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1.
Sci Adv ; 8(24): eabo4271, 2022 06 17.
Article in English | MEDLINE | ID: mdl-35704572

ABSTRACT

Infection is one of the major causes of mortality in patients with systemic lupus erythematosus (SLE). We previously found that CD38, an ectoenzyme that regulates the production of NAD+, is up-regulated in CD8+ T cells of SLE patients and correlates with the risk of infection. Here, we report that CD38 reduces CD8+ T cell function by negatively affecting mitochondrial fitness through the inhibition of multiple steps of mitophagy, a process that is critical for mitochondria quality control. Using a murine lupus model, we found that administration of a CD38 inhibitor in a CD8+ T cell-targeted manner reinvigorated their effector function, reversed the defects in autophagy and mitochondria, and improved viral clearance. We conclude that CD38 represents a target to mitigate infection rates in people with SLE.


Subject(s)
Lupus Erythematosus, Systemic , Virus Diseases , Animals , CD8-Positive T-Lymphocytes/metabolism , Humans , Lupus Erythematosus, Systemic/metabolism , Mice , Mitochondria , Mitophagy , Virus Diseases/metabolism
2.
J Med Chem ; 65(13): 9418-9446, 2022 07 14.
Article in English | MEDLINE | ID: mdl-35762533

ABSTRACT

CD38 is one of the major nicotinamide adenine dinucleotide (NAD+)- and nicotinamide adenine dinucleotide phosphate (NADP+)-consuming enzymes in mammals. NAD+, NADP+, and their reduced counterparts are essential coenzymes for numerous enzymatic reactions, including the maintenance of cellular and mitochondrial redox balance. CD38 expression is upregulated in age-associated inflammation as well as numerous metabolic diseases, resulting in cellular and mitochondrial dysfunction. Recent literature studies demonstrate that CD38 is activated upon ischemia/reperfusion (I/R), leading to a depletion of NADP+, which results in endothelial damage and myocardial infarction in the heart. Despite increasing evidence of CD38 involvement in various disease states, relatively few CD38 enzymatic inhibitors have been reported to date. Herein, we describe a CD38 enzymatic inhibitor (MK-0159, IC50 = 3 nM against murine CD38) that inhibits CD38 in in vitro assay. Mice treated with MK-0159 show strong protection from myocardial damage upon cardiac I/R injury compared to those treated with NAD+ precursors (nicotinamide riboside) or the known CD38 inhibitor, 78c.


Subject(s)
ADP-ribosyl Cyclase 1/antagonists & inhibitors , Membrane Glycoproteins/antagonists & inhibitors , NAD , Reperfusion Injury , Animals , Enzyme Inhibitors , Ischemia , Mammals/metabolism , Mice , NAD/metabolism , NADP/metabolism , Reperfusion Injury/drug therapy , Reperfusion Injury/prevention & control
3.
Skelet Muscle ; 10(1): 30, 2020 10 22.
Article in English | MEDLINE | ID: mdl-33092650

ABSTRACT

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder stemming from a loss of functional dystrophin. Current therapeutic options for DMD are limited, as small molecule modalities remain largely unable to decrease the incidence or mitigate the consequences of repetitive mechanical insults to the muscle during eccentric contractions (ECCs). METHODS: Using a metabolomics-based approach, we observed distinct and transient molecular phenotypes in muscles of dystrophin-deficient MDX mice subjected to ECCs. Among the most chronically depleted metabolites was nicotinamide adenine dinucleotide (NAD), an essential metabolic cofactor suggested to protect muscle from structural and metabolic degeneration over time. We tested whether the MDX muscle NAD pool can be expanded for therapeutic benefit using two complementary small molecule strategies: provision of a biosynthetic precursor, nicotinamide riboside, or specific inhibition of the NAD-degrading ADP-ribosyl cyclase, CD38. RESULTS: Administering a novel, potent, and orally available CD38 antagonist to MDX mice successfully reverted a majority of the muscle metabolome toward the wildtype state, with a pronounced impact on intermediates of the pentose phosphate pathway, while supplementing nicotinamide riboside did not significantly affect the molecular phenotype of the muscle. However, neither strategy sustainably increased the bulk tissue NAD pool, lessened muscle damage markers, nor improved maximal hindlimb strength following repeated rounds of eccentric challenge and recovery. CONCLUSIONS: In the absence of dystrophin, eccentric injury contributes to chronic intramuscular NAD depletion with broad pleiotropic effects on the molecular phenotype of the tissue. These molecular consequences can be more effectively overcome by inhibiting the enzymatic activity of CD38 than by supplementing nicotinamide riboside. However, we found no evidence that either small molecule strategy is sufficient to restore muscle contractile function or confer protection from eccentric injury, undermining the modulation of NAD metabolism as a therapeutic approach for DMD.


