Changes of phospholipase A2 inhibitory activity in the K+-sensitive actin gelation factor during the differentiation of myeloid leukemia cells

The phospholipase A2 inhibitory activity of a 38 kDa K+-sensitive actin gelation factor in a murine leukemia cell line (M1) was examined. A specific antibody against 38 kDa protein was found to cross-react with 37 kDa protein (lipocortin) in rat peritoneal exudates. Although the native 38 kDa protein from M1 cells did not block phospholipase A2 activity, pretreatment with alkaline phosphatase produced a form that did inhibit this enzyme. However, a purified 38 kDa protein from differentiated M1 cells blocked phospholipase A2 activity without pretreatment with alkaline phosphatase. Phospholipase A2 inhibitory activity of the 38 kDa protein was not altered by addition of actin. These findings suggest that the phospholipase A2 inhibitory of our 38 kDa protein was induced during differentiation. We also proposed that our 38 kDa protein has the same epitope as lipocortin.     

Synthesis of 5'-substituted fluoro-neplanocin A analogues: importance of a hydrogen bonding donor at 5'-position for the inhibitory activity of S-adenosylhomocysteine hydrolase

Four 5'-substituted fluoro-neplanocin A analogues 1a-d were designed and synthesized, using cyclopentenone derivative 2 as a key intermediate. The inhibitory activity against SAH was in the following order: NH(2)>SH>F, N(3), indicating a hydrogen bonding donor such as OH or NH(2) was essential for inhibitory activity. All the final compounds showed much less decreased cytotoxicity in two cancer cell lines (Col2 and A549), implying that phosphorylation of the 5'-hydroxyl group of fluoro-neplanocin A is closely related to its high cytotoxicity.     

Mapping the region of the alpha-type phospholipase A2 inhibitor responsible for its inhibitory activity

alpha-Type phospholipase A(2) inhibitory protein (PLIalpha) from the serum of the venomous snake Gloydius brevicaudus, GbPLIalpha,isone of the protective endogenous proteins that neutralizes its own venom phospholipase A(2) (PLA(2)), and it is a homotrimer of subunits having a C-type lectin-like domain. The nonvenomous snake Elaphe quadrivirgata has a homologous serum protein, EqPLIalpha-LP, that does not show any inhibitory activity against various snake venom PLA(2)s (Okumura, K., Inoue, S., Ikeda, K., and Hayashi, K. (2003) IUBMB Life 55, 539-545). By constructing GbPLIalpha-Eq- PLIalpha-LP chimeric proteins, we have mapped the residues important in conferring GbPLIalpha inhibitory activity on region 13-36 in the primary structure of GbPLIalpha. Noninhibitory EqPLIalpha-LP showed comparable inhibitory activity only when this region was replaced with that of GbPLIalpha. Further, mutational analysis of the candidate residues revealed that the individual GbPLIalpha to EqPLIalpha-LP residue substitutions N26K, K28E, D29N, and Y144S each produced a mutant GbPLIalpha protein with reduced inhibitory activity, with the single N26K substitution having the most significant effect. Residues 13-36 were suspected to be located in the helical neck region of the GbPLIalpha trimer. Therefore, the region of GbPLIalpha responsible for PLA(2) inhibition was distinct from the carbohydrate-binding site of the homologous C-type lectin.     

Indole Derivatives as Potent Inhibitors of Larval Settlement by the Barnacle,Balanus amphitrite

A potent inhibitor of larval settlement by the barnacle, Balanus amphitrite, was isolated as 2,5,6-tribromo-1-methylgraimne from a marine invertebrate. In comparative tests on the activity of related compounds, such compounds as 2-methylgramine and 2-methyl-3-(morpholinomethyl)-indole exhibited potent inhibitory activity. The inhibitory activity toward larval settlement was found not to be due to toxicity but to a repellent effect.     

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).     

