Binding of protein kinase C to N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide through its ATP binding site

Protein kinase C (PKC) is a Ca2+- and phospholipid-dependent protein kinase which has been implicated as a key enzyme in the regulation of cellular growth. The naphthalenesulfonamide W7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] is representative of a number of cationic amphiphilic inhibitors of PKC which appear to inhibit PKC by interacting with the acidic phospholipid cofactor of the enzyme, according to kinetic studies. In a previous report, we demonstrated that PKC binds directly to W7 when the naphthalenesulfonamide is immobilized on agarose. In the present report, we have defined the mechanism of the binding of PKC to W7-agarose, and its relevance to the inhibitory mechanism of the naphthalenesulfonamide. We demonstrate that PKC bound W7-agarose through the catalytic domain of the enzyme. An active catalytic fragment of PKC was generated by limited proteolysis, and we found that this fragment bound W7-agarose and coeluted with intact PKC upon the addition of Triton X-100. W7 inhibited PKC activity by two different mechanisms. As previously reported, W7 inhibited PKC by interacting with the phospholipid cofactor of the enzyme (IC50 = 260 microM). However, at higher concentrations of W7, we found that this naphthalenesulfonamide inhibited PKC by serving as a competitive inhibitor with respect to the substrate ATP, according to a kinetic analysis of the inhibition of the active catalytic fragment of PKC by W7. W7 inhibited the active catalytic fragment of PKC as well as PKC-catalyzed phosphorylation of protamine sulfate, a reaction which is independent of Ca2+ and phospholipid, with similar potencies. Consistent with the kinetic evidence that W7 serves as a competitive inhibitor of PKC with respect to ATP, we found that, in the presence of 10 mM MgCl2, 1 mM ATP was sufficient to elute PKC from W7-agarose. Thus, naphthalenesulfonamide PKC inhibitors may include both agents which primarily function by interacting with the phospholipid cofactor of the enzyme and agents which primarily serve as active site inhibitors of PKC.     

1H-NMR studies of calmodulin: the modifying effect of W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide) on the calcium-induced conformational changes of calmodulin

The effect of W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin antagonist, on the calcium-bound conformation of calmodulin was studied by 1H-NMR at 400 MHz. W-7 affected the resonances of Ile-27, Phe-68, Phe-92, Ile-100, His-107 and Val-142. The resonances of Met-71, Met-72, Met-76, Phe-89 and Phe-141 may be affected by W-7. These findings suggest that W-7 binds to hydrophobic amino acid residues, which almost occur in calcium-binding sites II, III and IV or their vicinity. The effect of W-7 on the structure of calmodulin was similar to that of other drugs, trifluoperazine, D600 and oxmetidine. Thus, those residues in the high-field methyl region, the methionine methyl region and the phenylalanine aromatic region of calmodulin, which were similarly affected by all four drugs, may be important at the interface for binding of calmodulin to the regulatory sites on target enzymes.     

Inhibition of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity and tumor promotion by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) in mouse skin

N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited epidermal ornithine decarboxylase (ODC) induction caused either by 12-O-tetradecanoylphorbol-13-acetate (TPA) or teleocidin in CD-1 mice. Inhibitory effect of W-7 on TPA-caused ODC induction was also observed in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated skin and even after repetitive TPA treatment. TPA-induced skin tumor promotion was also suppressed by W-7. Meanwhile, W-7 showed only slight inhibitory effects on calcium-activated, phospholipid-dependent protein kinase (protein kinase C) activity of mouse epidermis stimulated either by Ca2+ or TPA in the presence of phosphatidylserine. Thus, it is unlikely that the anti-ODC-inducing and anti-tumor-promoting actions of W-7 are due to its inhibitory effect on protein kinase C. It may be possible that a calmodulin-mediating process is involved in the mechanism of epidermal ODC induction and tumor promotion caused by tumor promoters such as TPA and teleocidin.     

