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1.
Acta Crystallogr D Biol Crystallogr ; 67(Pt 9): 763-73, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21904029

ABSTRACT

The mitochondrial pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA. PDC activity is tightly regulated by four members of a family of pyruvate dehydrogenase kinase isoforms (PDK1-4), which phosphorylate and inactivate PDC. Recently, the development of specific inhibitors of PDK4 has become an especially important focus for the pharmaceutical management of diabetes and obesity. In this study, crystal structures of human PDK4 complexed with either AMPPNP, ADP or the inhibitor M77976 were determined. ADP-bound PDK4 has a slightly wider active-site cleft and a more disordered ATP lid compared with AMPPNP-bound PDK4, although both forms of PDK4 assume open conformations with a wider active-site cleft than that in the closed conformation of the previously reported ADP-bound PDK2 structure. M77976 binds to the ATP-binding pocket of PDK4 and causes local conformational changes with complete disordering of the ATP lid. M77976 binding also leads to a large domain rearrangement that further expands the active-site cleft of PDK4 compared with the ADP- and AMPPNP-bound forms. Biochemical analyses revealed that M77976 inhibits PDK4 with increased potency compared with the previously characterized PDK inhibitor radicicol. Thus, the present structures demonstrate for the first time the flexible and dynamic aspects of PDK4 in the open conformation and provide a basis for the development of novel inhibitors targeting the nucleotide-binding pocket of PDK4.


Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Protein Kinases/chemistry , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Adenylyl Imidodiphosphate/chemistry , Adenylyl Imidodiphosphate/metabolism , Crystallography, X-Ray , Humans , Protein Kinases/metabolism
2.
J Clin Invest ; 118(5): 1785-95, 2008 May.
Article in English | MEDLINE | ID: mdl-18382762

ABSTRACT

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor gamma-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.


Subject(s)
Antibodies, Monoclonal , Blood Platelets/immunology , Cyclic AMP/metabolism , Endocytosis/physiology , Platelet Aggregation Inhibitors , Platelet Membrane Glycoproteins/metabolism , Receptors, IgG/metabolism , Amino Acid Sequence , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Autoantibodies/genetics , Autoantibodies/immunology , Female , Humans , Macaca fascicularis , Male , Mice , Molecular Sequence Data , Platelet Adhesiveness/physiology , Platelet Aggregation Inhibitors/immunology , Platelet Aggregation Inhibitors/therapeutic use , Platelet Membrane Glycoproteins/genetics , Receptors, IgG/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
3.
J Biol Chem ; 283(3): 1628-1636, 2008 Jan 18.
Article in English | MEDLINE | ID: mdl-18024956

ABSTRACT

Membrane phospholipids are susceptible to oxidation, which is involved in various pathological processes such as inflammation, atherogenesis, neurodegeneration, and aging. One enzyme that may help to remove oxidized phospholipids from cells is intracellular type II platelet-activating factor acetylhydrolase (PAF-AH (II)), which hydrolyzes oxidatively fragmented fatty acyl chains attached to phospholipids. Overexpression of PAF-AH (II) in cells or tissues was previously shown to suppress oxidative stress-induced cell death. In this study we investigated the functions of PAF-AH (II) by generating PAF-AH (II)-deficient (Pafah2(-/-)) mice. PAF-AH (II) was predominantly expressed in epithelial cells such as kidney proximal and distal tubules, intestinal column epithelium, and hepatocytes. Although PAF-AH activity was almost abolished in the liver and kidney of Pafah2(-/-) mice, Pafah2(-/-) mice developed normally and were phenotypically indistinguishable from wild-type mice. However, mouse embryonic fibroblasts derived from Pafah2(-/-) mice were more sensitive to tert-butylhydroperoxide treatment than those derived from wild-type mice. When carbon tetrachloride (CCl(4)) was injected into mice, Pafah2(-/-) mice showed a delay in hepatic injury recovery. Moreover, after CCl(4) administration, liver levels of the esterified form of 8-iso-PGF(2alpha), a known in vitro substrate of PAF-AH (II), were higher in Pafah2(-/-) mice than in wild-type mice. These results indicate that PAF-AH (II) is involved in the metabolism of esterified 8-isoprostaglandin F(2alpha) and protects tissue from oxidative stress-induced injury.


Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolism , Liver/enzymology , Liver/pathology , Oxidative Stress , Phospholipids/metabolism , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , Animals , Carbon Tetrachloride , Dinoprost/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryo, Mammalian/enzymology , Epithelium/drug effects , Epithelium/enzymology , Esterification/drug effects , Fibroblasts/drug effects , Fibroblasts/enzymology , Gene Targeting , Hydrolysis/drug effects , Liver/drug effects , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/enzymology , Mice , Mice, Knockout , Oxidation-Reduction/drug effects , Oxidative Stress/drug effects , Platelet Activating Factor/metabolism , tert-Butyl Alcohol/pharmacology
4.
Chem Pharm Bull (Tokyo) ; 55(2): 317-23, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17268108

ABSTRACT

In the course of development of factor Xa (FXa) inhibitors, we have found unique compounds containing an N,O- and an N,N-spiro acetal structure. It appeared that the difference in overall conformation due to the N,X-spiro acetal structure might be important for FXa inhibitory activity. Therefore, other N,X-spiro acetal structures, an N,S- and an N,SO2-spiro acetal, were developed as analogues of the N,X-spiro acetal structure. Compound 7b (N,S-spiro acetal structure) was found to have the strongest activity in these series of N,X-spiro acetal compounds, which had ever been synthesized.(4,5)).


Subject(s)
Acetals/chemistry , Antithrombin III/chemical synthesis , Nitrogen/chemistry , Oxygen/chemistry , Piperidines/chemical synthesis , Spiro Compounds/chemical synthesis , Anticoagulants/pharmacology , Antithrombin III/pharmacology , Molecular Conformation , Molecular Structure , Piperidines/pharmacology , Serine Proteinase Inhibitors/pharmacology , Spiro Compounds/pharmacology
5.
Chem Pharm Bull (Tokyo) ; 54(11): 1535-44, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17077550

ABSTRACT

We have already reported unique compounds containing a N,O-spiro acetal structure as an orally active factor Xa (FXa) inhibitor. This time, we described a N,N-spiro acetal structure as an analogue of the N,O-spiro acetal structure for an orally active FXa inhibitor. The synthesis of these analogues could be achieved in a similar fashion to the N,O-spiro acetal synthesis. Consequently, FXa inhibitory activity was increased and more active compounds could be found (M58163: IC50 = 0.61 nM, M58169: IC50 = 0.58 nM). Additionally, the absolute configuration could be determined by X-ray crystallography analysis (M58169: (R)-config.).


Subject(s)
Factor Xa Inhibitors , Piperazines/chemical synthesis , Piperazines/pharmacology , Piperidones/chemistry , Spiro Compounds/chemistry , Crystallography, X-Ray , Magnetic Resonance Spectroscopy/methods , Models, Molecular , Molecular Structure , Naphthalenes/chemical synthesis , Naphthalenes/chemistry , Naphthalenes/pharmacology , Piperazines/chemistry , Piperidones/chemical synthesis , Piperidones/pharmacology , Sensitivity and Specificity , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacology , Stereoisomerism , Structure-Activity Relationship
6.
Bioorg Med Chem Lett ; 16(11): 2986-90, 2006 Jun 01.
Article in English | MEDLINE | ID: mdl-16540315

ABSTRACT

Since beta-tryptase is considered a critical mediator of asthma, potent tryptase inhibitors may be useful as new agents for the treatment of asthma. We investigated 4-substituted benzylamine derivatives and obtained M58539 (15h) as a potent inhibitor of beta-tryptase (IC50 = 5.0 nM) with high selectivity against other serine proteases, low molecular weight, clog P value less than 5, lack of amidino and guanidino groups, and independence of Zn2+ ion.


Subject(s)
Benzylamines/chemistry , Benzylamines/pharmacology , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Benzylamines/chemical synthesis , Ions/chemistry , Molecular Structure , Naphthalenes/chemistry , Serine Proteinase Inhibitors/chemistry , Structure-Activity Relationship , Tryptases , Zinc/chemistry , Zinc/pharmacology
7.
Eur J Pharmacol ; 529(1-3): 164-71, 2006 Jan 04.
Article in English | MEDLINE | ID: mdl-16316651

