ABSTRACT
A pharmacophore model of the P1' site, specific for aggrecanase, was defined using the specificity studies of the matrix metalloproteinases and the similar biological activity of aggrecanase and MMP-8. Incorporation of the side chain of a tyrosine residue into compound 1 as the P1' group provided modest selectivity for aggrecanase over MMP-1, -2, and -9. A cis-(1S)(2R)-amino-2-indanol scaffold was incorporated as a tyrosine mimic (P2') to conformationally constrain 2. Further optimization resulted in compound 11, a potent, selective, and orally bioavailable inhibitor of aggrecanase.
Subject(s)
Asparagine/chemical synthesis , Endopeptidases/metabolism , Hydroxamic Acids/chemical synthesis , Protease Inhibitors/chemical synthesis , Administration, Oral , Animals , Asparagine/analogs & derivatives , Asparagine/chemistry , Asparagine/pharmacokinetics , Asparagine/pharmacology , Biological Availability , Dogs , Drug Design , Endopeptidases/chemistry , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacokinetics , Hydroxamic Acids/pharmacology , Matrix Metalloproteinase 1/chemistry , Matrix Metalloproteinase 2/chemistry , Matrix Metalloproteinase 8/chemistry , Matrix Metalloproteinase 9/chemistry , Models, Molecular , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacokinetics , Protease Inhibitors/pharmacology , Protein Binding , Stereoisomerism , Structure-Activity RelationshipABSTRACT
SAR exploration at P1' using an anti-succinate-based macrocyclic hydroxamic acid as a template led to the identification of several bulky biphenylmethyl P1' derivatives which confer potent porcine TACE and anti-TNF-alpha cellular activities with high selectivity versus most of the MMPs screened. Our studies demonstrate for the first time that TACE has a larger S1' pocket in comparison to MMPs and that potent and selective TACE inhibitors can be achieved by incorporation of sterically bulky P1' residues.
Subject(s)
Heterocyclic Compounds, 1-Ring/chemical synthesis , Hydroxamic Acids/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/chemical synthesis , Tumor Necrosis Factor-alpha/antagonists & inhibitors , ADAM Proteins , ADAM17 Protein , Binding Sites , Heterocyclic Compounds, 1-Ring/chemistry , Heterocyclic Compounds, 1-Ring/pharmacology , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacology , Lipopolysaccharides/pharmacology , Models, Molecular , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Protein Binding , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/metabolismABSTRACT
To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of =0.2 microM in whole blood assay (WBA). Although the P3' area can accommodate a broad array of structurally diversified functional groups including polar residues, hydrophobic residues, and amino and carboxylic acid moieties, in both the cyclophane series and the cyclic carbamate series, a glycine residue at P3' was identified as a critical structural component to achieve both good in vitro potency and good oral activity. With a glycine residue at P3', an N-methylamide at P4' provided the best cyclophane analogue, SL422 (WBA IC(50) = 0.22 microM, LPS-mouse ED(50) = 15 mg/kg, po), whereas a morpholinylamide at P4' afforded the most potent and most orally active cyclic carbamate analogue, SP057 (WBA IC(50) = 0.067 microM, LPS-mouse ED(50) = 2.3 mg/kg, po). Further profiling for SL422 and SP057 showed that these macrocyclic compounds are potent TACE inhibitors, with K(i) values of 12 and 4.2 nM in the porcine TACE assay, and are broad-spectrum MMP inhibitors. Pharmacokinetic studies in beagle dogs revealed that SL422 and SP057 are orally bioavailable, with oral bioavailabilities of 11% and 23%, respectively.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Hydroxamic Acids/chemical synthesis , Lactams/chemical synthesis , Metalloendopeptidases/antagonists & inhibitors , Tumor Necrosis Factor-alpha/antagonists & inhibitors , ADAM Proteins , ADAM17 Protein , Administration, Oral , Animals , Biological Availability , Carbamates/chemical synthesis , Carbamates/chemistry , Carbamates/pharmacokinetics , Carbamates/pharmacology , Dogs , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , Humans , Hydroxamic Acids/chemistry , Hydroxamic Acids/pharmacokinetics , Hydroxamic Acids/pharmacology , In Vitro Techniques , Lactams/chemistry , Lactams/pharmacokinetics , Lactams/pharmacology , Male , Mice , Stereoisomerism , Structure-Activity Relationship , Tumor Necrosis Factor-alpha/analysisABSTRACT
