Design and synthesis of new secretory phospholipase A2 inhibitor of a phospholipid analog

All stereoisomers of N-acyl-4,5-disubstituted oxazolidinone phospholipid analogs were synthesized by regio and stereoselective epoxide ring opening accompanied by introduction of an amino group. The (4R,5S)-derivative showed stronger inhibitory activity toward type II phospholipase A2 than the 4-substituted oxazolidinone phospholipid analog previously reported.     

Microtubule depolymerization selectively down-regulates the synthesis of proinflammatory secretory nonpancreatic phospholipase A2

Reports which analyzed the effects of secondary cytoreductive surgery at second-look laparotomy have often included small numbers of patients who have been treated with a variety of first-line chemotherapy regimens and those who may have progressed on first-line therapy. The purpose of this study was to analyze survival following secondary cytoreductive surgery at second-look laparotomy in patients with advanced ovarian cancer. Review of the surgical data of 153 patients allowed classification of tumor size found at second-look laparotomy and tumor size remaining after cytoreduction. Multivariate analysis evaluated multiple risk factors for survival. Of 153 patients, 124 had macroscopic tumor at second-look laparotomy and 29 had microscopic disease only. Fifteen of 69 (22%) patients were found to have tumor > 1 cm in diameter and were cytoreduced to microscopic residual and 18/69 (26%) were left with 1 cm tumor. Twenty-one of 55 (38%) patients with < or = 1 cm tumor were debulked to microscopic residual. The shortest survival relative to patients found to have microscopic disease at second-look laparotomy was observed among patients whose maximum tumor size remained > 1 cm following second-look laparotomy (relative risk = 3.1, P = 0.0004). No difference in survival was seen between patients found to have microscopic disease and those cytoreduced to microscopic disease (P = 0.24). The risk of death was lower among patients debulked to a lower category (< or = 1 cm debulked to microscopic, relative risk = 0.48, P = 0.02; > 1 cm reduced to < or = 1 cm, relative risk = 0.49, P = 0.02; > 1 cm reduced to microscopic, relative risk = 0.44, P = 0.01). Whether this apparent beneficial effect of cytoreductive surgery at second-look laparotomy reflects the biology of the tumor which allows surgical cytoreduction or the effects of cytoreduction can only be addressed in a randomized prospective trial.     

Melittin binds to secretory phospholipase A2 and inhibits its enzymatic activity

Synthetic melittin inhibited the enzymatic activity of secretory phospholipase A2 (PLA2) from various sources, including bee and snake venoms, bovine pancreas, and synovial fluid from rheumatoid arthritis patients, irrespective of substrate (e.g., [14C]-phosphatidylcholine or phosphatidylethanolamine vesicles and [3H]-oleic acid-labeled E.coli). A Lineweaver-Burk analysis showed that melittin was a noncompetitive inhibitor of bee venom PLA2, causing a change in Vmax from 200 to 50 units/min/mg of protein. The Km remained unchanged (0.75 nmole). Melittin inhibited approximately 50% of purified bee venom PLA2 activity in a 30:1 molar ratio (melittin:enzyme). Because the enzyme kinetics indicated a PLA2-melittin interaction, a melittin-sepharose affinity column was used to purify a PLA2 from human serum. Further, an enzyme-linked assay was developed to quantitate PLA2 activity in biological fluids using avidin-peroxidase and ELISA plates coated with biotinylated melittin. These observations may have potential therapeutic significance, as well as provide a convenient basis for the isolation and quantitation of PLA2.     

Differential activation of human neutrophil cytosolic phospholipase A2 and secretory phospholipase A2 during priming by 1,2-diacyl- and 1-O-alkyl-2-acylglycerols

