 were designed from aminoquinazolines 1, which had previously been demonstrated to exhibit potent inhibition of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminopyrimidine amides 3 possessing improved cellular potency and selectivity profiles relative to their aminoquinazoline predecessors 1. Orally bioavailable compound 13b inhibited the anti-CD3-induced production of interleukin-2 (IL-2) in mice in a dose-dependent manner (ED 50 = 9.4 mg/kg).


Subject(s)
Amides/pharmacology , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrimidines/pharmacology , T-Lymphocytes/drug effects , Administration, Oral , Amides/chemical synthesis , Amides/chemistry , Animals , Cell Proliferation/drug effects , Crystallography, X-Ray , Dose-Response Relationship, Drug , Drug Design , Enzyme Activation/drug effects , Female , Humans , Interleukin-2/antagonists & inhibitors , Interleukin-2/metabolism , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Lipopolysaccharides/pharmacology , Male , Mice , Mice, Inbred BALB C , Mice, Knockout , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrimidines/chemical synthesis , Pyrimidines/chemistry , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects , Signal Transduction/physiology , Stereoisomerism , Structure-Activity Relationship , T-Lymphocytes/metabolism
8.
J Med Chem ; 51(6): 1637-48, 2008 Mar 27.
Article in English | MEDLINE | ID: mdl-18278858

ABSTRACT

Lck, or lymphocyte specific kinase, is a cytoplasmic tyrosine kinase of the Src family expressed in T-cells and NK cells. Genetic evidence from knockout mice and human mutations demonstrates that Lck kinase activity is critical for T-cell receptor-mediated signaling, leading to normal T-cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T-cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the structure-guided design, synthesis, structure-activity relationships, and pharmacological characterization of 2-amino-6-phenylpyrimido[5',4':5,6]pyrimido[1,2- a]benzimidazol-5(6 H)-ones, a new class of compounds that are potent inhibitors of Lck. The most promising compound of this series, 6-(2,6-dimethylphenyl)-2-((4-(4-methyl-1-piperazinyl)phenyl)amino)pyrimido[5',4':5,6]pyrimido-[1,2- a]benzimidazol-5(6 H)-one ( 25), exhibits potent inhibition of Lck kinase activity. This activity translates into inhibition of in vitro cell-based assays and in vivo models of T-cell activation and arthritis, respectively.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Arthritis/drug therapy , Benzimidazoles/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Pyrimidinones/chemical synthesis , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , Crystallography, X-Ray , Disease Models, Animal , Drug Design , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Female , Injections, Intradermal , Interleukin-2/metabolism , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Rats , Rats, Inbred Lew , Rats, Sprague-Dawley , Reproducibility of Results , Stereoisomerism , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism
9.
Assay Drug Dev Technol ; 5(1): 105-15, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17355203

ABSTRACT

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.


Subject(s)
11-beta-Hydroxysteroid Dehydrogenase Type 1/analysis , 11-beta-Hydroxysteroid Dehydrogenase Type 1/chemistry , Electrochemistry/methods , Fluorescence Resonance Energy Transfer/methods , Hydrocortisone/analysis , Luminescent Measurements/methods , Microchemistry/methods , Humans , Radioisotope Dilution Technique , Reproducibility of Results , Sensitivity and Specificity
10.
Assay Drug Dev Technol ; 4(4): 397-409, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16945013

ABSTRACT

The complexity of the p38 mitogen-activated protein kinase (MAPK) signaling pathway presents challenges to understanding the efficacy of p38 inhibitors. Biochemical recombinant kinase assays and tumor necrosis factor alpha (TNFalpha) secretion assays are typically used to evaluate p38alpha inhibitors, but they do not provide insight into proximal intracellular events. Stimulation of the pathway evokes a cascade of phosphorylation events, accompanied by movement of molecules to different cellular compartments. Herein, we describe the profiling and potency comparison of a large set of p38alpha inhibitors with a pyrimidinone, imidazopyrimidine, or triazolopyrimidine core against biochemical recombinant p38alpha kinase activity, lipopolysaccharide (LPS)-mediated TNFalpha secretion by THP-1 cells, and a set of cellular imaging assays in SW1353 chondrocytes and baby hamster kidney cells. These pathway assays included p38 phosphorylation, MAPK-activated protein kinase 2 translocation, and heat shock protein (HSP) 27 phosphorylation. We established that HSP27 phosphorylation correlates well with LPS-induced TNFalpha secretion, validating our cellular imaging assays. We also found that the choice of cells and inducer can profoundly affect cellular potency results. High-content analysis may reveal signaling details, enriching our understanding of the mechanism of action of p38alpha inhibitors.