ATP inhibits pump activity of lymph vessels via adenosine A1 receptor-mediated involvement of NO- and ATP-sensitive K+ channels

We examined the effects of ATP on intrinsic pump activity in lymph vessels isolated from the rat. ATP caused significant dilation with a cessation of lymphatic pump activity. Removal of the endothelium or pretreatment with Nomega-nitro-L-arginine methyl ester (L-NAME) significantly reduced ATP-induced inhibitory responses of lymphatic pump activity, whereas reduction was not suppressed completely by 10(-6) M ATP. L-arginine significantly restored ATP-induced inhibitory responses in the presence of L-NAME. ATP-induced inhibitory responses in lymph vessels with endothelium were also significantly, but not completely, suppressed by pretreatment with glibenclamide. 8-Cyclopentyl-1,3-dipropylxanthine (a selective adenosine A1 receptor antagonist), but not suramine (a P2X and P2Y receptor antagonist) or 3,7-dimethyl-1-proparglyxanthine (a selective adenosine A2 receptor antagonist), significantly decreased ATP-induced inhibitory responses. alpha,beta-methylene ATP (a selective P2X and P2Y receptor agonist) had no significant effect on lymphatic pump activity. In some lymph vessels with endothelium (24 of 30 preparations), adenosine also caused dose-dependent dilation with a cessation of lymphatic pump activity. L-NAME significantly reduced the inhibitory responses induced by the lower (3 x 10(-8)-3 x 10(-7) M) concentrations of adenosine. Glibenclamide or 8-cyclopentyl-1,3-dipropylxanthine also significantly suppressed adenosine-induced inhibitory responses. These findings suggest that ATP-induced dilation and inhibition of pump activity of isolated rat lymph vessels are endothelium-dependent and -independent responses. ATP-mediated inhibitory responses may be, in part, related to production of endogenous nitric oxide, involvement of ATP-sensitive K+ channels, or activation of adenosine A1 receptors in lymphatic smooth muscle and endothelium.     

Effect of the photocatalytic activity of TiO(2) on plasmid DNA

We investigated the photodynamic DNA strand-breaking activity of TiO(2). A solution of super-coiled pBR 322 DNA was irradiated with 5 J/cm(2) of UVA in the presence of TiO(2) and the products were analyzed by using gel electrophoresis. The ratio of open-circular DNA to super-coiled circular DNA was calculated from the resulting peak areas as a DNA strand-breaking index (SBI). The SBI of anatase-structure TiO(2) (band gap=3.23 eV) was greater than that of rutile structure (band gap=3.06 eV), and the level of SBI correlated with the photocatalytic activity for degradation of 2-propanol. The inhibitory effects of active oxygen scavengers, including DMSO, glutathione and histidine, on the DNA strand-breaking activity were examined. All of the scavengers except ascorbic acid showed inhibitory effects, as did several polyhydric alcohols including mannitol, a well-known hydroxyl radical scavenger. These results suggest that the photodynamic DNA strand-breaking activity of TiO(2) is due to active oxygen species, especially hydroxyl radicals. Polyhydric alcohols showed an inverse correlation between the inhibitory effect on DNA strand-breaking activity and the octanol/water partition coefficient (logP).     

Demonstration of a phospholipase A2 inhibitor in human plasma and in plasma from the European hedgehog (Erinaceus europaeus)

1. Endogenous phospholipase A2 (PLA2) inhibitors in human plasma and in plasma from the hedgehog (Erinaceus europaeus) were demonstrated. 2. The PLA2 activity increased 45-fold in human plasma when a PLA2 binding factor was removed. Furthermore, two peaks of PLA2 inhibitory activity were found after DEAE-chromatography. 3. High levels of PLA2 inhibitory activity was found in plasma from the European hedgehog, E. europaeus. 4. The molecular weight was estimated to 140,000. 5. On DEAE-chromatography two peaks were found which were chromatographically similar to the PLA2 inhibitors in human plasma.     

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.     