The MAP kinase pathway has been well-characterized as a cascade of sequential protein phosphorylation events leading to the upregulation of a variety of genes in response to growth factors and mitogens. We are interested in the role of these kinases in inflammation and have thus examined their activity in vivo using TPA-induced ear edema in the mouse as a model of inflammation. We show that the activities of both ERK-1 and ERK-2 are upregulated in this model in response to TPA. Increased levels of ERK phosphorylation are measurable as early as 15 min poststimulation and reach a level 8-fold over controls at 4 h. In contrast, minimal activation of JNK or p38 is observed. Topical treatment of ears with the MEK inhibitor, U0126, prevents ERK phosphorylation and ear swelling in a dose-dependent manner in this model. These results suggest that the MEK/ERK pathway is important during an inflammatory response in vivo.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Animals , Butadienes/pharmacology , Butadienes/therapeutic use , Edema/prevention & control , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , JNK Mitogen-Activated Protein Kinases , MAP Kinase Kinase 1 , Male , Mice , Mitogen-Activated Protein Kinase 3 , Mitogen-Activated Protein Kinases/metabolism , Nitriles/pharmacology , Nitriles/therapeutic use , Phosphorylation , Protein Serine-Threonine Kinases/antagonists & inhibitorsABSTRACT
Aggrecan is responsible for the mechanical properties of cartilage. One of the earliest changes observed in arthritis is the depletion of cartilage aggrecan due to increased proteolytic cleavage within the interglobular domain. Two major sites of cleavage have been identified in this region at Asn(341)-Phe(342) and Glu(373)-Ala(374). While several matrix metalloproteinases have been shown to cleave at Asn(341)-Phe(342), an as yet unidentified protein termed "aggrecanase" is responsible for cleavage at Glu(373)-Ala(374) and is hypothesized to play a pivotal role in cartilage damage. We have identified and cloned a novel disintegrin metalloproteinase with thrombospondin motifs that possesses aggrecanase activity, ADAMTS11 (aggrecanase-2), which has extensive homology to ADAMTS4 (aggrecanase-1) and the inflammation-associated gene ADAMTS1. ADAMTS11 possesses a number of conserved domains that have been shown to play a role in integrin binding, cell-cell interactions, and extracellular matrix binding. We have expressed recombinant human ADAMTS11 in insect cells and shown that it cleaves aggrecan at the Glu(373)-Ala(374) site, with the cleavage pattern and inhibitor profile being indistinguishable from that observed with native aggrecanase. A comparison of the structure and expression patterns of ADAMTS11, ADAMTS4, and ADAMTS1 is also described. Our findings will facilitate the study of the mechanisms of cartilage degradation and provide targets to search for effective inhibitors of cartilage depletion in arthritic disease.
Subject(s)
Endopeptidases/genetics , Metalloendopeptidases/genetics , ADAM Proteins , ADAMTS5 Protein , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA, Complementary , Endopeptidases/isolation & purification , Endopeptidases/metabolism , Humans , Metalloendopeptidases/metabolism , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
In arthritic diseases there is a gradual erosion of cartilage that leads to a loss of joint function. Aggrecan, which provides cartilage with its properties of compressibility and elasticity, is the first matrix component to undergo measurable loss in arthritis. This loss of aggrecan appears to be due to an increased rate of degradation, that can be attributed to proteolytic cleavage of the core protein within the interglobular domain (IGD). Two major sites of cleavage have been identified within the IGD. One, between the amino acids Asn341-Phe342, where the matrix metalloproteinases (MMPs) have been shown to clip; and the other, between Glu373-Ala374, which is attributed to a novel protease, "aggrecanase." We have generated aggrecanase in conditioned media from IL-1-stimulated bovine nasal cartilage and have used an enzymatic assay to evaluate this proteinase activity. In these studies we follow the generation of aggrecanase and MMPs in response to IL-1 in this system and examine the contribution of these enzymes in aggrecan degredation. Our data suggest that aggrecanase is a key enzyme in cartilage aggrecan degradation that represents a novel target for cartilage protection therapy in arthritis.