Subject(s)
Drug Design , Image Processing, Computer-Assisted/methods , Mitogen-Activated Protein Kinase 14/metabolism , Protein Kinase Inhibitors/pharmacology , Signal Transduction/drug effects , Animals , Anisomycin/pharmacology , Cell Line , Cell Line, Tumor , Chondrocytes/drug effects , Chondrocytes/metabolism , Dose-Response Relationship, Drug , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , HSP27 Heat-Shock Proteins , Heat-Shock Proteins/metabolism , Humans , Interleukin-1beta/chemistry , Interleukin-1beta/metabolism , Intracellular Signaling Peptides and Proteins , Lipopolysaccharides/pharmacology , Mitogen-Activated Protein Kinase 14/antagonists & inhibitors , Mitogen-Activated Protein Kinase 14/genetics , Molecular Chaperones , Molecular Structure , Neoplasm Proteins/metabolism , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemistry , Protein Kinases/metabolism , Protein Serine-Threonine Kinases , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/metabolism
11.
J Med Chem ; 49(19): 5671-86, 2006 Sep 21.
Article in English | MEDLINE | ID: mdl-16970394

ABSTRACT

The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and natural killer (NK) cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. Selective inhibition of Lck is expected to offer a new therapy for the treatment of T-cell-mediated autoimmune and inflammatory disease. Screening of our kinase-preferred collection identified aminoquinazoline 1 as a potent, nonselective inhibitor of Lck and T cell proliferation. In this report, we describe the synthesis and structure-activity relationships of a series of novel aminoquinazolines possessing in vitro mechanism-based potency. Optimized, orally bioavailable compounds 32 and 47 exhibit anti-inflammatory activity (ED(50) of 22 and 11 mg/kg, respectively) in the anti-CD3-induced production of interleukin-2 (IL-2) in mice.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Benzamides/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Quinazolines/chemical synthesis , Administration, Oral , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Benzamides/chemistry , Benzamides/pharmacology , Biological Availability , Cell Proliferation/drug effects , Cells, Cultured , Female , Humans , In Vitro Techniques , Interleukin-2/biosynthesis , Male , Mice , Mice, Inbred BALB C , Models, Molecular , Quinazolines/chemistry , Quinazolines/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Necrosis Factor-alpha/biosynthesis
12.
J Med Chem ; 49(16): 4981-91, 2006 Aug 10.
Article in English | MEDLINE | ID: mdl-16884310

ABSTRACT

The lymphocyte-specific kinase (Lck) is a cytoplasmic tyrosine kinase of the Src family expressed in T cells and NK cells. Genetic evidence in both mice and humans demonstrates that Lck kinase activity is critical for signaling mediated by the T cell receptor (TCR), which leads to normal T cell development and activation. A small molecule inhibitor of Lck is expected to be useful in the treatment of T cell-mediated autoimmune and inflammatory disorders and/or organ transplant rejection. In this paper, we describe the synthesis, structure-activity relationships, and pharmacological characterization of 2-aminopyrimidine carbamates, a new class of compounds with potent and selective inhibition of Lck. The most promising compound of this series, 2,6-dimethylphenyl 2-((3,5-bis(methyloxy)-4-((3-(4-methyl-1-piperazinyl)propyl)oxy)phenyl)amino)-4-pyrimidinyl(2,4-bis(methyloxy)phenyl)carbamate (43) exhibits good activity when evaluated in in vitro assays and in an in vivo model of T cell activation.


Subject(s)
Aminopyridines/chemical synthesis , Anti-Inflammatory Agents/chemical synthesis , Carbamates/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Pyrimidines/chemical synthesis , Administration, Oral , Aminopyridines/chemistry , Aminopyridines/pharmacology , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Biological Availability , Carbamates/chemistry , Carbamates/pharmacology , Crystallography, X-Ray , Humans , In Vitro Techniques , Jurkat Cells , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Structure , Pyrimidines/chemistry , Pyrimidines/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/immunology
13.
Bioorg Med Chem Lett ; 15(9): 2409-13, 2005 May 02.
Article in English | MEDLINE | ID: mdl-15837335

ABSTRACT

Novel potent trisubstituted pyridazine inhibitors of p38 MAP (mitogen activated protein) kinase are described that have activity in both cell-based assays of cytokine release and animal models of rheumatoid arthritis. They demonstrated potent inhibition of LPS-induced TNF-alpha production in mice and exhibited good efficacy in the rat collagen induced arthritis model.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Pyridazines/chemical synthesis , Pyridazines/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , Binding Sites , Drug Design , Humans , Indicators and Reagents , Kinetics , Microsomes, Liver/enzymology , Models, Molecular , Molecular Structure , Protein Conformation , Structure-Activity Relationship , p38 Mitogen-Activated Protein Kinases/chemistry
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