Identification of a region of beta-amyloid precursor protein essential for its gelatinase A inhibitory activity

Because beta-amyloid precursor protein (APP) has the abilities both to interact with extracellular matrix and to inhibit gelatinase A activity, this molecule is assumed to play a regulatory role in the gelatinase A-catalyzed degradation of extracellular matrix. To determine a region of APP essential for the inhibitory activity, we prepared various derivatives of APP. Functional analyses of proteolytic fragments of soluble APP (sAPP) and glutathione S-transferase fusion proteins, which contain various COOH-terminal parts of sAPP, showed that a site containing residues 579-601 of APP(770) is essential for the inhibitory activity. Moreover, a synthetic decapeptide containing the ISYGNDALMP sequence corresponding to residues 586-595 of APP(770) had a gelatinase A inhibitory activity slightly higher than that of sAPP. Studies of deletion of the NH(2)- and COOH-terminal residues and alanine replacement of internal residues of the decapeptide further revealed that Tyr(588), Asp(591), and Leu(593) of APP mainly stabilize the interaction between gelatinase A and the inhibitor. We also found that the residues of Ile(586), Met(594), and Pro(595) modestly contribute to the inhibitory activity. The APP-derived decapeptide efficiently inhibited the activity of gelatinase A (IC(50) = 30 nm), whereas its inhibitory activity toward membrane type 1 matrix metalloproteinase was much weaker (IC(50) = 2 microm). The decapeptide had poor inhibitory activity toward gelatinase B, matrilysin, and stromelysin (IC(50) > 10 microm). The APP-derived inhibitor formed a complex with active gelatinase A but not with progelatinase A, and the complex formation was prevented completely by a hydroxamate-based synthetic inhibitor. Therefore, the decapeptide region of APP is likely an active site-directed inhibitor that has high selectivity toward gelatinase A.     

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.     

Extracellular ATP inhibits the small-conductance K channel on the apical membrane of the cortical collecting duct from mouse kidney

We have used the patch-clamp technique to study the effects of changing extracellular ATP concentration on the activity of the small-conductance potassium channel (SK) on the apical membrane of the mouse cortical collecting duct. In cell-attached patches, the channel conductance and kinetics were similar to its rat homologue. Addition of ATP to the bathing solution of split-open single cortical collecting ducts inhibited SK activity. The inhibition of the channel by ATP was reversible, concentration dependent (K(i) = 64 microM), and could be completely prevented by pretreatment with suramin, a specific purinergic receptor (P(2)) blocker. Ranking of the inhibitory potency of several nucleotides showed strong inhibition by ATP, UTP, and ATP-gamma-S, whereas alpha, beta-Me ATP, and 2-Mes ATP failed to affect channel activity. This nucleotide sensitivity is consistent with P(2)Y(2) purinergic receptors mediating the inhibition of SK by ATP. Single channel analysis further demonstrated that the inhibitory effects of ATP could be elicited through activation of apical receptors. Moreover, the observation that fluoride mimicked the inhibitory action of ATP suggests the activation of G proteins during purinergic receptor stimulation. Channel inhibition by ATP was not affected by blocking phospholipase C and protein kinase C. However, whereas cAMP prevented channel blocking by ATP, blocking protein kinase A failed to abolish the inhibitory effects of ATP. The reduction of K channel activity by ATP could be prevented by okadaic acid, an inhibitor of protein phosphatases, and KT5823, an agent that blocks protein kinase G. Moreover, the effect of ATP was mimicked by cGMP and blocked by L-NAME (N(G)-nitro-l-arginine methyl ester). We conclude that the inhibitory effect of ATP on the apical K channel is mediated by stimulation of P(2)Y(2) receptors and results from increasing dephosphorylation by enhancing PKG-sensitive phosphatase activity.     

Effect of phospholipase A2 inhibitors on the release of arachidonic acid and cell viability in cold-stored hepatocytes

We investigated the effect of phospholipase A(2) (PLA(2)) inhibitors on PLA(2) activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [(3)H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA(2) inhibitors. PLA(2) activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA(2) activity was also suppressed (58% of control) by trifluoperazine (50 microM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 microM) suppressed PLA(2) activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA(2) activity. Thus, the inhibitory activity of these agents appears unrelated to Ca-calmodulin-phospholipid interaction but to have an inhibitory effect on PLA(2) activity. To study the effects of PLA(2) inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs-Henseleit buffer for 2 h at 37 degrees C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA(2) inhibitors blocked PLA(2) activity, there was no suppression of LDH release. PLA(2) may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.     

