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1.
Am J Physiol Gastrointest Liver Physiol ; 321(5): G500-G512, 2021 11 01.
Article in English | MEDLINE | ID: mdl-34494462

ABSTRACT

Mouse and human data implicate the NOD1 and NOD2 sensors of the intestinal microbiome and the associated signal transduction via the receptor interacting protein kinase 2 (RIPK2) as a potential key signaling node for the development of inflammatory bowel disease (IBD) and an attractive target for pharmacological intervention. The TRUC mouse model of IBD was strongly indicated for evaluating RIPK2 antagonism for its effect on intestinal inflammation based on previous knockout studies with NOD1, NOD2, and RIPK2. We identified and profiled the BI 706039 molecule as a potent and specific functional inhibitor of both human and mouse RIPK2 and with favorable pharmacokinetic properties. We dosed BI 706039 in the spontaneous TRUC mouse model from age 28 to 56 days. Oral, daily administration of BI 706039 caused dose-responsive and significant improvement in colonic histopathological inflammation, colon weight, and terminal levels of protein-normalized fecal lipocalin (all P values <0.001). These observations correlated with dose responsively increasing systemic levels of the BI 706039 compound, splenic molecular target engagement of RIPK2, and modulation of inflammatory genes in the colon. This demonstrates that a relatively low oral dose of a potent and selective RIPK2 inhibitor can modulate signaling in the intestinal immune system and significantly improve disease associated intestinal inflammation.NEW & NOTEWORTHY The RIPK2 kinase at the apex of microbiome immunosensing is an attractive target for pharmacological intervention. A low oral dose of a RIPK2 inhibitor leads to significantly improved intestinal inflammation in the murine TRUC model of colitis. A selective and potent inhibitor of the RIPK2 kinase may represent a new class of therapeutics that target microbiome-driven signaling for the treatment of IBD.


Subject(s)
Colitis, Ulcerative/drug therapy , Colon/drug effects , Protein Kinase Inhibitors/pharmacology , Receptor-Interacting Protein Serine-Threonine Kinase 2/antagonists & inhibitors , Animals , Biological Availability , Cells, Cultured , Colitis, Ulcerative/enzymology , Colitis, Ulcerative/genetics , Colitis, Ulcerative/pathology , Colon/enzymology , Colon/pathology , Crohn Disease/enzymology , Crohn Disease/pathology , Cytokines/genetics , Cytokines/metabolism , DNA-Binding Proteins/genetics , Disease Models, Animal , Feces/chemistry , Humans , Inflammation Mediators/metabolism , Lipocalins/metabolism , Mice, Inbred BALB C , Mice, Knockout , Models, Biological , Monocytes/drug effects , Monocytes/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/genetics , Receptor-Interacting Protein Serine-Threonine Kinase 2/metabolism , T-Box Domain Proteins/genetics
2.
PLoS One ; 9(6): e100883, 2014.
Article in English | MEDLINE | ID: mdl-24967665

ABSTRACT

GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases.


Subject(s)
Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Myeloid Cells/drug effects , Myeloid Cells/immunology , Receptors, G-Protein-Coupled/agonists , Animals , CHO Cells , Cluster Analysis , Cricetulus , Cyclic AMP , Cytokines/biosynthesis , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Female , Gene Expression Profiling , Humans , Inflammation Mediators/metabolism , Lipopolysaccharides/immunology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Myeloid Cells/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism
3.
J Biomol Screen ; 19(3): 407-16, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24003058

ABSTRACT

Sphingosine-1-phosphate (S1P) is a bioactive metabolite with pleiotropic effects on multiple cellular processes in health and disease. Responses elicited by S1P are a result of binding to five specific G-protein-coupled receptors. We have developed multiple assays to systematically study the downstream signaling of these receptors, including early events such as direct receptor activation (GTPγS) as well as more distal events such as S1P1 receptor degradation. Employing such assays, we have characterized and compared multiple S1P1 agonists that are in clinical development including FTY720, BAF312, CS-0777, and other molecules from the S1P1 patent literature. Our parallel assessment has allowed us to compare their potency against S1P1, their selectivity against the four other S1P receptors, as well as species cross-reactivity. We note that all of the compounds studied signal in an identical manner through S1P1, leading to receptor degradation.