We have shown previously that both 1,2-diacylglycerol (AAG) and 1-O-alkyl-2-acylglycerol (EAG) prime neutrophil release of arachidonic acid via uncharacterized phospholipases A2. Therefore, we investigated the actions of EAG and AAG specifically on neutrophil cytosolic (cPLA2) and secretory (sPLA2) phospholipase A2s. We hypothesized that AAG as a protein kinase activator would activate cPLA2 via phosphorylation events. EAG is antagonistic to the AAG activation of PKC, thus it was not expected to act via phosphorylation of cPLA2. Neutrophils were primed with either AAG or EAG and then stimulated with fMLP. When neutrophils were primed with 5-20 microM 1,2-diacylglycerol, a shift was observed in cPLA2 migration on SDS-PAGE gels, consistent with phosphorylation of the protein. This gel shift was not seen after exposure to EAG. AAG also caused a parallel increase in enzymatic activity of cPLA2 that was not seen with EAG. We also investigated whether either diglyceride would cause similar priming or direct secretion of sPLA2. Both AAG and EAG directly caused significant secretion of neutrophil sPLA2. EAG also increased the release of sPLA2 in cells subsequently stimulated with fMLP. Thus, AAG activated cPLA2 and stimulated secretion of sPLA2. In contrast, EAG did not activate cPLA2, but directly activated secretion of sPLA2. We also demonstrated that human synovial fluid sPLA2 increased AA release from resting and fMLP-stimulated neutrophils. Given that diglycerides prime for release of AA, PAF, and LTB4, these current data support the hypothesis that such priming may be mediated by phosphorylation dependent (cPLA2) or phosphorylation independent (e.g. secretion of sPLA2) events.     

Potent inhibitory activities of hydrophobic aci-reductones (2-hydroxytetronic acid analogs) against membrane and human low-density lipoprotein oxidation

In a murine model of respiratory syncytial virus disease, prior sensitization to the attachment glycoprotein (G) leads to pulmonary eosinophilia and enhanced illness. Three different approaches were taken to dissect the region of G responsible for enhanced disease and protection against challenge. First, mutant viruses, containing frameshifts that altered the COOH terminus of the G protein, were used to challenge mice sensitized by scarification with recombinant vaccinia virus (rVV) expressing wild-type G. Second, cDNA expressing these mutated G proteins were expressed by rVV and used to vaccinate mice before challenge with wild-type respiratory syncytial virus (RSV). These studies identified residues 193-205 to be responsible for G-induced weight loss and lung eosinophilia and showed that this region was not was not necessary for induction of protective immunity. Third, mice were sensitized using an rVV that expressed only amino acids 124-203 of the G protein. Upon RSV challenge, mice sensitized with this rVV developed enhanced weight loss and eosinophilia. This is the first time that a region within RSV (amino acids 193-203) has been shown to be responsible for induction of lung eosinophilia and disease enhancement. Moreover, we now show that it is possible to induce protective immunity with an altered G protein without inducing a pathological response.     

Induction of release of secretory nonpancreatic phospholipase A2 from human articular chondrocytes

OBJECTIVE: Secretory nonpancreatic phospholipase A2 (sPLA2) is a known inducer/promoter of the inflammatory process in the joints. It correlates with disease activity in adult and juvenile rheumatoid arthritis. Synovial fluids contain high concentrations of sPLA2. We discovered that human articular cartilage contains large quantities of sPLA2 and that culture chondrocytes constitutively synthesize and release sPLA2. To test the mechanism controlling the release of sPLA2, we exposed cultured human articular chondrocytes to cytokines and other agents, known to induce sPLA2 in other cells. METHODS: Chondrocytes obtained from cartilage of normal appearance from rheumatoid and osteoarthritic joints, and from normal, neonatal joints were compared to rabbit articular chondrocytes. Radiolabeled Escherichia coli derived phospholipid assay and ELISA technique using monoclonal antibodies against recombinant human synovial type sPLA2 were employed. The cells were grown as monolayers as well as in alginate beads. RESULTS: Human articular chondrocytes from both arthritic and neonatal joints released sPLA2 constitutively but could not be further stimulated with interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), IL-6, oncostatin M, lipopolysaccharide (LPS), or forskolin. Marked stimulation was observed when the cells were exposed to 8-bromo cyclic adenosine monophosphate (cAMP). Growing the cells as monolayers or in alginate beads did not change the results. In contrast to human cells, rabbit chondrocytes responded to IL-1 beta and IL-1/TNF, but not to TNF-alpha alone, with a very marked increase in extracellular sPLA2 activity. CONCLUSION: Human articular chondrocytes synthesize and constitutively release sPLA2. Such continuous release is most probably responsible for the high concentration of sPLA2 in articular cartilage and may be the source of synovial fluid sPLA2. To our knowledge, human articular chondrocytes are the only sPLA2 producing cells tested to date that do not respond to cytokine stimulation with increased sPLA2 activity; yet enhancement was seen with 8-bromo cAMP. It seems therefore that, human articular chondrocytes possess signalling mechanisms for the release of sPLA2 unlike those from other mammalian cells. The significance of this observation remains to be elucidated.     