Evaluation of the inhibition of mushroom tyrosinase and cellular tyrosinase activities of oxyresveratrol: comparison with mulberroside A

The inhibitory effects of oxyresveratrol, the aglycone of mulberroside A, on mushroom and cellular tyrosinase activities and melanin synthesis were evaluated. Mulberroside A and oxyresveratrol showed inhibitory activity against mushroom tyrosinase, with oxyresveratrol demonstrating a greater inhibitory effect than that of mulberroside A. Oxyresveratrol and mulberroside A strongly inhibited melanin production in Streptomyces bikiniensis and exhibited dose-dependent inhibition of tyrosinase activity and inhibition of melanin synthesis in B16F10 melanoma cells. However, the compounds exhibited nearly similar inhibitory effects on the activity of cellular tyrosinase and melanin synthesis in murine melanocytes. The inhibition of melanin synthesis by mulberroside A and oxyresveratrol was involved in suppressing the expression level of melanogenic enzymes, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). These results indicate that the inhibition rate of mushroom tyrosinase might not provide an accurate estimate of the inhibition rate of melanin synthesis in melanocytes.     

Synthesis of novel ageladine A analogs showing more potent matrix metalloproteinase (MMP)-12 inhibitory activity than the natural product

By employing a previously established synthetic scheme, the synthesis described in the title was carried out in order to explore the substituent effects in the pyrrole ring of ageladine A on MMP-12 inhibitory activity. It became evident that a halogen atom (Br or Cl) at the 2-position and an additional bromine atom at the 4-position are highly effective for improving the inhibitory activity. These studies led us to discover three novel ageladine A analogs (4a, c, o) showing more potent MMP-12 inhibitory activity than the natural product.     

Synthesis, in vitro inhibitory activity towards COX-2 and haemolytic activity of derivatives of esculentoside A

Esculentoside A (EsA) has been reported to possess anti-inflammatory activity and selective inhibitory activity towards cyclooxygenase-2. A series of derivatives of EsA were synthesized by converting the C-28 carboxylic acid group into amides. The haemolytic activity and inhibitory activity towards cyclooxygenase-2 were evaluated in vitro. The SAR study of the derivatives was conducted and showed that introducing aromatic ring to EsA greatly enhanced its biological activity. Compound 23 showed higher inhibitory activity than Celecoxib and EsA, but lower haemolytic toxicity than EsA.     

Discovery of Ipragliflozin (ASP1941): a novel C-glucoside with benzothiophene structure as a potent and selective sodium glucose co-transporter 2 (SGLT2) inhibitor for the treatment of type 2 diabetes mellitus

A series of C-glucosides with various heteroaromatics has been synthesized and its inhibitory activity toward SGLTs was evaluated. Upon screening several compounds, the benzothiophene derivative (14a) was found to have potent inhibitory activity against SGLT2 and good selectivity versus SGLT1. Through further optimization of 14a, a novel benzothiophene derivative (14h; ipragliflozin, ASP1941) was discovered as a highly potent and selective SGLT2 inhibitor that reduced blood glucose levels in a dose-dependent manner in diabetic models KK-A(y) mice and STZ rats.     

Structural requirement of chalcones for the inhibitory activity of interleukin-5

Novel chalcones were found as potent inhibitors of interleukin (IL)-5. 1-(2-Benzyloxy-6-hydroxyphenyl)-3-(4-hydroxyphenyl)-2-propen-1-one (2b, 78.8% inhibition at 50microM, IC(50)=25.3microM) was initially identified as a potent inhibitor of IL-5. This shows the compatible activity with budesonide or sophoricoside. To identify structural requirements, 26 chalcones were prepared and their inhibitory activities were tested against IL-5. Among them, compound 4-[(E)-3-(2-cyclohexylmethoxy-6-hydroxyphenyl)-3-oxoprop-1-enyl]benzenesulfonamide (2w, 99.5% inhibition at 50microM, IC(50)=1.8microM) shows the most potent activity. The important structural requirements of these chalcone analogs exhibiting the inhibitory activity against IL-5 were recognized as the following. (1) The hydrophobic group such as benzyloxy or cyclohexylmethoxy at 6-position of A ring is necessary. (2) The existence of phenolic hydroxyl at 6-position of A ring is critical. (3) Propenone unit as alpha,beta-unsaturated ketone is essential. (4) Electron withdrawing groups with hydrogen acceptor property at 4-position of B ring enhance the activity and quantitative structure-activity relationship of 2 regarding these substituents was determined.