Subject(s)
Down-Regulation/drug effects , Receptors, Lysosphingolipid/agonists , Receptors, Lysosphingolipid/antagonists & inhibitors , Animals , CHO Cells , Calcium/metabolism , Cell Line , Cricetulus , Cyclic AMP/metabolism , Dose-Response Relationship, Drug , Drug Discovery , Humans , Mice , Organophosphates/pharmacology , Proteolysis/drug effects , Rats , Receptors, Lysosphingolipid/metabolism , Sphingosine/analogs & derivatives , Sphingosine/pharmacology
4.
Am J Clin Exp Immunol ; 2(2): 172-85, 2013.
Article in English | MEDLINE | ID: mdl-23885334

ABSTRACT

Vascular adhesion protein-1 (VAP-1) has been implicated in the pathogenesis of inflammatory diseases and is suggested to play a role in immune cell trafficking. It is not clear whether this effect is mediated by the oxidase activity or by other features of the protein such as direct adhesion. In order to study the role of VAP-1 oxidase activity in vivo, we have generated mice carrying an oxidase activity-null VAP-1 protein. We demonstrate that the VAP-1 oxidase null mutant mice have a phenotype similar to the VAP-1 null mice in animal models of sterile peritonitis and antibody induced arthritis suggesting that the oxidase activity is responsible for the inflammatory function of VAP-1.

5.
J Immunol ; 190(7): 3533-40, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23436932

ABSTRACT

Sphingosine-1-phosphate (S1P) receptors are critical for lymphocyte egress from secondary lymphoid organs, and S1P receptor modulators suppress lymphocyte circulation. However, the role of S1P receptors on monocytes is less clear. To elucidate this, we systematically evaluated monocytes in rats and mice, both in naive and inflammatory conditions, with S1P receptor modulators FTY720 and BAF312. We demonstrate that S1P receptor modulators reduce circulating monocytes in a similar time course as lymphocytes. Furthermore, total monocyte numbers were increased in the spleen and bone marrow, suggesting that S1P receptor modulation restricts egress from hematopoietic organs. Monocytes treated ex vivo with FTY720 had reduced CD40 expression and TNF-α production, suggesting a direct effect on monocyte activation. Similar reductions in protein expression and cytokine production were also found in vivo. Suppression of experimental autoimmune encephalomyelitis in mice and rats by FTY720 correlated with reduced numbers of lymphocytes and monocytes. These effects on monocytes were independent of S1P3, as treatment with BAF312, a S1P1,4,5 modulator, led to similar results. These data reveal a novel role for S1P receptors on monocytes and offer additional insights on the mechanism of action of S1P receptor modulators in disease.


Subject(s)
Monocytes/drug effects , Monocytes/metabolism , Propylene Glycols/pharmacology , Receptors, Lysosphingolipid/metabolism , Sphingosine/analogs & derivatives , Animals , Bone Marrow/drug effects , Bone Marrow/metabolism , Cell Movement/immunology , Cytokines/biosynthesis , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Female , Fingolimod Hydrochloride , Killer Cells, Natural/metabolism , Leukocyte Count , Mice , Monocytes/immunology , Neutrophils/metabolism , Rats , Sphingosine/pharmacology , Spleen/drug effects , Spleen/metabolism
6.
AIDS Res Hum Retroviruses ; 29(3): 493-500, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23062187

