ABSTRACT
The lipophilic 1-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, inhibitors of recombinant lipoprotein-associated phospholipase A(2), has been modified to give inhibitors of high potency in human plasma and enhanced physicochemical properties. Phenylpiperazineacetamide derivative 23 shows very promising oral activity.
Subject(s)
Enzyme Inhibitors/pharmacology , Phospholipases A/antagonists & inhibitors , Phospholipases A/blood , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Administration, Oral , Animals , Enzyme Inhibitors/chemical synthesis , Humans , Inhibitory Concentration 50 , Metabolic Clearance Rate/physiology , Microsomes, Liver/metabolism , Piperazines/chemical synthesis , Piperazines/pharmacology , Pyrimidinones/chemical synthesis , Pyrimidinones/pharmacology , Rabbits , RatsABSTRACT
Modification of the pyrimidone 5-substituent in a series of 1-((amidolinked)-alkyl)-pyrimidones, lipophilic inhibitors of lipoprotein-associated phospholipase A2, has given inhibitors of nanomolar potency and improved physicochemical properties. Compound 23 was identified as a potent, highly water soluble. CNS penetrant inhibitor suitable for intravenous administration.
Subject(s)
Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Phospholipases A/antagonists & inhibitors , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , Animals , Arteriosclerosis/drug therapy , Drug Administration Routes , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Humans , Male , Molecular Structure , Phospholipases A/metabolism , Phospholipases A2 , Pyrimidinones/chemical synthesis , Rabbits , Rats , Solubility , Water/chemistryABSTRACT
From two related series of 2-(alkylthio)-pyrimidones, a novel series of 1-((amidolinked)-alkyl)-pyrimidones has been designed as nanomolar inhibitors of human lipoprotein-associated phospholipase A2. These compounds show greatly enhanced activity in isolated plasma. Selected derivatives such as compounds 51 and 52 are orally active with a good duration of action.
Subject(s)
Enzyme Inhibitors/pharmacology , Lipoproteins/metabolism , Phospholipases A/antagonists & inhibitors , Pyrimidinones/pharmacology , Administration, Oral , Animals , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Molecular Structure , Phospholipases A/metabolism , Phospholipases A2 , Pyrimidinones/administration & dosage , Pyrimidinones/chemistry , RabbitsABSTRACT
The synthesis of analogues of SB-253514, a novel natural product derived inhibitor of lipoprotein associated phospholipase A2 (Lp-PLA2), is described together with their ability to inhibit Lp-PLA2.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemical synthesis , Enzyme Inhibitors/chemical synthesis , Phospholipases A/antagonists & inhibitors , Pyrans/chemical synthesis , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Enzyme Inhibitors/pharmacology , Phospholipases A2 , Pyrans/pharmacology , Structure-Activity RelationshipABSTRACT
Starting from two weakly active hits from high throughput screening, a novel series of 2-(alkylthio)-pyrimidin-4-ones with high potency and selectivity for lipoprotein-associated phospholipase A2 has been designed. In contrast to previously known inhibitors, these have been shown to act by a non-covalent and substrate competitive mechanism.
Subject(s)
Phospholipases A/antagonists & inhibitors , Pyrimidinones/chemistry , Pyrimidinones/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Phospholipases A2 , Pyrimidinones/chemical synthesis , Structure-Activity Relationship , Substrate SpecificityABSTRACT
A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.
