Synthesis of benzoyl phenyl benzoates as effective inhibitors for phospholipase A2 and hyaluronidase enzymes

Benzoylation of (hydroxy phenyl) phenyl methanone 2a-g to benzoyl phenyl benzoates 4a-g, a benzophenone analogue, was achieved in good yield. All the newly synthesized compounds were evaluated for their phospholipase A2 [E.C. 3.1.1.4] and hyaluronidase [E.C. 3.2.1.35] enzyme inhibitory activity in snake venom as source and their structure-activity relationship with respect to different groups is reported for the first time. The in vitro PLA2 enzyme inhibitory activity and in vivo anti-inflammatory activity studies of benzoyl phenyl benzoates are illustrated.     

Phospholipase A2 inhibitors synthesized by two entomopathogenic bacteria, Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata

The entomopathogenic bacteria Xenorhabdus nematophila and Photorhabdus temperata subsp. temperata suppress insect immune responses by inhibiting the catalytic activity of phospholipase A(2) (PLA(2)), which results in preventing biosynthesis of immune-mediating eicosanoids. This study identified PLA(2) inhibitors derived from culture broths of these two bacteria. Both X. nematophila and P. temperata subsp. temperata culture broths possessed significant PLA(2)-inhibitory activities. Fractionation of these bacterial metabolites in the culture broths using organic solvent and subsequent chromatography purified seven potent PLA(2) inhibitors, three of which (benzylideneacetone [BZA], proline-tyrosine [PY], and acetylated phenylalanine-glycine-valine [FGV]) were reported in a previous study. Four other compounds (indole, oxindole, cis-cyclo-PY, and p-hydroxyphenyl propionic acid) were identified and shown to significantly inhibit PLA(2). X. nematophila culture broth contained these seven compounds, while P. temperata subsp. temperata culture broth contained three compounds (BZA, acetylated FGV, and cis-cyclo-PY). BZA was detected in the largest amount among these PLA(2) compounds in both bacterial culture broths. All seven bacterial metabolites also showed significant inhibitory activities against immune responses, such as phenoloxidase activity and hemocytic nodulation; BZA was the most potent. Finally, this study characterized these seven compounds for their insecticidal activities against the diamondback moth, Plutella xylostella. Even though these compounds showed relatively low toxicities to larvae, they significantly enhanced the pathogenicity of Bacillus thuringiensis. This study reports bacterial-origin PLA(2) inhibitors, which would be applicable for developing novel insecticides.     

Inhibitory effect of synthetic C-C biflavones on various phospholipase A(2)s activity

Several prototypes of C-C biflavones (a-f) were synthesized and evaluated their inhibitory activity against phospholipase A(2)s (PLA(2)s) activity. The synthetic C-C biflavones (a-f) showed rather different inhibitory activity against various PLA(2)s. Most synthetic C-C biflavonoids exhibited potent and broad inhibitory activity against various sPLA(2)s and cPLA(2) tested regardless of their structural array. In particular, of natural and synthetic biflavonoids tested, the synthetic C-C biflavonoid (d) only showed inhibitory activity against sPLA(2) X. None of the natural and synthetic biflavonoids tested showed inhibitory activity against sPLA(2) IB. Further chemical modification of these basic structures will be carried out in order to investigate the synthetic C-C biflavones which possess more selective inhibitory activity against isozymes of PLA(2).     

PLA2/PGE2 are involved in the inhibitory effect of bradykinin on the angiotensin-(1-7)-stimulated Na(+)-ATPase activity of the proximal tubule