Subject(s)
Enzyme Inhibitors/pharmacology , Metabolome , Muscle, Skeletal/metabolism , Muscular Dystrophy, Duchenne/drug therapy , NAD/metabolism , Niacinamide/analogs & derivatives , Pyridinium Compounds/pharmacology , ADP-ribosyl Cyclase 1/antagonists & inhibitors , Animals , Dystrophin/deficiency , Enzyme Inhibitors/therapeutic use , Male , Membrane Glycoproteins/antagonists & inhibitors , Mice , Mice, Inbred C57BL , Mice, Inbred mdx , Muscle Contraction , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiology , Muscular Dystrophy, Duchenne/genetics , Muscular Dystrophy, Duchenne/metabolism , Niacinamide/pharmacology , Niacinamide/therapeutic use , Pyridinium Compounds/therapeutic use
4.
ACS Med Chem Lett ; 10(7): 1056-1060, 2019 Jul 11.
Article in English | MEDLINE | ID: mdl-31312408

ABSTRACT

Excess aldosterone production and signaling are primary contributors to numerous cardiovascular disorders including primary aldosteronism and resistant hypertension. Recently, inhibition of aldosterone synthesis via the enzyme aldosterone synthase (CYP11B2) has been pursued to ameliorate the negative effects of elevated aldosterone. Herein, we report the development of aldosterone synthase inhibitors using a pyrimidine-based metal binding group leading to the highly selective CYP11B2 inhibitor 22. Superior selectivity combined with robust pharmacokinetics afforded highly selective in vivo aldosterone suppression in a monkey model of adrenal steroidogenesis, demonstrating the potential for selective aldosterone lowering in humans with pyrimidine 22.

5.
J Med Chem ; 58(17): 7021-56, 2015 Sep 10.
Article in English | MEDLINE | ID: mdl-26267483

ABSTRACT

Starting from the micromolar 8-quinoline carboxamide high-throughput screening hit 1a, a systematic exploration of the structure-activity relationships (SAR) of the 4-, 6-, and 8-substituents of the quinoline ring resulted in the identification of approximately 10-100-fold more potent human CD38 inhibitors. Several of these molecules also exhibited pharmacokinetic parameters suitable for in vivo animal studies, including low clearances and decent oral bioavailability. Two of these CD38 inhibitors, 1ah and 1ai, were shown to elevate NAD tissue levels in liver and muscle in a diet-induced obese (DIO) C57BL/6 mouse model. These inhibitor tool compounds will enable further biological studies of the CD38 enzyme as well as the investigation of the therapeutic implications of NAD enhancement in disease models of abnormally low NAD.


Subject(s)
ADP-ribosyl Cyclase 1/antagonists & inhibitors , Amides/chemistry , Aminoquinolines/chemistry , NAD/metabolism , Quinolines/chemistry , Amides/chemical synthesis , Amides/pharmacology , Aminoquinolines/chemical synthesis , Aminoquinolines/pharmacology , Animals , Biological Availability , Crystallography, X-Ray , Humans , Hydrolysis , Liver/metabolism , Membranes, Artificial , Mice, Inbred C57BL , Models, Molecular , Muscle, Skeletal/metabolism , Obesity/metabolism , Permeability , Protein Conformation , Quinolines/chemical synthesis , Quinolines/pharmacology , Stereoisomerism , Structure-Activity Relationship
6.
PLoS One ; 10(8): e0134927, 2015.
Article in English | MEDLINE | ID: mdl-26287487