ABSTRACT

Cardiovascular disease (CVD) biomarkers were examined in a cohort of HIV-infected and HIV-uninfected adolescents who participated in Adolescent Trials Network study 083 utilizing samples from the Reaching for Excellence in Adolescent Care cohort, a longitudinal study of youth infected through adult risk behavior. Nonfasting blood samples from 97 HIV-infected and 81 HIV-uninfected adolescents infected by adult risk behaviors were analyzed for total cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), very low-density lipoprotein (VLDL), triglycerides, apolipoprotein A-I, high-sensitivity C-reactive protein (hsCRP), soluble vascular adhesion molecule-1 (sVCAM-1), myeloperoxidase, and neopterin at baseline and 18 months later. Results were analyzed using ANOVA, Wilcoxon signed-rank, and paired t tests. Among infected subjects 67 received antiretroviral therapy and 30 were treatment naive. The HIV-infected and HIV-uninfected subjects were similar in gender, ethnicity, and cardiovascular risk factors such as smoking and obesity. In all groups lipid parameters were within accepted guidelines for cardiovascular risk. Among HIV-infected youth on antiretroviral therapy (ART), HDL and apoprotein A-I were significantly lower when compared to uninfected youth. hsCRP was not elevated and thus not predictive for risk in any group. sVCAM-1 levels were significantly elevated in both HIV-infected groups: 1,435 ng/ml and 1,492 ng/ml in untreated and treated subjects, respectively, and 1,064 ng/ml in the uninfected group (p<0.0001). Across all groups neopterin correlated with sVCAM at 18 months (Spearman correlation coefficient 0.58, p<0.0001). Only 9% of ART-treated subjects fully suppressed virus. Lipid profiles and hsCRP, traditional markers of cardiovascular disease, are not abnormal among HIV-infected youth but elevated sVCAM may be an early marker of atherosclerosis.


Subject(s)
Biomarkers/blood , Cardiovascular Diseases/diagnosis , Endothelium, Vascular/pathology , HIV Infections/complications , HIV Infections/virology , HIV-1/pathogenicity , Vascular Cell Adhesion Molecule-1/blood , Adolescent , Cohort Studies , Female , Humans , Longitudinal Studies , Male
7.
J Biomol Screen ; 16(9): 1106-11, 2011 Oct.
Article in English | MEDLINE | ID: mdl-21844327

ABSTRACT

Vascular adhesion protein-1 (VAP-1), also known as semicarbazide-sensitive amine oxidase (SSAO) or copper-containing amine oxidase (AOC3, EC 1.4.3.6), catalyzes oxidative deamination of primary amines. One endogenous substrate has recently been described (Siglec 10), and although its mechanism of action in vivo is not completely understood, it is suggested to play a role in immune cell trafficking, making it a target of interest for autoimmune and inflammatory diseases. Much of the enzymology performed around this target has been conducted with absorbance, fluorescent, or radiometric formats that can have some limitations for high-throughput screening and subsequent compound profiling. The authors present the use of a bioluminescent assay, originally developed for monoamine oxidase enzymes, in a high-throughput format. It can be used for related SSAOs such as AOC1 given their substrate similarity with VAP-1. The authors also demonstrate that it is compatible with different sources of VAP-1, both purified recombinant and VAP-1 overexpressed on live cells.


Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , High-Throughput Screening Assays , Luminescent Measurements , Amine Oxidase (Copper-Containing)/antagonists & inhibitors , Animals , CHO Cells , Cell Line, Transformed , Cricetinae , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , HEK293 Cells , Humans , Inhibitory Concentration 50 , Mice , Rats
9.
Biochemistry ; 43(37): 11658-71, 2004 Sep 21.
Article in English | MEDLINE | ID: mdl-15362850

