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1.
J Lipid Res ; 51(12): 3559-67, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20805092

ABSTRACT

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGAT-expressing cells with stable isotope-labeled [¹³C18]oleic acid. The [¹³C18]oleoyl-incorporated TG and DAG lipid species were profiled. The TG synthesis pathway assay was optimized to a one-step extraction, followed by LC/MS/MS quantification. Further, we report a novel LC/MS/MS method for tracing hepatic TG synthesis and VLDL-TG secretion in vivo by administering [¹³C18]oleic acid to rats. The [¹³C18]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [¹³C18]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [¹³C18]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [¹³C18]oleoyl-incorporated VLDL-TG. This DGAT1-selective assay will enable researchers to discern differences between the roles of DGAT1 and DGAT2 in TG synthesis in vitro and in vivo.


Subject(s)
Diacylglycerol O-Acyltransferase/metabolism , Enzyme Assays/methods , Liver/enzymology , Animals , Carbon Radioisotopes/metabolism , Cells, Cultured , Chromatography, Liquid , Hepatocytes/cytology , Hepatocytes/enzymology , Humans , Insecta/cytology , Insecta/enzymology , Insecta/virology , Kidney/cytology , Kidney/embryology , Kidney/enzymology , Lipoproteins, VLDL/chemistry , Lipoproteins, VLDL/metabolism , Male , Oleic Acid/metabolism , Rats , Rats, Sprague-Dawley , Tandem Mass Spectrometry , Triglycerides/chemistry , Triglycerides/metabolism
2.
J Med Chem ; 53(4): 1843-56, 2010 Feb 25.
Article in English | MEDLINE | ID: mdl-20102150

ABSTRACT

We have identified RWJ-671818 (8) as a novel, low molecular weight, orally active inhibitor of human alpha-thrombin (K(i) = 1.3 nM) that is potentially useful for the acute and chronic treatment of venous and arterial thrombosis. In a rat deep venous thrombosis model used to assess antithrombotic efficacy, oral administration of 8 at 30 and 50 mg/kg reduced thrombus weight by 87 and 94%, respectively. In an anesthetized rat antithrombotic model, where electrical stimulation of the carotid artery created a thrombus, 8 prolonged occlusion time 2- and 3-fold at 0.1 and 1.0 mg/kg, i.v., respectively, and more than doubled activated clotting time and activated partial thromboplastin time at the higher dose. This compound had excellent oral bioavailability of 100% in dogs with an estimated half-life of approximately 3 h. On the basis of its noteworthy preclinical data, 8 was advanced into human clinical trials and successfully progressed through phase 1 studies.


Subject(s)
Anticoagulants/chemical synthesis , Fibrinolytic Agents/chemical synthesis , Guanidines/chemical synthesis , Pyrazines/chemical synthesis , Thrombin/antagonists & inhibitors , Amino Acid Motifs , Animals , Anticoagulants/pharmacokinetics , Anticoagulants/pharmacology , Blood Pressure/drug effects , Caco-2 Cells , Crystallography, X-Ray , Cytochrome P-450 CYP3A , Cytochrome P-450 CYP3A Inhibitors , Dogs , Double-Blind Method , Electrocardiography , Female , Fibrinolytic Agents/pharmacokinetics , Fibrinolytic Agents/pharmacology , Guanidines/pharmacokinetics , Guanidines/pharmacology , Guinea Pigs , Heart Rate/drug effects , Hemodynamics/drug effects , Humans , In Vitro Techniques , Male , Microsomes, Liver/metabolism , Models, Molecular , Pyrazines/pharmacokinetics , Pyrazines/pharmacology , Rats , Rats, Sprague-Dawley , Recombinant Proteins/antagonists & inhibitors , Structure-Activity Relationship , Thrombin/chemistry , Venous Thrombosis/blood , Venous Thrombosis/drug therapy
3.
J Cardiovasc Pharmacol ; 55(5): 459-68, 2010 May.
Article in English | MEDLINE | ID: mdl-20051879