ABSTRACT

Nicotinamide adenine dinucleotide (NAD+) is a key cofactor required for essential metabolic oxidation-reduction reactions. It also regulates various cellular activities, including gene expression, signaling, DNA repair and calcium homeostasis. Intracellular NAD+ levels are tightly regulated and often respond rapidly to nutritional and environmental changes. Numerous studies indicate that elevating NAD+ may be therapeutically beneficial in the context of numerous diseases. However, the role of NAD+ on skeletal muscle exercise performance is poorly understood. CD38, a multi-functional membrane receptor and enzyme, consumes NAD+ to generate products such as cyclic-ADP-ribose. CD38 knockout mice show elevated tissue and blood NAD+ level. Chronic feeding of high-fat, high-sucrose diet to wild type mice leads to exercise intolerance and reduced metabolic flexibility. Loss of CD38 by genetic mutation protects mice from diet-induced metabolic deficit. These animal model results suggest that elevation of tissue NAD+ through genetic ablation of CD38 can profoundly alter energy homeostasis in animals that are maintained on a calorically-excessive Western diet.


Subject(s)
ADP-ribosyl Cyclase 1/genetics , ADP-ribosyl Cyclase 1/metabolism , Diet, Western/adverse effects , Metabolic Diseases/genetics , Metabolic Diseases/metabolism , Physical Conditioning, Animal/physiology , ADP-ribosyl Cyclase/metabolism , Animals , Cyclic ADP-Ribose/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Muscle, Skeletal/metabolism , NAD/metabolism , Oxidation-Reduction
7.
J Med Chem ; 58(8): 3548-71, 2015 Apr 23.
Article in English | MEDLINE | ID: mdl-25828863

ABSTRACT

A series of thiazoloquin(az)olinones were synthesized and found to have potent inhibitory activity against CD38. Several of these compounds were also shown to have good pharmacokinetic properties and demonstrated the ability to elevate NAD levels in plasma, liver, and muscle tissue. In particular, compound 78c was given to diet induced obese (DIO) C57Bl6 mice, elevating NAD > 5-fold in liver and >1.2-fold in muscle versus control animals at a 2 h time point. The compounds described herein possess the most potent CD38 inhibitory activity of any small molecules described in the literature to date. The inhibitors should allow for a more detailed assessment of how NAD elevation via CD38 inhibition affects physiology in NAD deficient states.


Subject(s)
ADP-ribosyl Cyclase 1/antagonists & inhibitors , Quinolones/chemistry , Quinolones/pharmacology , Thiazoles/chemistry , Thiazoles/pharmacology , ADP-ribosyl Cyclase 1/metabolism , Animals , Cell Line , Dogs , Drug Discovery , Humans , Liver/drug effects , Liver/metabolism , Mice, Inbred C57BL , Molecular Docking Simulation , Muscles/drug effects , Muscles/metabolism , NAD/analysis , NAD/blood , NAD/metabolism , Obesity/drug therapy , Obesity/metabolism , Quinolones/chemical synthesis , Quinolones/pharmacokinetics , Thiazoles/chemical synthesis , Thiazoles/pharmacokinetics
8.
Am J Physiol Endocrinol Metab ; 306(2): E150-6, 2014 Jan 15.
Article in English | MEDLINE | ID: mdl-24253050

ABSTRACT

A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed "mechano-growth factor" (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.


Subject(s)
Insulin-Like Growth Factor I/metabolism , Myoblasts/drug effects , Stem Cells/drug effects , Animals , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Humans , Insulin-Like Growth Factor I/chemistry , Insulin-Like Growth Factor I/pharmacology , Mice , Myoblasts/physiology , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Primary Cell Culture , Protein Processing, Post-Translational , Protein Structure, Tertiary , Stem Cells/physiology
9.
J Biol Chem ; 285(34): 26335-40, 2010 Aug 20.
Article in English | MEDLINE | ID: mdl-20554517

ABSTRACT

Met, the tyrosine kinase receptor for the hepatocyte growth factor is a prominent regulator of cancer cell invasiveness and has emerged as a promising therapeutic target. Binding of the anti-Met monoclonal antibody DN30 to its epitope induces the proteolytic cleavage of Met, thereby impairing the invasive growth of tumors. The molecular mechanism controlling this therapeutic shedding process has so far been unknown. Here, we report that A Disintegrin And Metalloproteinase (ADAM)-10, but not ADAM-17, is required for DN30-induced Met shedding. Knockdown of ADAM-10 in different tumor cell lines or abrogation of its proteolytic activity by natural or synthetic inhibitors abolished Met down-regulation on the cell surface as well as reduction of Met activation. Moreover, hepatocyte growth factor-induced tumor cell migration and invasion were impaired upon ADAM-10 knockdown. Thus, the therapeutic effect of DN30 involves ADAM-10-dependent Met shedding, linking for the first time a specific metalloprotease to target therapy against a receptor tyrosine kinase.