Subject(s)
Cartilage/enzymology , Endopeptidases/metabolism , Matrix Metalloproteinase 3/metabolism , Animals , Cartilage/drug effects , Cattle , Endopeptidases/genetics , Interleukin-1/pharmacology , Kinetics , Matrix Metalloproteinase 3/genetics , Metalloendopeptidases/metabolism , Nasal Septum , Organ Culture Techniques , Time FactorsABSTRACT
We purified, cloned, and expressed aggrecanase, a protease that is thought to be responsible for the degradation of cartilage aggrecan in arthritic diseases. Aggrecanase-1 [a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4)] is a member of the ADAMTS protein family that cleaves aggrecan at the glutamic acid-373-alanine-374 bond. The identification of this protease provides a specific target for the development of therapeutics to prevent cartilage degradation in arthritis.
Subject(s)
Extracellular Matrix Proteins , Metalloendopeptidases/chemistry , Metalloendopeptidases/genetics , ADAM Proteins , ADAMTS1 Protein , ADAMTS4 Protein , Aggrecans , Amino Acid Sequence , Arthritis/drug therapy , Cartilage/metabolism , Catalytic Domain , Cloning, Molecular , Disintegrins/chemistry , Disintegrins/metabolism , Humans , Hydroxamic Acids/pharmacology , Interleukin-1/pharmacology , Lectins, C-Type , Metalloendopeptidases/isolation & purification , Metalloendopeptidases/metabolism , Molecular Sequence Data , Procollagen N-Endopeptidase , Protease Inhibitors/pharmacology , Protein Sorting Signals , Proteoglycans/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence AnalysisABSTRACT
The discovery of terphenyl derivatives as highly selective COX-2 inhibitors resulted from our efforts to overcome poor pharmacokinetics demonstrated by the COX-2 selective diarylthiophene DuP 697 [2-bromo-4-(4'-sulfonylmethyl)phenyl-5-(4'-fluoro)phenylthiophe ne]. Detailed SAR related to the ortho-biphenyls and variants of the central ring are described herein.
Subject(s)
Cyclooxygenase Inhibitors/chemistry , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacokinetics , Isoenzymes/drug effects , Molecular Structure , Prostaglandin-Endoperoxide Synthases/drug effects , Structure-Activity Relationship , Thiophenes/pharmacokineticsABSTRACT
Activation of the extracellular signal-regulated kinase (ERK) pathway has been shown to occur in monocytes following stimulation with LPS. However, the importance of this event for monocyte function is not clear. To address this issue, we used the novel MAP/ERK kinase (MEK) inhibitor, U0126. Stimulation of monocytes with LPS resulted in activation of the mitogen-activated protein kinase (MAPK) family members ERK, Jun NH2-terminal kinase (JNK), and p38. Treatment of monocytes with LPS in the presence of U0126 blocked the activation of ERK1 and ERK2. However, the activation of Jun NH2-terminal kinase and p38 family members was not affected by the compound, confirming the selectivity of U0126. To examine the effects of MEK inhibition on monocyte function, we measured production of the cytokines IL-1, IL-8, and TNF, as well as PGE2. Monocytes treated with LPS in the presence of U0126 failed to release IL-1, IL-8, TNF, or PGE2. The failure to secrete IL-1 and TNF was due to decreased levels of mRNA. These results demonstrate that activation of MEK/ERK is critical for cytokine and PGE2 production by monocytes in response to LPS.