Recently, we demonstrated that bradykinin (BK) counteracts the stimulatory effect of Ang-(1-7) on the Na(+)-ATPase activity from basolateral membrane of the proximal tubule through B2 receptor. In the present paper, the signaling pathway involved in the inhibitory response of the Na(+)-ATPase activity to BK was investigated. The following results indicate that the phospholipase A2 (PLA2)/COX/prostaglandin E (PGE2) pathway is implicated in this process: (1) The inhibitory effect of BK on Ang-(1-7)-stimulated enzyme is abolished in a dose-dependent manner by quinacrine (10(-9)-10(-6)M), a nonspecific PLA2 inhibitor, and by PACOCF3 (10(-7)M), an inhibitor of a Ca(2+)-independent PLA2. However, AACOCF3 (2 x 10(-4) M), an inhibitor of the cytosolic PLA2, does not modify the inhibitory effect of BK. (2) The inhibitory effect of BK on the Ang-(1-7)-stimulated enzyme is reversed by cyclooxygenase (COX) inhibitors diclofenac (10(-12) M) and indomethacin (10(-12) M). (3) PGE2 (10(-12)-10(-5) M) inhibits the Na(+)-ATPase activity in a dose dependent manner. (4)The inhibitory effects of PGE2 and BK on the Na(+)-ATPase activity are not cumulative. (5) PGE2 (10(-12)-10(-8) M) counteracts the stimulatory effect of Ang-(1-7) on the enzyme activity in a dose-dependent manner.     

Development of a second generation of inhibitors of microsomal prostaglandin E synthase 1 expression bearing the gamma-hydroxybutenolide scaffold

Petrosaspongiolide M (PM), a marine sesterterpene metabolite bearing the gamma-hydroxybutenolide scaffold and displaying a potent inhibitory activity toward PLA(2) enzyme, was selected by us as an attractive target in order to explore its mechanism of action at molecular level. In the course of our investigations we decided to synthetically modify the parent compound to clarify the structural determinants responsible for the activity; in fact, very recently, our research group reported the synthesis and the pharmacological properties of a first collection of PM analogues generated by Ludi approach. The synthesized compounds showed a poor or moderate activity toward PLA(2) enzymes, nevertheless we discovered a potent and selective modulator of the expression of microsomal prostaglandin E synthase 1 (mPGES-1), an enzyme highly involved in the inflammatory response, which represents an interesting target for the development of a new class of anti-inflammatory agents. In this paper we report the synthesis of a further collection of nine analogues, having the same scaffold of PM, the gamma-hydroxybutenolide, and bearing, as side chain, more complex aromatic portions, in substitution of the sesterterpene moiety. Their pharmacological behavior against PLA(2) enzymes as well as to modulate the expression of inducible cyclooxygenase 2 (COX-2) and mPGES-1 enzymes is also described.     

Studies on the measurement of phospholipase A2 (PLA2) and PLA2 inhibitor activities in ram semen.

Extraction with Tris-citrate or Tris-NaCl-EGTA improved the yield of phospholipase A2 (PLA2) from ram semen by 40-50 fold over the previously recommended method of extraction by dilute (0.18 N) sulphuric acid. The enzyme activity in the citrate extract deteriorated more rapidly than in Tris-NaCl-EGTA. The semen PLA2 activity was optimum at pH 8.0, heat sensitive at 70 degrees C for 30 min, activated by Ca2+ (although approximately 60% activity was also found in the absence of calcium) and did not exist as a pro-enzyme. The semen PLA2 activity was equally distributed among the sperm and seminal plasma (SP) components of ram semen. However, the low levels of PLA2 activity in the SP of vasectomised rams tend to suggest that PLA2 in the SP fraction may have originated from testicular or epididymal secretions or leakage, from sperm. PLA, in sperm exists as a large molecular weight aggregate, whereas in SP it is present as a smaller aggregate. In addition to PLA2, semen also contained PLA2 inhibitor activities. Inhibition was observed against PLA2s from bee venom, pig pancreas and oviductal extracts. The inhibitory activity is presumed to be due to a large molecular weight protein as the inhibitor activity was not extracted in a chloroform:methanol (2:1; v/v) mixture, it was non-dialysable, precipitated by 10% trichloroacetic acid and destroyed by proteases. The inhibitor activity was distributed in various molecular weight fractions of sperm, SP and SP from vasectomised rams.     