Symmetric covalent linkage of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) results in novel derivatives with increased inhibitory activities against calcium/calmodulin complex

A useful calmodulin (CaM) antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), was invented by Hidaka et al. in 1978 (J. Pharmacol. Exp. Ther. 207, 8-15). Here, we have designed new CaM antagonists on the basis of the three-dimensional structure of Ca2+/CaM complexed with W-7. Eleven new compounds all share a similar architecture, in which two W-7 molecules are linked between their aminohexyl termini by a linker with different functionalities. A wide range of inhibitory activities against Ca2+/CaM-dependent protein kinase I (CaM kinase I) has been observed with these self-crosslinked W-7 analogs, (W-7)2. In vitro competitive CaM kinase I assays using CaM kinase I and nuclear magnetic resonance studies indicated that one (W-7)2 molecule binds to one CaM molecule as expected, with the two chloronaphthalene rings of (W-7)2 being anchored separately to the N- and C-terminal hydrophobic pockets of Ca2+/CaM. The most potent compound, N,N'-bis[6-(5-chloro-1-naphthalenesulfonyl)-amino-1-hexyl]-p-xylen e-diamine ((W-7)2 - 10), inhibits CaM kinase I activity at an IC50 value of 0.23 microM; about 75 times more effectively than W-7. The length and basicity of the linker sequence in (W-7)2 significantly contribute to inhibitory activity. The present study opens an avenue for developing powerful CaM antagonists that could be used at low doses in vivo.     

6-(1-溴乙基)-4-氯-5-氟嘧啶合成新方法

以氟乙酸乙酯为起始原料,改进抗真菌药物伏立康唑中间体6-(1-溴乙基)-4-氯-5-氟嘧啶的合成方法.该方法简化了生产工艺,降低了反应成本,反应条件温和,适合工业化生产,总收率为41.7%.     

The antifungal activities of some 6-[N-(halophenyl)amino]-7-chloro-5,8-quinolinediones againstCandida species

A series of 6-[N-(halophenyl)amino]-7-chloro-5,8-quinolinedione derivatives 1-10 were tested for antifungal susceptibilities, in vitro, against pathogenic Candida species such as C. albicans, C. glabrata, C. krusei, C. parapsilosis and C. tropicalis. The MICs were determined by the standard macrodilution techniques, according to the NCCLS 1992 guidelines. The 6-[N-(halophenyl)amino]-7-chloro-5,8-quinolinedione derivatives showed generally potent antifungal activities against pathogenic Candida species. Among them, derivative 1, 2, 5 and 7 showed more potent antifungal activities than ketoconazole. All derivatives 1-10 had specially potent activities against C. tropicalis. Derivative 1 and 2 containing (N-3,4-dihalo-phenyl)amino moiety exhibited the potent antifungal activities. Derivative 2 with (3,4-dichlorophenyl)amino substituent was the most effective in preventing the growth of Candida species at MICs 4 micrograms/ml respectively.     

Extraneuronal noradrenaline transport (uptake2) in a human cell line (Caki-1 cells)

Summary This study describes for the first time an experimental system for the extraneuronal transport mechanism of noradrenaline (uptake₂) which is based on a clonal cell line (Caki-1). Caki-1 cells were originally derived from a human renal cell carcinoma. The conclusion that these cells express uptake₂ is supported by several experimental findings. (1) The initial rate of ³H-noradrenaline uptake in Caki-1 cells is saturable, the K _m being 450 mol/l. (2) Inhibitors of uptake₂ such as corticosterone (1 mol/l) and O-methyl-isoprenaline (100 Emol/l) largely inhibit ³H-noradrenaline uptake in Caki-1 cells. Whereas inhibitors of the neuronal transport mechanism for noradrenaline (uptake₁) such as desipramine (1 mol/l) and cocaine (10 mol/l) do not reduce it. (3) Depolarization of Caki-1 cells by the elevation of extracellular potassium inhibits ³H-noradrenaline uptake. (4) There is a highly significant correlation between the IC₅₀'s of various compounds for the inhibition of ³H-noradrenaline uptake in Caki-1 cells and rabbit aorta known to possess uptake₂.Interestingly enough, uptake₂ in Caki-1 cells and rabbit aorta is inhibited by cimetidine, quinidine and procainamide which are substrates of the renal transport mechanism for organic cations. Moreover, ³H-cimetidine is shown to be a substrate of uptake₂ in the isolated perfused rat heart. These results indicate a striking similarity between uptake₂ and the renal transport mechanism for organic cations. Send offprint requests to E. Schömig at the above addressSupported by the Deutsche Forschungsgemeinschaft (SFB 176, Scho 373) and the Dr. Robert Pfleger Stiftung     