Subject(s)
Cytokines/biosynthesis , Dinoprostone/biosynthesis , Lipopolysaccharides/pharmacology , Mitogen-Activated Protein Kinases , Monocytes/enzymology , Monocytes/metabolism , Protein Kinase Inhibitors , Butadienes/pharmacology , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cells, Cultured , Cyclooxygenase 2 , Cyclooxygenase 2 Inhibitors , Cyclooxygenase Inhibitors/pharmacology , Cytokines/antagonists & inhibitors , Cytokines/genetics , Dinoprostone/antagonists & inhibitors , Enzyme Activation/immunology , Humans , Isoenzymes/biosynthesis , Isoenzymes/genetics , JNK Mitogen-Activated Protein Kinases , Macrophage Activation/drug effects , Macrophage Activation/immunology , Membrane Proteins , Mitogen-Activated Protein Kinase Kinases , Monocytes/immunology , Nitriles/pharmacology , Phosphorylation/drug effects , Prostaglandin-Endoperoxide Synthases/biosynthesis , Prostaglandin-Endoperoxide Synthases/genetics , Transcription, Genetic/drug effects , Transcription, Genetic/immunology , Up-Regulation/drug effects , Up-Regulation/genetics , p38 Mitogen-Activated Protein KinasesABSTRACT
The compound U0126 (1,4-diamino-2,3-dicyano-1, 4-bis[2-aminophenylthio]butadiene) was identified as an inhibitor of AP-1 transactivation in a cell-based reporter assay. U0126 was also shown to inhibit endogenous promoters containing AP-1 response elements but did not affect genes lacking an AP-1 response element in their promoters. These effects of U0126 result from direct inhibition of the mitogen-activated protein kinase kinase family members, MEK-1 and MEK-2. Inhibition is selective for MEK-1 and -2, as U0126 shows little, if any, effect on the kinase activities of protein kinase C, Abl, Raf, MEKK, ERK, JNK, MKK-3, MKK-4/SEK, MKK-6, Cdk2, or Cdk4. Comparative kinetic analysis of U0126 and the MEK inhibitor PD098059 (Dudley, D. T., Pang, L., Decker, S. J., Bridges, A. J., and Saltiel, A. R. (1995) Proc. Natl. Acad. Sci U. S. A. 92, 7686-7689) demonstrates that U0126 and PD098059 are noncompetitive inhibitors with respect to both MEK substrates, ATP and ERK. We further demonstrate that the two compounds bind to deltaN3-S218E/S222D MEK in a mutually exclusive fashion, suggesting that they may share a common or overlapping binding site(s). Quantitative evaluation of the steady state kinetics of MEK inhibition by these compounds reveals that U0126 has approximately 100-fold higher affinity for deltaN3-S218E/S222D MEK than does PD098059. We further tested the effects of these compounds on the activity of wild type MEK isolated after activation from stimulated cells. Surprisingly, we observe a significant diminution in affinity of both compounds for wild type MEK as compared with the deltaN3-S218E/S222D mutant enzyme. These results suggest that the affinity of both compounds is mediated by subtle conformational differences between the two activated MEK forms. The MEK affinity of U0126, its selectivity for MEK over other kinases, and its cellular efficacy suggest that this compound will serve as a powerful tool for in vitro and cellular investigations of mitogen-activated protein kinase-mediated signal transduction.