ABSTRACT

OBJECTIVE: Torcetrapib, a prototype cholesteryl ester transfer protein (CETP) inhibitor with potential for decreasing atherosclerotic disease, increased cardiovascular events in clinical trials. The identified hypertensive and aldosterone-elevating actions of torcetrapib may not fully account for this elevated cardiovascular risk. Therefore, we evaluated the effects of torcetrapib on endothelial mediated vasodilation in vivo. METHODS AND RESULTS: In vivo endothelial mediated vasodilation was assessed using ultrasound imaging of acetylcholine-induced changes in rabbit central ear artery diameter. Torcetrapib, in addition to producing hypertension and baseline vasoconstriction, markedly inhibited acetylcholine-induced vasodilation. A structurally distinct CETP inhibitor, JNJ-28545595, did not affect endothelial function despite producing similar degrees of CETP inhibition and high-density lipoprotein elevation. Nitroprusside normalized torcetrapib's basal vasoconstriction and elicited dose-dependent vasodilation of norepinephrine preconstricted arteries in torcetrapib-treated animals, indicating torcetrapib did not impair smooth muscle function. CONCLUSIONS: Torcetrapib significantly impairs endothelial function in vivo, independent of CETP inhibition and high-density lipoprotein elevation. Given the well-documented association of endothelial dysfunction with cardiovascular disease and risk, this activity of torcetrapib may have contributed to increased cardiovascular risk in clinical trials.


Subject(s)
Anticholesteremic Agents/adverse effects , Cardiovascular Diseases/chemically induced , Cholesterol Ester Transfer Proteins/antagonists & inhibitors , Endothelium, Vascular/drug effects , Quinolines/adverse effects , Vasodilation/drug effects , Administration, Oral , Animals , Anticholesteremic Agents/administration & dosage , Anticholesteremic Agents/pharmacokinetics , Cardiovascular Diseases/metabolism , Cardiovascular Diseases/physiopathology , Dose-Response Relationship, Drug , Injections, Intravenous , Male , Molecular Structure , Quinolines/administration & dosage , Quinolines/pharmacokinetics , Rabbits
4.
Blood Coagul Fibrinolysis ; 21(2): 128-34, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20010091

ABSTRACT

Whereas heparin functions as an antithrombotic agent by promoting antithrombin III-based inhibition of thrombin and factor Xa, there is less appreciation for the combination behavior with small-molecule, direct inhibitors of these proteases. We conducted a study in a high-shear arterial environment to explore the potential for a cooperative antithrombotic effect with a thrombin inhibitor (argatroban), a factor Xa inhibitor (YM-60828), and a dual thrombin/factor Xa inhibitor (RWJ-445167). We employed a platelet-dependent vascular injury model in which rats were subjected to an acute electrical injury to the carotid artery. Antithrombotic efficacy was measured for thrombin inhibitor argatroban and factor Xa inhibitor YM-60828 administered alone or in combination. The results indicate that there is a cooperative antithrombotic effect in vivo when both thrombin and factor Xa are inhibited simultaneously. The dual thrombin/factor Xa inhibitor RWJ-445167 was found to have potent antithrombotic activity in this high-shear environment. A comparison of results for RWJ-445167 and argatroban showed additional efficacy with RWJ-445167, suggestive of drug synergy.


Subject(s)
Factor Xa Inhibitors , Guanidines/pharmacology , Naphthalenes/pharmacology , Pipecolic Acids/pharmacology , Piperidines/pharmacology , Sulfonamides/pharmacology , Thrombin/antagonists & inhibitors , Animals , Arginine/analogs & derivatives , Blood Coagulation/drug effects , Drug Synergism , Factor Xa/metabolism , Humans , Male , Rats , Rats, Sprague-Dawley , Thrombin/metabolism
5.
6.
J Med Chem ; 51(2): 282-97, 2008 Jan 24.
Article in English | MEDLINE | ID: mdl-18159923

ABSTRACT

We have developed a novel series of potent and selective factor Xa inhibitors that employ a key 7-fluoroindazolyl moiety. The 7-fluoro group on the indazole scaffold replaces the carbonyl group of an amide that is found in previously reported factor Xa inhibitors. The structure of a factor Xa cocrystal containing 7-fluoroindazole 51a showed the 7-fluoro atom hydrogen-bonding with the N-H of Gly216 (2.9 A) in the peptide backbone. Thus, the 7-fluoroindazolyl moiety not only occupied the same space as the carbonyl group of an amide found in prior factor Xa inhibitors but also maintained a hydrogen bond interaction with the protein's beta-sheet domain. The structure-activity relationship for this series was consistent with this finding, as the factor Xa inhibitory potencies were about 60-fold greater (DeltaDelta G approximately 2.4 kcal/mol) for the 7-fluoroindazoles 25a and 25c versus the corresponding indazoles 25b and 25d. Highly convergent synthesis of these factor Xa inhibitors is also described.