Subject(s)
Azetidines/pharmacology , Enzyme Inhibitors/pharmacology , Lipoproteins, LDL/metabolism , Phospholipases A/metabolism , Sulfoxides/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Aryldialkylphosphatase , Chemotaxis, Leukocyte/drug effects , Esterases/antagonists & inhibitors , Humans , Oxidation-Reduction , Phosphatidylcholine-Sterol O-Acyltransferase/antagonists & inhibitors , Phospholipases A/antagonists & inhibitors , Phospholipases A2 , Protein BindingABSTRACT
Investigation of the inhibition of LDL-associated phospholipase A2 by monocyclic beta-lactams has shown that LDL phospholipase A2 is capable of hydrolyzing monocyclic-beta-lactams by a mechanism which shares many similarities to the hydrolysis of beta-lactams by beta-lactamases. We believe that this is the first demonstration of a serine-dependent lipase being able to hydrolyze an amide bond. Although 4-(phenylthio)-N-(4-phenyl-2-oxobutyl)azetidin-2-one, SB-216477, and its enantiomers are relatively modest covalent inactivators with kobs/[I] = 46 M-1 s-1 for the R enantiomer, analysis of the kinetics of inactivation and reactivation shows that these compounds act as slow-turnover substrates, presumably via an acylation-deacylation mechanism. The detection of a suprastoichiometric burst indicates that the pathway must be branched with the branching giving rise to the slow reactivation via a more stable covalent intermediate. Study of the two enantiomers of SB-216477 shows that LDL-associated phospholipase A2 is sensitive to the beta-lactam stereochemistry at C4. However, a common achiral intermediate is formed along the turnover pathway, and this must be at or immediately prior to the branch point.
Subject(s)
Azetidines/pharmacology , Lactams , Lipoproteins, LDL/metabolism , Phospholipases A/antagonists & inhibitors , beta-Lactams/pharmacology , 1-Alkyl-2-acetylglycerophosphocholine Esterase , Azetidines/metabolism , Chromatography, High Pressure Liquid , Enzyme Activation/drug effects , Kinetics , Phospholipases A/metabolism , Phospholipases A2 , Spectrum Analysis , Stereoisomerism , Substrate Specificity , beta-Lactams/metabolismABSTRACT
Using expressed sequence tag (EST) homology screening, a new human serine dependent phospholipase A2 (HSD-PLA2) was identified that has 40% amino acid identity with human low density lipoprotein-associated phospholipase A2 (LDL-PLA2). HSD-PLA2 has very recently been purified and cloned from brain tissue but named PAF-AH II. However, because the homology with LDL-PLA2 suggested a broader substrate specificity than simply platelet activating factor (PAF), we have further characterized this enzyme using baculovirus-expressed protein. The recombinant enzyme, which was purified 21-fold to homogeneity, had a molecular mass of 44kDa and possessed a specific activity of 35 micromol min-1 mg-1 when assayed against PAF. Activity could also be measured using 1-decanoyl-2-(4-nitrophenylglutaryl) phosphate (DNGP) as substrate. Like LDL-PLA2, HSD-PLA2 was able to hydrolyse oxidatively modified phosphatidylcholines when supplemented to human LDL prior to copper-stimulated oxidation. A GXSXG motif evident from sequence information and inhibition of its activity by 3,4, dichloroisocoumarin, diisopropyl fluorophosphate (DFP) and diethyl p-nitrophenyl phosphate (DENP) confirm that the enzyme is serine dependent. Moreover, sequence comparison indicates the HSD-PLA2 probable active site triad positions are shared with LDL-PLA2 and a C. elegans homologue, suggesting that these sequences comprise members of a new enzyme family. Although clearly structurally related with similar substrate specificities further work reported here shows HSD-PLA2 and LDL-PLA2 to be different with respect to chromosomal localization and tissue distribution.
Subject(s)
Phospholipases A/metabolism , Phospholipids/metabolism , Platelet Activating Factor/metabolism , Amino Acid Sequence , Animals , Brain/enzymology , Caenorhabditis elegans , Cell Line , Chromatography, Affinity , Chromatography, Gel , Chromatography, Ion Exchange , Cloning, Molecular , Consensus Sequence , Humans , Kinetics , Molecular Sequence Data , Organ Specificity , Oxidation-Reduction , Phospholipases A/chemistry , Phospholipases A/isolation & purification , Phospholipases A2 , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Spodoptera , Substrate Specificity , TransfectionABSTRACT
A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 micromol.min-1.mg-1 and 12 micromol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.