Subject(s)
Butadienes/pharmacology , Enzyme Inhibitors/pharmacology , Nitriles/pharmacology , Protein Kinase Inhibitors , Animals , Butadienes/chemistry , COS Cells , DNA/metabolism , Enzyme Inhibitors/chemistry , Flavonoids/pharmacology , Gene Expression Regulation/drug effects , Kinetics , MAP Kinase Kinase 1 , Mitogen-Activated Protein Kinase Kinases , Nitriles/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-fos/antagonists & inhibitors , Proto-Oncogene Proteins c-jun/antagonists & inhibitors , Proto-Oncogene Proteins c-raf/metabolism , Signal Transduction/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factor AP-1/metabolismABSTRACT
Three mitogen-activated protein kinase pathways are up-regulated during the activation of T lymphocytes, the extracellular signal-regulated kinase (ERK), Jun NH2-terminal kinase, and p38 mitogen-activated protein kinase pathways. To examine the effects of blocking the ERK pathway on T cell activation, we used the inhibitor U0126, which has been shown to specifically block mitogen-activated protein kinase/ERK kinase (MEK), the kinase upstream of ERK. This compound inhibited T cell proliferation in response to antigenic stimulation or cross-linked anti-CD3 plus anti-CD28 Abs, but had no effect on IL-2-induced proliferation. The block in T cell proliferation was mediated by down-regulating IL-2 mRNA levels. Blocking Ag-induced proliferation by inhibiting MEK did not induce anergy, unlike treatments that block entry into the cell cycle following antigenic stimulation. Surprisingly, induction of anergy in T cells exposed to TCR cross-linking in the absence of costimulation was also not affected by blocking MEK, unlike cyclosporin A treatment that blocks anergy induction. These results suggest that inhibition of MEK prevents T cell proliferation in the short term, but does not cause any long-term effects on either T cell activation or induction of anergy. These findings may help determine the viability of using mitogen-activated protein kinase inhibitors as immune suppressants.
Subject(s)
Lymphocyte Activation , Protein Kinases/immunology , Signal Transduction/immunology , T-Lymphocytes/immunology , Animals , CD28 Antigens/immunology , CD3 Complex/immunology , Cell Division/immunology , Clone Cells , Interleukin-2/immunology , Mice , Mice, Inbred BALB C , Mitogen-Activated Protein Kinase Kinases , T-Lymphocytes/cytologyABSTRACT
Examination of the S1 area of the active site of pro-stromelysin has led us to the design of novel and potent inhibitors of matrix metalloproteinases containing constrained quaternary-hydroxy group at P1. The synthesis and biological activity of these compounds with variations at P1', P2', and P3' will be described.
Subject(s)
Hydroxamic Acids/chemistry , Metalloendopeptidases/antagonists & inhibitors , Protease Inhibitors/chemistry , Catalytic Domain , Collagenases/chemistry , Computer Simulation , Drug Design , Enzyme Precursors/antagonists & inhibitors , Enzyme Precursors/chemistry , Hydrogen Bonding , Hydroxamic Acids/chemical synthesis , Hydroxamic Acids/pharmacology , Indicators and Reagents , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 3/chemistry , Matrix Metalloproteinase Inhibitors , Metalloendopeptidases/chemistry , Models, Molecular , Molecular Conformation , Molecular Structure , Protease Inhibitors/chemical synthesis , Protease Inhibitors/pharmacology , Protein Conformation , Structure-Activity RelationshipABSTRACT
The structure-activity relationships of some tetracyclic heterocycles related to Brequinar were explored. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction are related to ring system, heteroatom placement, and pendant ring substitution.
Subject(s)
Biphenyl Compounds/chemistry , Heterocyclic Compounds/chemistry , Heterocyclic Compounds/pharmacology , Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Oxidoreductases Acting on CH-CH Group Donors , Dihydroorotate Dehydrogenase , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Lymphocyte Culture Test, Mixed , Oxidoreductases/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Structure-activity relationships were explored for some analogs of Brequinar having a linking atom between the 2-biphenyl substituent and the quinoline ring. Activities as inhibitors of dihydroorotate dehydrogenase and the mixed lymphocyte reaction were related to the overall shape and lipophilicity of the 2-substituent.
Subject(s)
Immunosuppressive Agents/chemistry , Immunosuppressive Agents/pharmacology , Biphenyl Compounds/chemistry , Carbon/chemistry , Lymphocyte Culture Test, Mixed , Structure-Activity RelationshipABSTRACT
In search of antiinflammatory drugs with a new mechanism of action, U0126 was found to functionally antagonize AP-1 transcriptional activity via noncompetitive inhibition of the dual specificity kinase MEK with an IC50 of 0.07 microM for MEK 1 and 0.06 microM for MEK 2. U0126 can undergo isomerization and cyclization reactions to form a variety of products, both chemically and in vivo, all of which exhibit less affinity for MEK and lower inhibition of AP-1 activity than parent, U0126.