Subject(s)
ADAM Proteins/physiology , Amyloid Precursor Protein Secretases/physiology , Antibodies/therapeutic use , Membrane Proteins/physiology , Proto-Oncogene Proteins c-met/metabolism , Receptors, Growth Factor/metabolism , ADAM10 Protein , Antibodies/pharmacology , Cell Line, Tumor , Hepatocyte Growth Factor/pharmacology , Humans , Neoplasm Invasiveness/prevention & control , Neoplasm Metastasis/drug therapy , Neoplasm Metastasis/prevention & control , Proto-Oncogene Proteins c-met/drug effects , Receptors, Growth Factor/drug effects
10.
J Med Chem ; 52(24): 7962-5, 2009 Dec 24.
Article in English | MEDLINE | ID: mdl-19902954

ABSTRACT

A series of 3-aryl-4-isoxazolecarboxamides identified from a high-throughput screening campaign as novel, potent small molecule agonists of the human TGR5 G-protein coupled receptor is described. Subsequent optimization resulted in the rapid identification of potent exemplars 6 and 7 which demonstrated improved GLP-1 secretion in vivo via an intracolonic dose coadministered with glucose challenge in a canine model. These novel TGR5 receptor agonists are potentially useful therapeutics for metabolic disorders such as type II diabetes and its associated complications.


Subject(s)
Isoxazoles/pharmacology , Receptors, G-Protein-Coupled/agonists , Amides/chemistry , Amides/pharmacokinetics , Amides/pharmacology , Animals , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/metabolism , Disease Models, Animal , Dogs , Glucagon-Like Peptide 1/metabolism , Glucose/administration & dosage , Humans , Isoxazoles/chemistry , Isoxazoles/pharmacokinetics , Rats
11.
Bioorg Med Chem Lett ; 18(15): 4360-3, 2008 Aug 01.
Article in English | MEDLINE | ID: mdl-18606544

ABSTRACT

We report the synthesis and in vitro activity of a series of novel pyrrolidinyl pyridones and pyrazinones as potent inhibitors of prolyl oligopeptidase (POP). Within this series, compound 39 was co-crystallized within the catalytic site of a human chimeric POP protein which provided a more detailed understanding of how these inhibitors interacted with the key residues within the catalytic pocket.


Subject(s)
Pyridones/chemical synthesis , Pyridones/pharmacology , Pyrrolidines/chemical synthesis , Pyrrolidines/pharmacology , Serine Endopeptidases/drug effects , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Brain/drug effects , Brain/metabolism , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Drug Design , Humans , Molecular Conformation , Molecular Structure , Prolyl Oligopeptidases , Pyridones/blood , Pyridones/chemistry , Pyrrolidines/blood , Pyrrolidines/chemistry , Serine Proteinase Inhibitors/blood , Serine Proteinase Inhibitors/chemistry
12.
Am J Physiol Endocrinol Metab ; 293(6): E1810-9, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17911345

ABSTRACT

Fat depot sizes peak in middle age but decrease by advanced old age. This phenomenon is associated with ectopic fat deposition, decreased adipocyte size, impaired differentiation of preadipocytes into fat cells, decreased adipogenic transcription factor expression, and increased fat tissue inflammatory cytokine generation. To define the mechanisms contributing to impaired adipogenesis with aging, we examined the release of TNFalpha, which inhibits adipogenesis, and the expression of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), which blocks activity of adipogenic C/EBP family members, in preadipocytes cultured from young, middle-aged, and old rats. Medium conditioned by fat tissue, as well as preadipocytes, from old rats impeded lipid accumulation by preadipocytes from young animals. More TNFalpha was released by preadipocytes from old than young rats. Differences in TNFalpha-converting enzyme, TNFalpha degradation, or the presence of macrophages in cultures were not responsible. TNFalpha induced rat preadipocyte CHOP expression. CHOP was higher in undifferentiated preadipocytes from old than younger animals. Overexpression of CHOP in young rat preadipocytes inhibited lipid accumulation. TNFalpha short interference RNA reduced CHOP and partially restored lipid accumulation in old rat preadipocytes. CHOP normally increases during late differentiation, potentially modulating the process. This late increase in CHOP was not affected substantially by aging: CHOP was similar in differentiating preadipocytes and fat tissue from old and young animals. Hypoglycemia, which normally causes an adaptive increase in CHOP, was less effective in inducing CHOP in preadipocytes from old than younger animals. Thus increased TNFalpha release by undifferentiated preadipocytes with elevated basal CHOP contributes to impaired adipogenesis with aging.