Phospholipase A2 from Trypanosoma brucei gambiense and Trypanosoma brucei brucei: inhibition by organotins

Activity and kinetics of phospholipase A2 (PLA2) from Trypanosoma brucei gambiense (Wellcome strain) and Trypanosoma brucei brucei (GUTat 3.1) were examined using two different fluorescent substrates. The activity in the supernatants of sonicated parasites was Ca2+-independent, strongly stimulated by Triton X-100 with optimum activity at 37 degrees C and pH 6.5-8.5. To encourage a possible interaction between the parasite enzyme and organotin compounds, fatty acid derivatives of dibutyltin dichloride were synthesized and evaluated as potential inhibitors of PLA2. The enzyme from the two-trypanosome species differ with respect to kinetic parameters and are noncompetitively inhibited by the organotin compounds. The Michaelis constant (KM) for PLA2 from T. b. brucei is 63.87 and 30.90 microM while for T. b. gambiense it is 119.64 and 32.91 microM for the substrates 1,2-bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (PBGPC) and 2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBDC12-HPC), respectively.     

Synthesis of benzophenone oxime analogues as inhibitor of secretory phospholipase A(2) with anti-inflammatory activity

The title compound have been synthesized and tested for structure activity relationship for Phospholipase A(2) (PLA(2)) [E.C. 3.1.1.4] enzyme inhibition. The in vitro PLA(2) enzyme inhibitory activity of benzophenone oxime analogue and in vivo anti-inflammatory activity studies using mice are highlighted.     

Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A2 by acylamino phospholipid analogues.

The competitive inhibition of human pancreatic and a mutant human platelet phospholipase A2 (PLA2) was investigated using acylamino phospholipid analogues, which are potent competitive inhibitors of porcine pancreatic PLA2 [De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257]. Both the mutant platelet PLA2 and the human pancreatic PLA2 are effectively inhibited by these compounds. The enzyme from platelets is most strongly inhibited by compounds with a negatively charged phosphoglycol headgroup. Compounds with a neutral phosphocholine headgroup are only weak inhibitors, whereas an inhibitor with a phosphoethanolamine headgroup shows an intermediate inhibitory capacity. The platelet PLA2 is most effectively inhibited by negatively charged inhibitors having a relatively short (four or more carbon atoms) alkylchain on position one and a acylamino chain of 14 carbon atoms on position two. For the pancreatic enzyme an inhibitor with a phosphoethanolamine headgroup was more effective than inhibitors with either a phosphocholine or a phosphoglycol headgroup. The chainlength preference of the pancreatic enzyme resembles that of the platelet PLA2. The largest discrimination in inhibition between the human platelet and the human pancreatic PLA2 is obtained with inhibitors with a negatively charged phosphoglycol headgroup, an alkyl chain of four carbon atoms on position one and a long acylamino chain of 14-16 carbon atoms on position two. Because the platelet PLA2 is thought to have several biological functions, specific inhibitors of this enzyme could have important implications in the design of pharmaceutically interesting compounds.     

The caveolin scaffolding domain modifies 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor binding properties by inhibiting phospholipase A2 activity

Activation of the enzyme phospholipase (PLA 2) has been proposed to be part of the molecular mechanism involved in the alteration of 2-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor responsiveness during long term changes in synaptic plasticity (long term potentiation). This study assesses the effect of the caveolin-1 scaffolding domain (CSD) on the activity of the regulatory enzyme PLA2. Caveolin-1 is a 22-kDa cholesterol-binding membrane protein known to inhibit the activity of most of its interacting partners. Our results show that the calcium-dependent cytosolic form of PLA2 (cPLA2) and caveolin-1 co-localized in mouse primary hippocampal neuron cultures and that they were co-immunoprecipitated from mouse hippocampal homogenates. A peptide corresponding to the scaffolding domain of caveolin-1 (Cav-(82-101)) dramatically inhibited cPLA2 activity in purified hippocampal synaptoneurosomes. Activation of endogenous PLA2 activity with KCl or melittin increased the binding of [3H]AMPA to its receptor. This effect was almost completely abolished by the addition of the CSD peptide to these preparations. Moreover, we demonstrated that the inhibitory action of the CSD peptide on AMPA receptor binding properties is specific (because a scrambled version of this peptide failed to have any effect) and that it is mediated by an inhibition of PLA2 enzymatic activity (because the CSD peptide failed to have an effect in membrane preparations lacking endogenous PLA2 activity). These results raised the possibility that caveolin-1, via the inhibition of cPLA2 enzymatic activity, may interfere with synaptic facilitation and long term potentiation formation in the hippocampus.     