Subject(s)
Factor Xa Inhibitors , Indazoles/chemical synthesis , Serine Proteinase Inhibitors/chemical synthesis , Caco-2 Cells , Cell Membrane Permeability , Crystallography, X-Ray , Factor Xa/chemistry , Humans , Hydrogen Bonding , In Vitro Techniques , Indazoles/chemistry , Indazoles/pharmacology , Microsomes, Liver/enzymology , Models, Molecular , Protein Conformation , Serine Proteinase Inhibitors/chemistry , Serine Proteinase Inhibitors/pharmacology , Structure-Activity Relationship , Thermodynamics
7.
Chem Biol Drug Des ; 68(1): 29-36, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16923023

ABSTRACT

Compound 2 (RWJ-445167; 3DP-10017), a dual inhibitor of thrombin and factor Xa, was advanced into human clinical studies. However, its oral bioavailability in humans proved to be below acceptable limits. To address this issue, we explored a prodrug approach involving numerous guanidine derivatives. Prodrug candidates of classes A (carbamate derivatives), B (imidate derivatives), and C (alkyl and acyl derivatives), compounds 3-6, were synthesized and evaluated for anticoagulant activity at 2 h after oral administration to rats. In comparison to the parent drug (2), little worthwhile improvement was observed for the prodrug candidates.


Subject(s)
Anticoagulants/pharmacology , Factor Xa Inhibitors , Guanidines/pharmacology , Prodrugs/pharmacology , Sulfonamides/pharmacology , Thrombin/antagonists & inhibitors , Administration, Oral , Animals , Anticoagulants/chemical synthesis , Anticoagulants/pharmacokinetics , Biological Availability , Carbamates/chemical synthesis , Dogs , Guanidine/analogs & derivatives , Guanidine/chemistry , Guanidines/chemical synthesis , Guanidines/pharmacokinetics , Imidoesters/chemical synthesis , Male , Prodrugs/chemical synthesis , Prodrugs/pharmacokinetics , Rats , Rats, Sprague-Dawley , Sulfonamides/chemical synthesis , Sulfonamides/pharmacokinetics , Time Factors
8.
J Biol Chem ; 280(18): 18001-7, 2005 May 06.
Article in English | MEDLINE | ID: mdl-15741158

ABSTRACT

Certain leukocytes release serine proteases that sustain inflammatory processes and cause disease conditions, such as asthma and chronic obstructive pulmonary disease. We identified beta-ketophosphonate 1 (JNJ-10311795; RWJ-355871) as a novel, potent dual inhibitor of neutrophil cathepsin G (K(i) = 38 nm) and mast cell chymase (K(i) = 2.3 nm). The x-ray crystal structures of 1 complexed with human cathepsin G (1.85 A) and human chymase (1.90 A) reveal the molecular basis of the dual inhibition. Ligand 1 occupies the S(1) and S(2) subsites of cathepsin G and chymase similarly, with the 2-naphthyl in S(1), the 1-naphthyl in S(2), and the phosphonate group in a complex network of hydrogen bonds. Surprisingly, however, the carboxamido-N-(naphthalene-2-carboxyl)piperidine group is found to bind in two distinct conformations. In cathepsin G, this group occupies the hydrophobic S(3)/S(4) subsites, whereas in chymase, it does not; rather, it folds onto the 1-naphthyl group of the inhibitor itself. Compound 1 exhibited noteworthy anti-inflammatory activity in rats for glycogen-induced peritonitis and lipopolysaccharide-induced airway inflammation. In addition to a marked reduction in neutrophil influx, 1 reversed increases in inflammatory mediators interleukin-1alpha, interleukin-1beta, tissue necrosis factor-alpha, and monocyte chemotactic protein-1 in the glycogen model and reversed increases in airway nitric oxide levels in the lipopolysaccharide model. These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cathepsins/antagonists & inhibitors , Cathepsins/metabolism , Leukocytes/enzymology , Organophosphonates/pharmacology , Piperidines/pharmacology , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/pharmacology , Acute Disease , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cathepsin G , Chymases , Crystallography, X-Ray , Humans , Leukocytes/drug effects , Male , Mast Cells/enzymology , Organophosphonates/administration & dosage , Organophosphonates/chemistry , Peritonitis/drug therapy , Peritonitis/enzymology , Piperidines/chemistry , Pulmonary Disease, Chronic Obstructive/drug therapy , Pulmonary Disease, Chronic Obstructive/enzymology , Rats , Rats, Sprague-Dawley , Serine Proteinase Inhibitors/administration & dosage , Serine Proteinase Inhibitors/chemistry
9.
J Med Chem ; 48(6): 1984-2008, 2005 Mar 24.
Article in English | MEDLINE | ID: mdl-15771442