Subject(s)
Adipocytes/metabolism , Adipogenesis/drug effects , Aging/physiology , Transcription Factor CHOP/metabolism , Tumor Necrosis Factor-alpha/metabolism , ADAM Proteins/metabolism , ADAM17 Protein , Adipocytes/cytology , Adipocytes/drug effects , Adipose Tissue/cytology , Adipose Tissue/metabolism , Animals , Blotting, Western , Cells, Cultured , Coculture Techniques , Culture Media, Conditioned/pharmacology , Epididymis/cytology , Kidney/cytology , Male , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/genetics , Rats , Rats, Inbred BN , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor CHOP/genetics , Transfection , Tumor Necrosis Factor-alpha/genetics
13.
J Cell Sci ; 120(Pt 6): 943-52, 2007 Mar 15.
Article in English | MEDLINE | ID: mdl-17344430

ABSTRACT

Kit ligand (Kitl), the ligand for the Kit receptor tyrosine kinase, plays important roles in hematopoiesis, gametogenesis and melanogenesis. Kitl is synthesized as a membrane-anchored precursor that can be processed to produce the soluble growth factor. Here, we evaluated the role of ADAM (a disintegrin and metalloprotease) metalloproteases in ectodomain shedding of Kitl. We found that both ADAM17 and ADAM19 affect Kitl1 shedding, albeit in different ways. Overexpression of ADAM19 resulted in decreased levels of Endo-H-resistant mature Kitl1, thereby reducing the amount of Kitl that is shed from cells following stimulation with phorbol esters. ADAM17 was identified as the major phorbol-ester-stimulated sheddase of Kitl1, whereas ADAMs 8, 9, 10, 12 and 15 were not required for this process. ADAM17 also emerged as the major constitutive and phorbol-ester-stimulated sheddase of Kitl2 in mouse embryonic fibroblasts. Mutagenesis of the juxtamembrane domain of Kitl2 showed no stringent sequence requirement for cleavage by ADAM17, although two nonadjacent stretches of four amino acid residues were identified that are required for Kitl2 shedding. Taken together, this study identifies a novel sheddase, ADAM17, for Kitl1 and Kitl2, and demonstrates that ADAM19 can reduce ADAM17-dependent phorbol-ester-stimulated Kitl1 ectodomain shedding.


Subject(s)
ADAM Proteins/metabolism , Protein Precursors/metabolism , Stem Cell Factor/metabolism , Tetradecanoylphorbol Acetate/pharmacology , ADAM17 Protein , Alternative Splicing , Amino Acid Sequence , Animals , COS Cells , Cells, Cultured , Chlorocebus aethiops , Mice , Molecular Sequence Data , Protein Processing, Post-Translational , Protein Structure, Tertiary
14.
Nat Immunol ; 7(12): 1293-8, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17072319

ABSTRACT

CD23, the low-affinity immunoglobulin E receptor, is an important modulator of the allergic response and of diseases such as rheumatoid arthritis. The proteolytic release of CD23 from cells is considered a key event in the allergic response. Here we used loss-of-function and gain-of-function experiments with cells lacking or overexpressing candidate CD23-releasing enzymes (ADAM8, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM19 and ADAM33), ADAM-knockout mice and a selective inhibitor to identify ADAM10 as the main CD23-releasing enzyme in vivo. Our findings provide a likely target for the treatment of allergic reactions and set the stage for further studies of the involvement of ADAM10 in CD23-dependent pathologies.