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2)

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.     

Sera of patients suffering from inflammatory diseases contain group IIA but not group V phospholipase A(2)

During recent years, the high phospholipase A(2) (PLA(2)) concentrations at sites of inflammation and in circulation in several life-threatening diseases, such as sepsis, multi-organ dysfunction and acute respiratory distress syndrome, has generally been ascribed to the non-pancreatic group IIA PLA(2). Recently the family of secreted low molecular mass PLA(2) enzymes has rapidly expanded. In some cases, a newly described enzyme appeared to be cross-reactive with antibodies against the group IIA enzyme. For this reason, reports describing the expression of group IIA PLA(2) during inflammatory conditions need to be reevaluated. Here we describe the identification of the PLA(2) activity in sera of acute chest syndrome patients and in sera of trauma victims. In both cases, the PLA(2) activity was identified as group IIA. This classification was based upon cross-reactivity with monoclonal antibodies against group IIA PLA(2) which do not recognize the recombinant human group V enzyme. Moreover, purification of the enzymatic activity from the two sera followed by N-terminal amino acid sequence analyses revealed only the presence of group IIA enzyme.     

Purification and characterization of a membrane-associated 48-kilodalton phospholipase A(2) in leaves of broad bean

Several lines of evidence indicate that phospholipase A(2) (PLA(2)) plays a crucial role in plant cellular responses through production of linolenic acid, the precursor of jasmonic acid, from membrane phospholipids. Here we report the purification and characterization of a 48-kD PLA(2) from the membrane fractions of leaves of broad bean (Vicia faba). The plant PLA(2) was purified to near homogeneity by sequential column chromatographies from the membrane extracts. The purified 48-kD protein migrated as a single band on a SDS-PAGE gel and its density correlated with the PLA(2) activity. It was further confirmed that this 48-kD protein is the PLA(2) enzyme based on immunoprecipitating the activity with a monoclonal antibody against it and purifying the enzyme to homogeneity with the antibody affinity column. The purified plant PLA(2) preferred 2-linolenoyl-sn-glycerol-3-phosphocholine (GPC) to 2-linoleoyl-GPC, 2-palmitoyl-GPC and 2-arachidonyl-GPC as substrates with a pH optimum at pH 7.0 to 8.0. The plant PLA(2) was activated by calmodulin and inhibited by pretreatment of 5,8,11, 14-eicosatetraynoic acid known as an inhibitor of mammalian PLA(2)s. The enzyme was characterized as a Ca(2+)-independent PLA(2) different from mammalian PLA(2)s. This membrane-associated and Ca(2+)-independent PLA(2) is suggested to play an important role in the release of linolenic acid, the precursor of jasmonic acid, through a signal transduction pathway.     

Synthesis, structure, molecular docking, and structure-activity relationship analysis of enamines: 3-aryl-4-alkylaminofuran-2(5H)-ones as potential antibacterials

Thirty-one 3-aryl-4-alkylaminofuran-2(5H)-ones were designed, prepared and tested for their antibacterial activity. Some of them showed significant antibacterial activity against Gram-positive organisms, especially against Staphylococcus aureus ATCC 25923, but all were inactive against Gram-negative organisms. Out of these compounds, 3-(4-bromophenyl)-4-(2-(4-nitrophenyl)hydrazinyl)furan-2(5H)-one (4a11) showed the most potent antibacterial activity against S. aureus ATCC 25923 with MIC(50) of 0.42 μg/mL. The enzyme assay revealed that the possible antibacterial mechanism of the synthetic compounds might be due to their inhibitory activity against tyrosyl-tRNA synthetase. Molecular dockings of 4a11 into S. aureus tyrosyl-tRNA synthetase active site were also performed. This inhibitor snugly fitting the active site might well explain its excellent inhibitory activity. Meanwhile, this modeling disclosed that a more suitable optimization strategy might be to modify the benzene ring at 3-position of furanone with hydrophilic groups.     