ABSTRACT

A novel inhibitor of p38 mitogen-activated protein kinase (p38), CMPD1, identified by high-throughput screening, is characterized herein. Unlike the p38 inhibitors described previously, this inhibitor is substrate selective and noncompetitive with ATP. In steady-state kinetics experiments, CMPD1 was observed to prevent the p38alpha-dependent phosphorylation (K(i)(app) = 330 nM) of the splice variant of mitogen-activated protein kinase-activated protein kinase 2 (MK2a) that contains a docking domain for p38alpha and p38beta, but it did not prevent the phosphorylation of ATF-2 (K(i)(app) > 20 microM). In addition to kinetic studies, isothermal titration calorimetry and surface plasmon resonance experiments were performed to elucidate the mechanism of inhibition. While isothermal titration calorimetry analysis indicated that CMPD1 binds to p38alpha, CMPD1 was not observed to compete with ATP for p38alpha, nor was it able to interrupt the binding of p38alpha to MK2a observed by surface plasmon resonance. Therefore, deuterium exchange mass spectrometry (DXMS) was employed to study the p38alpha.CMPD1 inhibitory complex, to provide new insight into the mechanism of substrate selective inhibition. The DXMS data obtained for the p38alpha.CMPD1 complex were compared to the data obtained for the p38alpha.MK2a complex and a p38alpha.active site binding inhibitor complex. Alterations in the DXMS behavior of both p38alpha and MK2a were observed upon complex formation, including but not limited to the interaction between the carboxy-terminal docking domain of MK2a and its binding groove on p38alpha. Alterations in the D(2)O exchange of p38alpha produced by CMPD1 suggest that the substrate selective inhibitor binds in the vicinity of the active site of p38alpha, resulting in perturbations to regions containing nucleotide binding pocket residues, docking groove residues (E160 and D161), and a Mg(2+) ion cofactor binding residue (D168). Although the exact mechanism of substrate selective inhibition by this novel inhibitor has not yet been disclosed, the results suggest that CMPD1 binding in the active site region of p38alpha induces perturbations that may result in the suboptimal positioning of substrates and cofactors in the transition state, resulting in selective inhibition of p38alpha activity.


Subject(s)
Biphenyl Compounds/metabolism , Enzyme Inhibitors/metabolism , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Protein Structure, Tertiary , Activating Transcription Factor 2 , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Animals , Binding Sites , Biphenyl Compounds/chemistry , Calorimetry , Cyclic AMP Response Element-Binding Protein/metabolism , Enzyme Inhibitors/chemistry , Intracellular Signaling Peptides and Proteins , Mice , Mitogen-Activated Protein Kinase 14 , Mitogen-Activated Protein Kinases/metabolism , Models, Molecular , Molecular Sequence Data , Molecular Structure , Phosphorylation , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Substrate Specificity , Surface Plasmon Resonance , Transcription Factors/metabolism
10.
Biochemistry ; 43(31): 9950-60, 2004 Aug 10.
Article in English | MEDLINE | ID: mdl-15287722

ABSTRACT

The p38 mitogen-activated protein kinase (p38) pathway is required for the production of proinflammatory cytokines (TNFalpha and IL-1) that mediate the chronic inflammatory phases of several autoimmune diseases. Potent p38 inhibitors, such as the slow tight-binding inhibitor BIRB 796, have recently been reported to block the production of TNFalpha and IL-1beta. Here we analyze downstream signaling complexes and molecular mechanisms, to provide new insight into the function of p38 signaling complexes and the development of novel inhibitors of the p38 pathway. Catalysis, signaling functions, and molecular interactions involving p38alpha and one of its downstream signaling partners, mitogen-activated protein kinase-activated protein kinase 2 (MK2), have been explored by steady-state kinetics, surface plasmon resonance, isothermal calorimetry, and stopped-flow fluorescence. Functional 1/1 signaling complexes (Kd = 1-100 nM) composed of activated and nonactivated forms of p38alpha and a splice variant of MK2 (MK2a) were characterized. Catalysis of MK2a phosphorylation and activation by p38alpha was observed to be efficient under conditions where substrate is saturating (kcat(app) = 0.05-0.3 s(-1)) and nonsaturating (kcat(app)/KM(app) = 1-3 x 10(6) M(-1) s(-1)). Specific interactions between the carboxy-terminal residues of MK2a (370-400) and p38alpha precipitate formation of a high-affinity complex (Kd = 20 nM); the p38alpha-dependent MK2a phosphorylation reaction was inhibited by the 30-amino acid docking domain peptide of MK2a (IC50 = 60 nM). The results indicate that the 30-amino acid docking domain peptide of MK2a is required for the formation of a tight, functional p38alpha.MK2a complex, and that perturbation of the tight-docking interaction between these signaling partners prevents the phosphorylation of MK2a. The thermodynamic and steady-state kinetic characterization of the p38alpha.MK2a signaling complex has led to a clear understanding of complex formation, catalysis, and function on the molecular level.