Subject(s)
ADAM Proteins/immunology , ADAM Proteins/metabolism , Amyloid Precursor Protein Secretases/immunology , Amyloid Precursor Protein Secretases/metabolism , Membrane Proteins/immunology , Membrane Proteins/metabolism , Receptors, IgE/immunology , Receptors, IgE/metabolism , ADAM10 Protein , Animals , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Fibroblasts/immunology , Fibroblasts/metabolism , Flow Cytometry , Humans , Immunoblotting , Mice , Mice, Knockout , Transfection
15.
Comb Chem High Throughput Screen ; 8(4): 327-39, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16101009

ABSTRACT

We report a systematic analysis of the P1' and P2' substrate specificity of TNF-alpha converting enzyme (TACE) using a peptide library and a novel analytical method, and we use the substrate specificity information to design novel reverse hydroxamate inhibitors. Initial truncation studies, using the amino acid sequence around the cleavage site in precursor-TNF-alpha, showed that good turnover was obtained with the peptide DNP-LAQAVRSS-NH2. Based on this result, 1000 different peptide substrates of the form Biotin-LAQA-P1'-P2'-SSK(DNP)-NH2 were prepared, with 50 different natural and unnatural amino acids at P1' in combination with 20 different amino acids at P2'. The peptides were pooled, treated with purified microsomal TACE, and the reaction mixtures were passed over a streptavidin affinity column to remove unreacted substrate and the N-terminal biotinylated product. C-terminal cleavage products not binding to streptavidin were subjected to liquid chromatography/mass spectrometry analysis where individual products were identified and semiquantitated. 25 of the substrates were resynthesized as discrete peptides and assayed with recombinant TACE. The experiments show that recombinant TACE prefers lipophilic amino acids at the P1' position, such as phenylglycine, homophenylalanine, leucine and valine. At the P2' position, TACE can accommodate basic amino acids, such as arginine and lysine, as well as certain non-basic amino acids such as citrulline, methionine sulfoxide and threonine. These substrate preferences were used in the design of novel reverse hydroxamate TACE inhibitors with phenethyl and 5-methyl-thiophene-methyl side-chains at P1', and threonine and nitro-arginine at P2'.


Subject(s)
Drug Evaluation, Preclinical/methods , Metalloendopeptidases/antagonists & inhibitors , Metalloendopeptidases/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Interaction Mapping/methods , ADAM Proteins , ADAM17 Protein , Binding Sites , Biotin/chemistry , Chromatography, Liquid/methods , Drug Design , Image Processing, Computer-Assisted , Mass Spectrometry/methods , Metalloendopeptidases/genetics , Models, Molecular , Peptide Library , Peptides/chemistry , Peptides/pharmacology , Protein Conformation , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Structure-Activity Relationship , Substrate Specificity , Tumor Necrosis Factor-alpha/metabolism
16.
Comb Chem High Throughput Screen ; 8(2): 161-71, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15777180

ABSTRACT

The transmembrane metzinkin-proteases of the ADAM (a disintegrin and a metalloproteinase)-family ADAM10 and ADAM 17 are both implicated in the ectodomain shedding of various cell surface molecules including the IL6-receptor and the transmembrane chemokines CX3CL1 and CXCL16. These molecules are constitutively released from cultured cells, a process that can be rapidly enhanced by cell stimulation with phorbol esters such as PMA. Recent research supports the view that the constitutive cleavage predominantly involves ADAM10 while the inducible one is mediated to a large extent by ADAM17. We here describe the discovery of hydroxamate compounds with different potency against ADAM10 and ADAM17 and different ability to block constitutive and inducible cleavage of IL6R, CX3CL1 and CXCL16 by the two proteases. By screening a number of hydroxamate inhibitors for the inhibition of recombinant metalloproteinases, a compound was found inhibiting ADAM10 with more than 100-fold higher potency than ADAM17, which may be explained by an improved fit of the compound to the S1' specificity pocket of ADAM10 as compared to that of ADAM17. In cell-based cleavage experiments this compound (GI254023X) potently blocked the constitutive release of IL6R, CX3CL1 and CXCL16, which was in line with the reported involvement of ADAM10 but not ADAM17 in this process. By contrast, the compound did not affect the PMA-induced shedding, which was only blocked by GW280264X, a potent inhibitor of ADAM17. As expected, GI254023X did not further decrease the residual release of CX3CL1 and CXCL16 in ADAM10-deficient cells verifying that the compound's effect on the constitutive shedding of these molecules was exclusively due to the inhibition of ADAM10. Thus, GI254023X may by of use as a preferential inhibitor of constitutive shedding events without effecting the inducible shedding in response to agonists acting similar to PMA.