Human retinal pigment epithelium secretes a phospholipase A2 and contains two novel intracellular phospholipases A2.

The sensitivity of different phospholipase A2 (PLA2)-active fractions eluted from cation-exchange chromatography to para-bromophenacylbromide (pBPB), Ca2+-EGTA, DTT, heat, and H2SO4 indicates that human cultured retinal pigment epithelial (hRPE) cells probably contain two different intracellular PLA2 enzymes. Control experiments using "back-and-forth" thin-layer chromatography confirmed that, in our assay conditions, the generation of free fatty acids originated solely from PLA2 activity. Together with immunoblot experiments where no cross-reactivity was observed between the hRPE cytosolic PLA2 enzymes and several antisera directed against secretory PLA2s (sPLA2s) and cytosolic PLA2 (cPLA2), these findings suggest that intracellular hRPE PLA2s are different from well-known sPLA2s, cPLA2, and Ca2+-independent PLA2s. We also report an additional hRPE-PLA2 enzyme that is secreted and that exhibits sensitivity to pBPB, Ca2+-EGTA, DTT, heat, and H2SO4, which is characteristic of sPLA2 enzymes. This approximately 22-kDa PLA2 cross-reacted weakly with an antiserum directed against porcine pancreatic group I sPLA2 but strongly with an antiserum directed against N-terminal residues 1-14 of human synovial group II sPLA2, suggesting that this extracellular enzyme is a member of the sPLA2 class of enzymes. We thus conclude that there are three distinct PLA2 enzymes in cultured hRPE cells, including two novel intracellular PLA2s and a 22-kDa secreted sPLA2 enzyme.     

QSAR studies on structurally similar 2-(4-methanesulfonylphenyl)pyran-4-ones as selective COX-2 inhibitors: a Hansch approach

QSAR analysis based on classical Hansch approach was adopted on two recently reported novel series of 2-phenylpyran-4-ones as selective cyclooxygenase-2 (COX-2) inhibitors. The 6-methyl derivatives of title compounds bifurcate as 3-phenoxypyran-4-ones (subset A) and 3-phenylpyran-4-ones (subset B) among series 1. Series 2 consists of 5-chloro derivatives of title compounds. Various regression equations were derived to study the influence of phenoxy and phenyl ring substituents of series 1 compounds on COX-2, COX-1 and selective COX-2 over COX-1 inhibitory activity. The best triparametric equation derived for 36 compounds of series 1 explains the hydrophobic, electronic and steric requirements for improved COX-2 inhibitory activity. QSAR model derived to explore the selective COX-2 over COX-1 inhibition showed that selectivity could be influenced by size and lipophilicity of substituents. The size of the first atom of 2 substituents appears to have negative effect on selectivity, whereas highly polar 3 substituents at R are favorable for improved selectivity. QSAR investigations on series 2 compounds revealed some interesting correlation of COX-2 inhibitory activity with calculated physicochemical properties of whole molecules. The positive logP confirms the hydrophobic interaction of series 2 compounds with COX-2 enzyme. The positive MR term indicates that an overall increase in size and polarizabilty of the molecules increases COX-2 inhibitory activity. The positive contribution of structural variable suggests biphenyl analogs are extremely potent COX-2 inhibitors.     

Bacterial expression and characterization of starfish phospholipase A(2)

Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyranoside. The recombinant PLA(2) produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris--HCl buffer (pH 8.0). Renatured PLA(2) was purified by subsequent column chromatographies on DEAE--cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca(2+) requirement.     