High-affinity aptamers selectively inhibit human nonpancreatic secretory phospholipase A2 (hnps-PLA2)

Evidence is growing that reactive oxygen species (ROS), by-products of (normal) cellular aerobic metabolism, are involved in the pathogenesis of neurodegenerative diseases. One of these diseases is amyotrophic lateral sclerosis (ALS), in which motoneurons die, leading to paralysis and death. It remains uncertain whether ROS are the cause of (apoptotic) motoneuron death in ALS. To further understand the role of ROS in motoneuron death, we investigated the effects of ROS on isolated spinal rat motoneurons in culture. ROS were generated with a combination of iron(III) and ascorbate, or with hydrogen peroxide. Both toxic treatments resulted in a dose-dependent motoneuron death. Iron(III)/ascorbate toxicity was completely prevented with the hydrogen peroxide detoxifying enzyme catalase and partially prevented with the antioxidant vitamin E. SOD1, the enzyme that removes superoxide, did not protect against iron(III)/ascorbate toxicity. ROS treatment caused apoptotic motoneuron death: low doses of iron(III)/ ascorbate or hydrogen peroxide resulted in complete apoptosis ending in nuclear fragmentation, while high doses of ROS resulted in incomplete apoptosis (nuclear condensation). Thus, depending on the dose of ROS, the motoneurons complete the apoptotic pathway (low dose) or are stopped somewhere during this route (high dose).     

A new series of C3-aza carbocyclic influenza neuraminidase inhibitors: synthesis and inhibitory activity

The synthesis and influenza neuraminidase inhibitory activity of a new series of C3-aza carbocyclic neuraminidase inhibitors are described. Analogues 3c and 3j, bearing a 3-pentyl group, exhibit influenza A inhibitory activities comparable to that of 1.     

Studies on the synthesis of cholecystokinin A receptor antagonists. II. Synthesis and cholecystokinin A receptor inhibitory activities of sulfur-containing amide-carboxylic acid derivatives

A number of sulfur-containing amide-carboxylic acid derivatives were synthesized and tested for cholecystokinin A (CCK-A) receptor inhibitory activity in order to study structure-activity relationships. Significant CCK-A receptor inhibitory activities were found in only two series, that is, sulfoxide-carboxylic acid derivatives (9) and sulfone-carboxylic acid derivatives (10). As the most preferred compound, 5-(3,4-dichlorophenylsulfonyl)-4-(N,N-dipentylcarbamoyl)pent anoic acid (10n) was selected.     

New phospholipase A2 inhibitor: synthesis and inhibition mechanism of oxazolidinone phospholipid analog

(R)-3-Dodecanoyl-4-phosphatidylcholino-hydroxymethyl-2-oxazolidino ne (7), which is a new glycerophospholipid analog, was synthesized starting from (S)-glycidol through a 4-alkylsilyoxymethyl derivative and N-acyl-4-hydroxymethyl derivative. The cyclic amide analog 7 showed strong inhibitory activity toward both Group I and II PLA25, but the inhibitory potency of 7 was slightly weaker than that of the linear amide analog (R)-1, which had been developed by de Haas et al. (Biochem. Biophys. Acta 1990, 1043, 67). The interactions of 7 with human secretory PLA2 was investigated by computer modeling in comparison with those of the linear amide analog 1. The results of the computer modeling were very compatible with those of the inhibitory activities toward PLA2S, and the both results showed that the binding mode of the oxazolidinone analog 7 was very similar to that of the genuine substrate and was different from that of the linear amide analog 1.     

Prostaglandin E2 amplifies cytosolic phospholipase A2- and cyclooxygenase-2-dependent delayed prostaglandin E2 generation in mouse osteoblastic cells. Enhancement by secretory phospholipase A2

We used the MC3T3-E1 cell line, which originates from C57BL/6J mouse that is genetically type IIA secretory phospholipase A2 (sPLA2)-deficient, to reveal the type IIA sPLA2-independent route of the prostanglandin (PG) biosynthetic pathway. Kinetic and pharmacological studies showed that delayed PGE2 generation by this cell line in response to interleukin (IL)-1beta and tumor necrosis factor alpha (TNFalpha) was dependent upon cytosolic phospholipase A2 (cPLA2) and cyclooxygenase (COX)-2. Expression of these two enzymes was reduced by cPLA2 or COX-2 inhibitors and restored by adding exogenous arachidonic acid or PGE2, indicating that PGE2 produced by these cells acted as an autocrine amplifier of delayed PGE2 generation through enhanced cPLA2 and COX-2 expression. Exogenous addition or enforced expression of type IIA sPLA2 significantly increased IL-1beta/TNFalpha-initiated PGE2 generation, which was accompanied by increased expression of both cPLA2 and COX-2 and suppressed by inhibitors of these enzymes. Thus, our results revealed a particular cross-talk between the two PLA2 enzymes and COX-2 for delayed PGE2 biosynthesis by a type IIA sPLA2-deficient cell line. cPLA2 is responsible for initiating COX-2-dependent delayed PGE2 generation, and sPLA2, if introduced, enhances PGE2 generation by increasing cPLA2 and COX-2 expression via endogenous PGE2.     