ABSTRACT

Thrombin inhibitors are potentially useful in medicine for their anticoagulant and antithrombotic effects. We synthesized and evaluated diverse heterocycle-activated ketones based on the d-Phe-Pro-Arg, and related thrombin active-site recognition motifs, as candidate inhibitors. The peptide-based alpha-ketoheterocycles were typically prepared by either an imidate or a Weinreb amide route (Schemes 1 and 2), the latter of which proved to be more general. Test compounds were generally assayed for inhibition of human alpha-thrombin and bovine trypsin. From a structure-based design standpoint, the heterocycle allows one to explore and adjust interactions within the S1' subsite of thrombin. The preferred alpha-ketoheterocycle is a pi-rich 2-substituted azole with at least two heteroatoms proximal to the carbon bearing the keto group, and a preferred thrombin inhibitor is 2-ketobenzothiazole 3, with a potent K(i) value of 0.2 nM and ca. 15-fold selectivity over trypsin. 2-Ketobenzothiazole 13 exhibited exceedingly potent thrombin inhibition (K(i) = 0.000 65 nM; slow tight binding). Several alpha-ketoheterocycles had thrombin K(i) values in the range 0.1-400 nM. The "Arg" unit in the alpha-ketoheterocycles can be sensitive to stereomutation under mildy basic conditions. For example, 2-ketothiazoles 4 and 59 readily epimerize at pH 7.4, although they are fairly stable stereochemically at pH 3-4; thus, suitable conditions had to be selected for the enzymatic assays. Lead d-Phe-Pro-Arg 2-benzothiazoles 3, 4, and 68 displayed good selectivity for thrombin over other key coagulation enzymes (e.g., factor Xa, plasmin, protein Ca, uPA, tPA, and streptokinase); however, their selectivity for thrombin over trypsin was modest (<25-fold). Compounds 3, 4, and 68 exhibited potent in vitro antithrombotic activity as measured by inhibition of gel-filtered platelet aggregation induced by alpha-thrombin (IC(50) = 30-40 nM). They also proved to be potent anticoagulant/antithrombotic agents in vivo on intravenous administration, as determined in the canine arteriovenous shunt (ED(50) = 0.45-0.65 mg/kg) and the rabbit deep vein thrombosis (ED(50) = 0.1-0.4 mg/kg) models. Intravenous administration of 3, and several analogues, to guinea pigs caused hypotension and electrocardiogram abnormalities. Such cardiovascular side effects were also observed with some nonguanidine inhibitors and inhibitors having recognition motifs other than d-Phe-Pro-Arg. 2-Benzothiazolecarboxylates 4 and 68 exhibited significantly diminished cardiovascular side effects, and benzothiazolecarboxylic acid 4 had the best profile with respect to therapeutic index. The X-ray crystal structures of the ternary complexes 3-thrombin-hirugen and 4-thrombin-hirugen depict novel interactions in the S(1)' region, with the benzothiazole ring forming a hydrogen bond with His-57 and an aromatic stacking interaction with Trp-60D of thrombin's insertion loop. The benzothiazole ring of 3 displaces the Lys-60F side chain into a U-shaped gauche conformation, whereas the benzothiazole carboxylate of 4 forms a salt bridge with the side chain of Lys-60F such that it adopts an extended anti conformation. Since 3 has a 10-fold greater affinity for thrombin than does 4, any increase in binding energy resulting from this salt bridge is apparently offset by perturbations across the enzyme (viz. Figure 4). The increased affinity and selectivity of 2-ketobenzothiazole inhibitors, such as 3, may be primarily due to the aromatic stacking interaction with Trp-60D. However, energy contour calculations with the computer program GRID also indicate a favorable interaction between the benzothiazole sulfur atom and a hydrophobic patch on the surface of thrombin.


Subject(s)
Anticoagulants/chemical synthesis , Fibrinolytic Agents/chemical synthesis , Ketones/chemical synthesis , Oligopeptides/chemical synthesis , Thiazoles/chemical synthesis , Thrombin/antagonists & inhibitors , Animals , Anticoagulants/chemistry , Anticoagulants/pharmacology , Binding Sites , Cattle , Crystallography, X-Ray , Dogs , Drug Design , Electrocardiography/drug effects , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/pharmacology , Guinea Pigs , Humans , Hydrogen-Ion Concentration , Hypotension/chemically induced , In Vitro Techniques , Ketones/chemistry , Ketones/pharmacology , Models, Molecular , Molecular Structure , Oligopeptides/chemistry , Oligopeptides/pharmacology , Platelet Aggregation/drug effects , Rabbits , Stereoisomerism , Structure-Activity Relationship , Thiazoles/chemistry , Thiazoles/pharmacology , Thrombin/chemistry , Trypsin/chemistry , Venous Thrombosis/prevention & control
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