Structural and functional effects of tryptophans inserted into the membrane-binding and substrate-binding sites of human group IIA phospholipase A2

Phospholipase A(2) (PLA(2)) enzymes become activated by binding to biological membranes and hydrolyze phospholipids to free fatty acids and lyso-phospholipids, the precursors of inflammatory mediators. To understand the functional significance of amino acid residues at key positions, we have studied the effects of the substitution of Val(3) (membrane binding surface) and Phe(5) (substrate binding pocket) of human group IIA PLA(2) by tryptophan on the structure and function of the enzyme. Despite the close proximity of the sites of mutations, the V3W mutation results in substantial enhancement of the enzyme activity, whereas the F5W mutant demonstrates significantly suppressed activity. A structural analysis of all three proteins free in buffer and bound to membranes indicates that large differences in activities result from distinct conformational changes in PLA(2)s upon membrane binding. Although PLA(2) and the V3W mutant demonstrate a decrease in helical content and an increase in helix flexibility, the F5W mutant experiences partial distortion of the alpha-helical structure presumably resulting from the tendency of Trp(5) to insert into the membrane. Furthermore, whereas the PLA(2) and the V3W mutant bind to the membrane at similar and apparently productive-mode orientation, the F5W mutant binds to membranes with a distinctly different orientation. It is suggested that both the stimulatory effect of the V3W mutation and the inhibitory effect of the F5W mutation result from the high affinity of Trp for the membrane-water interface. Although Trp(3) at the membrane binding face of PLA(2) facilitates the proper membrane binding of the enzyme, Trp(5) in the internal substrate binding site causes partial unwinding of the N-terminal helix in order to interact with the membrane.     

Transglutaminase 2 and phospholipase A2 interactions in the inflammatory response in human Thp-1 monocytes

Several experimental approaches have demonstrated that transglutaminase 2 (TG2) increased activity is involved in monocyte activation and inflammatory response. Preliminary results also demonstrate a TG-mediated post-translational modification of phospholipase A₂ (PLA2), which catalyzes the release of arachidonic acid from its lipid storage sites. The control of PLA2-mediated production of eicosanoids has been found to be of great benefit for inflammatory disease treatment. However, the identification of the mechanisms of PLA2 activation is a very complex issue, because of the presence of multiple PLA2 forms. The aim of this study was to characterize the interactions between TG2 and sPLA2 in LPS-stimulated THP-1 cells, which were treated with TPA to induce early differentiated macrophage-type model. We demonstrated that increases in TG2 enzyme activity and protein expression may be considered an early event in monocyte/macrophage activation by LPS. Under these conditions, TG2 protein was co-immunoprecipitated with PLA2 by monoclonal antibody directed against the secretory form of the enzyme (sPLA2-V). Concomitantly, the PLA2 enzyme activity increased in TPA-treated cells exposed to LPS; these high levels of enzyme activity were significant reduced by R283, a site-specific inhibitor of TG2. Moreover, confocal laser scanning microscopy analysis of double-immunostained cytochemical specimens confirmed a co-localization of BAPA-labeled proteins and sPLA2-V in LPS-treated cells. These findings give evidence of a complex TG2/sPLA2-V, suggesting the possibility that sPLA2-V is a substrate for TG2. These results demonstrated that TG2 increases produced a sustained activation of PLA2 activity, suggesting a functional interaction between these enzymes in the regulation of inflammatory response.     

Design,synthesis and biological evaluation of type-II VEGFR-2 inhibitors based on quinoxaline scaffold

In an effort to develop ATP-competitive VEGFR-2 selective inhibitors, a series of new quinoxaline-based derivatives was designed and synthesized. The target compounds were biologically evaluated for their inhibitory activity against VEGFR-2. The design of the target compounds was accomplished after a profound study of the structure activity relationship (SAR) of type-II VEGFR-2 inhibitors. Among the synthesized compounds, 1-(2-((4-methoxyphenyl)amino)-3-oxo-3,4 dihydroquinoxalin-6-yl)-3-phenylurea (VIIa) displayed the highest inhibitory activity against VEGFR-2. Molecular modeling study involving molecular docking and field alignment was implemented to interpret the variable inhibitory activity of the newly synthesized compounds.