Subject(s)
Butadienes/chemistry , Enzyme Inhibitors/chemistry , Nitriles/chemistry , Protein Serine-Threonine Kinases/antagonists & inhibitors , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacokinetics , Anti-Inflammatory Agents/pharmacology , Biotransformation , Butadienes/pharmacokinetics , Butadienes/pharmacology , Cyclization , Enzyme Inhibitors/pharmacokinetics , Enzyme Inhibitors/pharmacology , NF-kappa B/antagonists & inhibitors , Nitriles/pharmacokinetics , Nitriles/pharmacology , Rats , Transcription Factor AP-1/antagonists & inhibitorsABSTRACT
A series of isothiazolones that inhibit pro-(matrix metallo-proteinase) (proMMP) activation but do not inhibit the active enzyme are effective as cartilage protectants in bovine nasal cartilage organ culture, preventing interleukin-1 (IL-1)-induced proteoglycan (aggrecan) degradation without affecting its synthesis. These compounds were found to bind to prostromelysin (proMMP-3) in a non-dialysable and stoichiometric manner. Preincubation with cartilage-protectant isothiazolones prevented the binding of [14C]iodoacetamide to Cys75 of the MMP-3 propeptide, suggesting that the activity of these compounds involves their binding to the Cys75 of the MMP zymogen. Studies following chymotrypsin activation of proMMP-3 by SDS/PAGE indicated that altered processing of the 57 kDa zymogen to the active form occurred in the presence of compound. The 53 kDa intermediate seen on normal activation was not formed; instead a different intermediate appeared with a molecular mass of approx. 46 kDa. N-terminal sequence analysis indicated that this intermediate was formed by cleavage at the putative 4-aminophenylmercuric acid cleavage site. Importantly the 45 kDa active MMP-3 species formed in the presence of compound was one amino acid residue shorter than the native MMP-3. These results suggest that the inhibition of cartilage proteoglycan degradation by isothiazolones might be due to their ability to bind to the Cys75 in the propeptide region of the MMP zymogen and interfere with its normal activation process.
Subject(s)
Cartilage/metabolism , Enzyme Precursors/metabolism , Matrix Metalloproteinase 3/metabolism , Protease Inhibitors/pharmacology , Proteoglycans/metabolism , Thiazoles/pharmacology , Amino Acid Sequence , Animals , Cattle , Chromatography, High Pressure Liquid , Enzyme Activation , Enzyme Precursors/antagonists & inhibitors , Enzyme Precursors/chemistry , Kinetics , Matrix Metalloproteinase 3/chemistry , Matrix Metalloproteinase Inhibitors , Molecular Sequence Data , Nose , Organ Culture Techniques , Protein Processing, Post-Translational , Structure-Activity RelationshipABSTRACT
A series of 2,5-diarylisothiazolones is reported that inhibit the IL-1 beta-induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, nonpeptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease. These compounds are relatively resistant to reductive metabolism by liver microsomal preparations and appear to inhibit cartilage breakdown by interfering with the proteolytic activation of matrix metalloproteinases.
Subject(s)
Cartilage/drug effects , Enzyme Inhibitors/pharmacology , Interleukin-1/metabolism , Thiazoles/pharmacology , Animals , Cartilage/metabolism , Cattle , Hydrolysis , Interleukin-1/antagonists & inhibitors , Magnetic Resonance Spectroscopy , Organ Culture Techniques , Osteoarthritis/metabolism , Osteoarthritis/prevention & control , Spectrophotometry, Infrared , Structure-Activity Relationship , Thiazoles/chemistryABSTRACT
The active metabolite of leflunomide. A771726, is a novel immunosuppressive compound that has been shown to be a powerful antiproliferative agent for mononuclear and T-cells. The molecular mechanism of action for this compound has not been clearly established. In vitro cellular and enzymatic assays, however, demonstrate that leflunomide is an inhibitor of several protein tyrosine kinases, with IC50 values between 30 and 100 microM. The in vivo properties of A771726 are reminiscent of another immunosuppressive agent, brequinar sodium, which has been shown to be a nonomolar inhibitor (Ki = 10-30 nM) of the enzyme dihydroorotate dehydrogenase (DHODase). On the basis, we have investigated the effects of leflunomide and A771726 on the activity of purified recombinant human DHODase. We find that A771726 is a potent inhibitor of DHODase (Ki = 179 +/- 19 nM), while the parent compound, leflunomide, had no inhibitory effect at concentrations as high as 1 microM. Studies of the dependence of inhibition on the concentrations of the substrates ubiquinone and dihydroorotate demonstrate that A771726 is a competitive inhibitor of the ubiquinone binding site and is noncompetitive with respect to dihydroorotate. The potency of A771726 as a DHODase inhibitor is thus 100-100-fold greater than that reported for its inhibition of protein tyrosine kinases. These data suggest that an alternative explanation for the immunosuppressive efficacy of A771726 may be the potent inhibition of DHODase by this compound.