Inhibition of prothrombinase by human secretory phospholipase A2 involves binding to factor Xa

Human group II secretory phospholipase A2 (hsPLA2) exhibits significant anticoagulant activity that does not require its enzymatic activity. We examined which coagulation factor was targeted by hsPLA2 and analyzed which region of the protein may be involved in this inhibition. Prothrombin time coagulation assays indicated that hsPLA2 did not inhibit activated factor V (FVa) activity, whereas activated factor X (FXa) one-stage coagulation assays suggested that FXa was inhibited. The inhibitory effect of hsPLA2 on prothrombinase activity of FXa, FV, phospholipids, and Ca2+ complex was markedly enhanced upon preincubation of hsPLA2 with FXa but not with FV. Prothrombinase activity was also strongly inhibited by hsPLA2 in the absence of PL. High concentrations of FVa in the prothrombinase generation assay reversed the inhibitory effect of hsPLA2. By using isothermal titration calorimetry, we demonstrated that hsPLA2 binds to FXa in solution with a 1:1 stoichiometry and a Kd of 230 nM. By using surface plasmon resonance we determined the rate constants, kon and koff, of the FXa/hsPLA2 interaction and analyzed the Ca2+ effect on these constants. When preincubated with FXa, synthetic peptides comprising residues 51-74 and 51-62 of hsPLA2 inhibited prothrombinase assays, providing evidence that this part of the molecule, which shares similarities with a region of FVa that binds to FXa, is likely involved in the anticoagulant interaction of hsPLA2 with FXa. In conclusion, we propose that residues 51-62 of hsPLA2 bind to FXa at a FVa-binding site and that hsPLA2 decreases the prothrombinase generation by preventing FXa.FVa complex formation.     

Increased expression of human type IIa secretory phospholipase A2 antigen in arthritic synovium

OBJECTIVE: To determine the localisation and level of expression of human type IIa secretory phospholipase A2 (sPLA2) in the synovium of rheumatoid arthritis (RA), osteoarthritis (OA), and non-arthritic (NA) patients and to examine the relation between sPLA2 and histological features of inflammation. METHODS: Immunoperoxidase staining using the anti-sPLA2 monoclonal antibody 9C1 was performed on frozen sections of knee synovium of 10 RA, 10 OA, and 10 NA patients. sPLA2 positive cells were scored on a scale of 0-3 in 10 fields of a representative tissue section from each case. Double labelling immunofluorescence confocal microscopy with antibodies to CD14 or CD45 and 9C1 was used to determine cell type specificity. Inflammation was assessed by semiquantitative scoring of lining layer thickness and mononuclear cell infiltrates (MC) and a cumulative inflammation score, generated by summing the two parameters. Scores in each group were compared using non-parametric statistical analysis. RESULTS: sPLA2 was localised to endothelium (EC), vascular smooth muscle (VSM), and mast cells (M) in all tissue sections. In RA and OA sections, staining was seen in both macrophage-like and fibroblast-like cells in the synovial lining layer (LL) and subsynovial lining layer (SLL). Perineural cells stained positively. Subintimal lymphoid aggregates (LA) were negative in all sections. The RA group showed significantly greater staining in extravascular synovial tissue (median 3.6, range 1.5-6.0) than the OA (median 1.95, range 0-5.3) or NA (median 0, range 0-5.9) groups (p < 0.05). LL staining was significantly higher in RA than both OA and NA sections (p < 0.05). The OA group showed a trend to higher staining scores than the NA group that did not reach significance. There was a significant correlation between the sPLA2 staining score and inflammation score within the RA patient group (p < 0.05). CONCLUSIONS: The synovium is a site of increased expression of sPLA2 antigen in both RA and OA relative to NA. Its presence in both fibroblast and macrophage-like cells in the LL and SLL of synovial tissue in RA and OA, but not NA, indicates that the enzyme is specifically induced in these regions in both conditions with expression in the LL being particularly characteristic of RA. The widespread expression of sPLA2 in synovium suggests it is likely to play a significant part in synovial pathology.     