Subject(s)
Endopeptidases/metabolism , Hydroxamic Acids/pharmacology , Membrane Proteins/metabolism , Metalloendopeptidases/metabolism , Phorbol Esters/pharmacology , Protease Inhibitors/pharmacology , ADAM Proteins , ADAM17 Protein , Amyloid Precursor Protein Secretases , Cells, Cultured , Chemokine CX3CL1 , Chemokines/metabolism , Chemokines, CX3C/metabolism , Chemokines, CXC/metabolism , Drug Interactions , Endopeptidases/drug effects , Hydroxamic Acids/chemistry , Interleukin-6/metabolism , Membrane Proteins/drug effects , Metalloendopeptidases/antagonists & inhibitors , Phorbol Esters/metabolism
17.
J Biol Chem ; 279(6): 4241-9, 2004 Feb 06.
Article in English | MEDLINE | ID: mdl-14638693

ABSTRACT

Protein ectodomain shedding, the proteolytic release of the extracellullar domain of membrane-tethered proteins, can dramatically affect the function of cell surface receptors, growth factors, cytokines, and other proteins. In this study, we evaluated the activities involved in ectodomain shedding of p75NTR, a neurotrophin receptor with critical roles in neuronal differentiation and survival. p75NTR is shed in a variety of cell types, including dorsal root ganglia cells and PC12 cells. In Chinese hamster ovary cells, inhibitors of the MEK/ERK and p38 MAP kinase pathways uncovered distinct signaling pathways required for the constitutive and stimulated shedding of p75NTR. Stimulated p75NTR shedding is abrogated in M2 mutant Chinese hamster ovary cells that lack functional tumor necrosis factor-alpha converting enzyme (TACE, also referred to as ADAM17) and in cells isolated from adam17-/- mice, but not in cells from adam9/12/15-/- or adam10-/- mice. Stimulated p75(NTR) shedding is strongly reduced by deletion of 15 amino acid residues in its extracellular membrane-proximal stalk domain. However, similar to other shed proteins, point mutations and overlapping shorter deletions within this region have little or no effect on shedding. Because ectodomain shedding of p75NTR releases a soluble ectodomain and could also be a prerequisite for its regulated intramembrane proteolysis, these findings may have important implications for the functional regulation of p75NTR.


Subject(s)
Metalloendopeptidases/metabolism , Receptors, Nerve Growth Factor/metabolism , ADAM Proteins , ADAM17 Protein , Amino Acid Sequence , Animals , Binding Sites , CHO Cells , Cells, Cultured , Cricetinae , In Vitro Techniques , MAP Kinase Signaling System , Metalloendopeptidases/chemistry , Metalloendopeptidases/deficiency , Metalloendopeptidases/genetics , Mice , Mice, Knockout , Molecular Sequence Data , Mutation , PC12 Cells , Protein Structure, Tertiary , Rats , Receptor, Nerve Growth Factor , Receptors, Nerve Growth Factor/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid
18.
Drug Metab Dispos ; 31(7): 870-7, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12814963

ABSTRACT

A series of N-hydroxyformamide tumor necrosis factor-alpha converting enzyme (TACE)/matrix metalloprotease (MMP) inhibitors were evaluated for their potential to induce human cytochrome P450 3A (CYP3A). Two in vitro assays were used: 1) a cell-based reporter gene assay for activation of the pregnane X receptor (PXR), and 2) a primary "sandwich" culture of human hepatocytes. Approximately 50 TACE/MMP inhibitors were evaluated in the human PXR assay. A range of PXR activation was observed, 0 to 150% of the activation of the known human CYP3A inducer rifampicin. Three TACE/MMP inhibitors were evaluated in rat and human hepatocytes. Significantly higher PXR activation/CYP3A induction was observed in PXR/hepatocyte models, respectively, for (2R,3S) 3-(formyl-hydroxyamino)-2-(2-methyl-1-propyl)-4-methylpentanoic acid [(1S,2S)-2-methyl-1-(2-pyridylcarbamoyl)-1-butyl]amide (GW3333) compared with (2R,3S)-6,6,6-trifluoro-3-[formyl(hydroxy)amino]-2-isobutyl-N-[(1S,2R)-2-methoxy-1-[(1,3-thiazol-2-ylamino)carbonyl]propyl]hexanamide (GW6495) and (2R)-N-[(1S)-2,2-dimethyl-1-[(methylamino)carbonyl]-propyl]-2-[(1S)-1-[formyl(hydroxy)amino]ethyl]-5-phenylpentanamide (GI4023). The CYP3A induction level achieved with GW3333 at a concentration of approximately 10 microM in human hepatocytes was comparable to that achieved with rifampicin at a concentration of 10 microM. The extent of rodent CYP3A induction caused by GW3333 was confirmed in vivo after daily oral administration for 14 days to rats. In conclusion, GW3333 is a potential inducer of CYP3A expression in vivo in humans, but other N-hydroxyformamides are less likely to induce CYP3A.