Subject(s)
MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/chemistry , Mitogen-Activated Protein Kinases/physiology , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/physiology , Alternative Splicing , Animals , Calorimetry , Catalysis , Fluorescein-5-isothiocyanate/metabolism , Humans , Intracellular Signaling Peptides and Proteins , Isoenzymes/antagonists & inhibitors , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/physiology , Kinetics , MAP Kinase Signaling System/genetics , Mice , Mitogen-Activated Protein Kinase 14 , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/genetics , Peptide Fragments/physiology , Phosphorylation , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Structure, Tertiary/genetics , Spectrometry, Fluorescence , Structure-Activity Relationship , Surface Plasmon Resonance , Thermodynamics , Ultracentrifugation
11.
J Med Chem ; 46(8): 1337-49, 2003 Apr 10.
Article in English | MEDLINE | ID: mdl-12672234

ABSTRACT

The tyrosine kinase p56lck (lck) is essential for T cell activation; thus, inhibitors of lck have potential utility as autoimmune agents. Our initial disclosure of a new class of lck inhibitors based on the phenylaminoimidazoisoquinolin-9-one showed reasonable cellular activity but did not work in vivo upon oral administration. Our current work highlights the further use of rational drug design and molecular modeling to produce a series of lck inhibitors that demonstrate cellular activity below 100 nM and are as efficacious as cyclosporin A in an in vivo mouse model of anti-CD3-induced IL-2 production.


Subject(s)
Benzimidazoles/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Immunosuppressive Agents/chemical synthesis , Isoquinolines/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Administration, Oral , Animals , Antibodies, Monoclonal/pharmacology , Benzimidazoles/chemistry , Benzimidazoles/pharmacology , CD3 Complex/immunology , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Female , Humans , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Interleukin-2/antagonists & inhibitors , Interleukin-2/biosynthesis , Interleukin-2/blood , Isoquinolines/chemistry , Isoquinolines/pharmacology , Jurkat Cells , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Conformation , Protein Binding , Structure-Activity Relationship
12.
J Med Chem ; 45(16): 3394-405, 2002 Aug 01.
Article in English | MEDLINE | ID: mdl-12139450

ABSTRACT

An imidazo[4,5-h]isoquinolin-7,9-dione (1) was identified as an adenosine 5'-triphosphate competitive inhibitor of lck by high throughput screening. Initial structure-activity relationship studies identified the dichlorophenyl ring and the imide NH as important pharmacophores. A binding model was constructed to understand how 1 binds to a related kinase, hck. These results suggested that removing the gem-dimethyl group and flattening the ring would enhance activity. This was realized by converting 1 to the imidazo[4,5-h]isoquinolin-9-one (20), resulting in an 18-fold improvement in potency against lck and a 50-fold increase in potency in a cellular assay.


Subject(s)
Enzyme Inhibitors/chemical synthesis , Imidazoles/chemical synthesis , Isoquinolines/chemical synthesis , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/antagonists & inhibitors , Animals , Antibodies/pharmacology , Binding Sites , CD3 Complex/immunology , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Female , Humans , Imidazoles/chemistry , Imidazoles/pharmacology , Interleukin-2/biosynthesis , Isoquinolines/chemistry , Isoquinolines/pharmacology , Jurkat Cells , Mice , Mice, Inbred BALB C , Models, Molecular , Molecular Conformation , Structure-Activity Relationship , T-Lymphocytes/drug effects , T-Lymphocytes/metabolism
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