Potent and selective bicyclic lactam inhibitors of thrombin: Part 2: P1 modifications

The synthesis and antithrombotic activity of a series of nonpeptide bicyclic thrombin inhibitors is described. We have explored the SAR with modifications to the P1 site. The introduction of arginine mimetics at the P1 site led to potent and selective thrombin inhibitors.     

The functions of five distinct mammalian phospholipase A2S in regulating arachidonic acid release. Type IIa and type V secretory phospholipase A2S are functionally redundant and act in concert with cytosolic phospholipase A2

We examined the relative contributions of five distinct mammalian phospholipase A2 (PLA2) enzymes (cytosolic PLA2 (cPLA2; type IV), secretory PLA2s (sPLA2s; types IIA, V, and IIC), and Ca2+-independent PLA2 (iPLA2; type VI)) to arachidonic acid (AA) metabolism by overexpressing them in human embryonic kidney 293 fibroblasts and Chinese hamster ovary cells. Analyses using these transfectants revealed that cPLA2 was a prerequisite for both the calcium ionophore-stimulated immediate and the interleukin (IL)-1- and serum-induced delayed phases of AA release. Type IIA sPLA2 (sPLA2-IIA) mediated delayed AA release and, when expressed in larger amounts, also participated in immediate AA release. sPLA2-V, but not sPLA2-IIC, behaved in a manner similar to sPLA2-IIA. Both sPLA2s-IIA and -V, but not sPLA2-IIC, were heparin-binding PLA2s that exhibited significant affinity for cell-surface proteoglycans, and site-directed mutations in residues responsible for their membrane association or catalytic activity markedly reduced their ability to release AA from activated cells. Pharmacological studies using selective inhibitors as well as co-expression experiments supported the proposal that cPLA2 is crucial for these sPLA2s to act properly. The AA-releasing effects of these sPLA2s were independent of the expression of the M-type sPLA2 receptor. Both cPLA2, sPLA2s-IIA, and -V were able to supply AA to downstream cyclooxygenase-2 for IL-1-induced prostaglandin E2 biosynthesis. iPLA2 increased the spontaneous release of fatty acids, and this was further augmented by serum but not by IL-1. Finally, iPLA2-derived AA was not metabolized to prostaglandin E2. These observations provide evidence for the functional cross-talk or segregation of distinct PLA2s in mammalian cells in regulating AA metabolism and phospholipid turnover.     

Lipoproteins are substrates for human secretory group IIA phospholipase A2: preferential hydrolysis of acute phase HDL

Group IIA secretory phospholipase A2 is an acute phase enzyme, co-expressed with serum amyloid A protein. Both are present in atherosclerotic lesions. We report that human normal and acute phase high density lipoproteins and low density lipoprotein are effective substrates for human group IIA phospholipase A2. The enzyme hydrolyzed choline and ethanolamine glycerophospholipids at the sn -2 position resulting in an accumulation of the corresponding lysophospholipids, including the unhydrolyzed alkyl and alkenyl ether derivatives. The hydrolysis of acute phase high density lipoprotein was 2- to 3-fold more rapid and intensive than of normal high density lipoprotein. The hydrolysis of lipoproteins was noted at enzyme concentration as low as 0.05 microgram/mg protein, which was within the range observed in the circulation in acute and chronic inflammatory diseases. The enzyme hydrolyzed the different molecular species of the residual glycerophospholipids in proportion to their mass, showing no preference for the release of arachidonic acid. Group IIA phospholipase A2 preferentially attacked the hydroxy and hydroperoxy linoleates and possibly other oxygenated fatty acids, which were released from the glycerophospholipids at early times of incubation. There was no effect on the content or molecular species composition of the sphingomyelins or neutral lipids of the lipoproteins. In conclusion, human plasma lipoproteins are the first reported natural biological substrates for human group IIA phospholipase A2. The enhanced hydrolysis of acute phase high density lipoproteins is probably due to its association with serum amyloid A protein, which enhances the activity of the enzyme and may promote its penetration to the lipid monolayer. As sPLA2-induced hydrolysis of the lipoproteins leads to accumulation of lysophosphatidylcholine and potentially toxic oxygenated fatty acids, overexpression of this enzyme may be proatherogenic.