Subject(s)
Aryl Hydrocarbon Hydroxylases/biosynthesis , Formamides/pharmacology , Hepatocytes/drug effects , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/antagonists & inhibitors , Oxidoreductases, N-Demethylating/biosynthesis , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/metabolism , ADAM Proteins , ADAM17 Protein , Administration, Oral , Amides/administration & dosage , Amides/pharmacokinetics , Aminopyridines/administration & dosage , Aminopyridines/blood , Aminopyridines/pharmacokinetics , Animals , Aryl Hydrocarbon Hydroxylases/drug effects , Cell Culture Techniques , Cytochrome P-450 CYP3A , Dipeptides/administration & dosage , Dipeptides/blood , Dipeptides/pharmacokinetics , Dose-Response Relationship, Drug , Drug Evaluation , Drug Evaluation, Preclinical , Enzyme Induction , Formamides/chemistry , Hepatocytes/metabolism , Humans , Male , Matrix Metalloproteinases/administration & dosage , Matrix Metalloproteinases/pharmacokinetics , Oxidoreductases, N-Demethylating/drug effects , Pregnane X Receptor , Rats , Rats, Wistar , Receptors, Cytoplasmic and Nuclear/drug effects , Receptors, Steroid/drug effects , Thiazoles/administration & dosage , Thiazoles/pharmacokinetics
20.
J Biol Chem ; 278(25): 22331-40, 2003 Jun 20.
Article in English | MEDLINE | ID: mdl-12682046

ABSTRACT

ADAMs are membrane-anchored glycoproteins with functions in fertilization, heart development, neurogenesis, and protein ectodomain shedding. Here we report an evaluation of the catalytic activity of recombinantly expressed soluble forms of ADAM19, a protein that is essential for cardiovascular morphogenesis. Proteolytic activity of soluble forms of ADAM19 was first demonstrated by their autocatalytic removal of a purification tag (Myc-His) and their ability to cleave myelin basic protein and the insulin B chain. The metalloprotease activity of ADAM19 is sensitive to the hydroxamic acid-type metalloprotease inhibitor BB94 (batimastat) but not to tissue inhibitors of metalloproteases (TIMPs) 1-3. Moreover, ADAM19 cleaves peptides corresponding to the known cleavage sites of tumor necrosis factor-alpha (TNF-alpha), TNF-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin ligand), and kit ligand-1 (KL-1) in vitro. Although ADAM19 is not required for shedding of TNFalpha and TRANCE in mouse embryonic fibroblasts, its overexpression in COS-7 cells results in strongly increased TRANCE shedding. This suggests a potential role for ADAM19 in shedding TRANCE in cells where both molecules are highly expressed, such as in osteoblasts. Interestingly, our results also indicate that ADAM19 can function as a negative regulator of KL-1 shedding in both COS-7 cells and mouse embryonic fibroblasts, instead of acting directly on KL-1. The identification of potential in vitro substrates offers the basis for further functional studies of ADAM19 in cells and in mice.


Subject(s)
Disintegrins , Membrane Proteins/metabolism , Metalloendopeptidases , Metalloproteases , Muscle Proteins/metabolism , ADAM Proteins , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Catalysis , Cell Line , Chlorocebus aethiops , Fibroblasts/cytology , Fibroblasts/metabolism , Kinetics , Membrane Proteins/chemistry , Membrane Proteins/deficiency , Membrane Proteins/genetics , Mice , Mice, Knockout , Molecular Sequence Data , Muscle Proteins/chemistry , Muscle Proteins/deficiency , Muscle Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spodoptera , Transfection
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