Subject(s)
Aniline Compounds/pharmacology , Enzyme Inhibitors/pharmacology , Hydroxybutyrates/pharmacology , Immunosuppressive Agents/pharmacology , Isoxazoles/pharmacology , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/antagonists & inhibitors , Crotonates , Dihydroorotate Dehydrogenase , Humans , Immunosuppressive Agents/metabolism , Isoxazoles/metabolism , Kinetics , Leflunomide , Nitriles , Oxidoreductases/genetics , Recombinant Proteins/antagonists & inhibitors , ToluidinesABSTRACT
Selected 15-, 32-, and 15,32-substituted lanosterol analogs are shown here to display time-dependent inactivation and lanosterol 14 alpha-methyl demethylase. These molecules are competitive with respect to substrate and require NADPH and O2 in order to display time dependence, thus supporting the premise that they are mechanism-based inactivators. Structural features required for lanosterol demethylation by the lanosterol demethylase such as nuclear double bond location and availability of an abstractable 15 alpha-proton are also essential elements for time-dependent inactivation. 32-(S)-Vinyllanost-8-en-3 beta,32-diol is a potent time-dependent inactivator (Kinact/Ki = 0.36 min-1 microM-1), while the 32-(R)-vinyllanost-8-en-3 beta,32-diol functions solely as a competitive demethylase inhibitor. These results support the premise that stereoselective oxidation occurs during lanosterol demethylation and that the 32-pro-S proton is abstracted during the demethylation reaction.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors , Lanosterol/analogs & derivatives , Oxidoreductases/antagonists & inhibitors , Binding, Competitive , Kinetics , Lanosterol/chemistry , Lanosterol/pharmacology , NADP/pharmacology , Oxygen/pharmacology , Stereoisomerism , Sterol 14-Demethylase , Structure-Activity RelationshipABSTRACT
A series of 15-, 32-, and 15,32-substituted lanost-8-en-3 beta-ols is described which function as inhibitors of cholesterol biosynthesis. These agents inhibit lanosterol 14 alpha-methyl demethylase activity as well as suppress HMG-CoA reduction activity in cultured cells. Several of these agents are extremely potent as both demethylase inhibitors and reductase suppressors, while others are more selective in their activities. Selected regio double bond isomers show preference for demethylase inhibition with the following order: delta 8 > delta 7 > delta 6 = unsaturated sterols. Comparisons also show that 4,4-dimethyl sterols are always more potent demethylase inhibitors and reductase suppressors than their 4,4-bisnomethyl counterparts. However, evaluation of an extensive oxylanosterol series leads us to conclude that demethylase inhibition and reductase suppression are not parallel in the same molecule. In addition, the oxylanosterols, but not the oxycholesterols, are able to disrupt coordinate regulation of HMG-CoA reductase from the LDL receptor. Thus, oxylanosterol treatment at levels which suppress reductase activity enhances LDL receptor activity. These results demonstrate that compounds can be made which (1) are selective reductase suppressors enabling dissection of the dual inhibitor nature of these compounds and (2) maximize reductase suppression and LDL receptor induction without demethylase inhibition which could lead to novel agents for serum cholesterol lowering.