ABSTRACT

Factor Xa plays an important role in blood coagulation and is widely regarded as an attractive target for antithrombotic drug development. M55551 and M55165 (1-arylsulfonyl-3-piperazinone derivatives) are novel synthetic factor Xa inhibitors. In vitro, M55551 and M55165 competitively inhibited factor Xa with K(i) values of 3.2 nM and 2.3 nM, respectively, and prolonged clotting time in human and rat plasma. Pharmacokinetic analysis of these compounds revealed that M55551 was intravenously active with a short half-life (0.2 h) and that M55165 exhibited good bioavailability (31%) with a long half-life (3.9 h). Therefore, the antithrombotic effects of M55551 and M55165 were compared with those of the intravenous anticoagulant argatroban and the oral anticoagulant warfarin. Intravenous administration of M55551 and oral administration of M55165 inhibited thrombus formation at 0.3 mg/kg and 10 mg/kg, respectively, without significant prolongation of bleeding time. In contrast, although argatroban (0.3 mg/kg) and warfarin (1 mg/kg) also inhibited thrombus formation, significant prolongation of bleeding time was observed at dosages of 3 mg/kg and 1 mg/kg, respectively. These results suggest that M55551 and M55165 are potent factor Xa inhibitors that are active upon intravenous and oral administration, respectively, and that may prove clinically useful for the treatment of thrombosis while minimizing bleeding risks.


Subject(s)
Anticoagulants , Factor Xa Inhibitors , Piperazines , Venous Thrombosis/drug therapy , Administration, Oral , Animals , Anticoagulants/pharmacokinetics , Anticoagulants/pharmacology , Anticoagulants/therapeutic use , Biological Availability , Bleeding Time , Disease Models, Animal , Half-Life , Humans , Injections, Intravenous , Male , Piperazines/pharmacokinetics , Piperazines/pharmacology , Piperazines/therapeutic use , Rats , Rats, Wistar , Venous Thrombosis/blood
8.
Chem Pharm Bull (Tokyo) ; 52(4): 406-12, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15056953

ABSTRACT

In the course of development of factor Xa (FXa) inhibitor in an investigation involving the synthesis of 1-arylsulfonyl-3-piperazinone derivatives, we found new compounds containing a unique spiro skeleton. Among such compounds, (-)-7-[(6-chloro-2-naphthalenyl)sulfonyl]tetrahydro-8a-(methoxymethyl)-1'-(4-pyridinyl)-spiro[5H-oxazolo[3,2-a]pyrazine-2(3H),4'-piperidin]-5-one (28, M55529) had activity more favorable than those of previously reported compounds. The inhibitory activity of M55529 for FXa is IC(50)=2 nM, with high selectivity for FXa over thrombin and trypsin.


Subject(s)
Factor Xa Inhibitors , Pyridines/chemical synthesis , Pyridines/pharmacology , Spiro Compounds/chemical synthesis , Spiro Compounds/pharmacology , Crystallography, X-Ray , Cyclization , Magnetic Resonance Spectroscopy , Mass Spectrometry , Structure-Activity Relationship , Thrombin/antagonists & inhibitors , Trypsin Inhibitors/chemical synthesis , Trypsin Inhibitors/pharmacology
9.
Chem Pharm Bull (Tokyo) ; 52(4): 459-62, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15056966

ABSTRACT

Compounds containing an ethylenediamine structure in place of the piperazine ring of M55113 (1) and M55551 (2) were synthesized to investigate the effects of a piperazine moiety and evaluated for activity as factor Xa (FXa) inhibitors. Most such compounds, however, exhibited lower activity (1/10-1/100) than that of M55113 and M55551 as FXa inhibitors.


Subject(s)
Factor Xa Inhibitors , Piperazines/chemical synthesis , Piperazines/pharmacology , Indicators and Reagents , Magnetic Resonance Spectroscopy , Spectrophotometry, Infrared , Structure-Activity Relationship
10.
Chem Pharm Bull (Tokyo) ; 50(9): 1187-94, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12237534

ABSTRACT

Intravascular clot formation is an important event in a number of cardiovascular diseases. The prevention of blood coagulation has become a major target for new therapeutic agents. Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in the blood coagulation cascade and represents an attractive target for anticoagulant drug development. We have investigated substituents in the central part of a lead compound (3: M55113), and discovered that compound M55551 (34: (R)-4-[(6-Chloro-2-naphthalenyl)sulfonyl]-6-oxo-1-[[1-(4-pyridinyl)-4-piperidinyl]methyl]-2-piperazinecarboxylic acid) is a potent inhibitor of FXa (IC(50)=0.006 microM), with high selectivity for FXa over trypsin and thrombin. The activity of this compound is ten times more powerful than the lead compound.


Subject(s)
Factor Xa Inhibitors , Arylsulfonates/chemical synthesis , Arylsulfonates/pharmacology , Crystallization , Hydrogen Bonding , Indicators and Reagents , Oxidation-Reduction , Piperazines/chemical synthesis , Piperazines/pharmacology , Piperidines/chemical synthesis , Piperidines/pharmacology , Structure-Activity Relationship , Thrombin/antagonists & inhibitors , Trypsin Inhibitors/chemical synthesis , Trypsin Inhibitors/pharmacology
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