1-(6-氯-3,4-亚甲二氧基苄基)-2-羧基-5-(2,6-二氯苄氧基)吲哚的合成工艺改进

目的寻找脂肪酸缩合酶Ⅲ(FabH)抑制剂1-(6-氯-3,4-亚甲-二氧基苄基)-2羧基-5-(2,6-二氯苄氧基)吲哚的新合成路线。方法以对乙酰氨基苯酚为起始原料,制备4-(2,6-二氯苄氧基)苯胺,用经典的Fischer吲哚合成法制备吲哚片断,随后引入6-氯-3,4-亚甲二氧基苄基,最后酯水解得到目标化合物。结果与结论目标化合物经1H-NMR1、3C-NMR和MS确证,中间体经1H-NMR确证。该路线成本低,操作简单可行,总收率14.2%,可顺利得到目标化合物。     

6-[(N-2,3-dichlorophenyl)amino]-7-chloro-5,8-quinolinedione treatment of candidiasis in normal mice

6-[(N-2,3-Dichlorophenyl)amino]-7-chloro-5,8-quinolinedione (RCK11) was tested forin vivo antifungal activities in the treatment of systemic infection withCandida albicans in normal mice compared with ketoconazole. The therapeutic potential of RCK11 had been assessed by evaluating their activities (survival rates) against systemic infections in normal mice. ED₅₀ of intraperitoneally administered RCK11 was 0.10±0.01 mg/kg but that of ketoconazole had 8. 00±0.73 mg/kg respectively. When RCK11 was administered intravenously at the ED₅₀ (0.10 mg/kg), the colony counts ofCandida albicans in the liver after 7 days and 14 days were reduced as likely as ketoconazole at the ED₅₀ (8.00 mg/kg), and the better survival rates than ketoconazole were achieved after 14 days. The results suggest that RCK11 may be a potent antifungal agent.     

Polarized efflux of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein from cultured epithelial cell monolayers.

We have investigated the polarity of the efflux of the intracellular pH fluorochrome 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) from layers of epithelial Madin-Darby canine kidney (MDCK, Strains I and II) and human intestinal (Caco-2, HCT-8 and T84) cells grown on porous membranes. In Strain I MDCK cells, BCECF efflux was effectively reduced by indomethacin (50% inhibition with 100 microM) and 5-nitro-2-(3-phenylpropyl-amino)-benzoate (NPPB; 50% inhibition with 10 microM). Replacement of external Cl- with bromide, iodide or nitrate did not alter BCECF efflux, while substitution with methanesulphonate resulted in a small but significant reduction. All five cell lines form confluent epithelial layers when grown on porous membranes. Efflux of BCECF from Strain I MDCK epithelial layers into the apical solution was approximately three times greater than into the basal solution. Addition of indomethacin to the apical solution attenuated efflux into the apical but not the basal solution, while basal indomethacin was effective against basal efflux. NPPB has a similar specificity of action. Adrenaline, a stimulant of electrogenic Cl- secretion, did not alter the pattern of BCECF efflux. BCECF efflux was also polarized, with apical efflux greater than basal efflux, in MDCK Strain II and Caco-2 epithelial layers. In contrast, BCECF efflux into the basal and apical media was equivalent in layers formed from HCT-8 and T84 cells. However, indomethacin reduced efflux in all five epithelial lines, although the relative sensitivities of the apical and basal efflux rates to indomethacin varied, as did the sensitivity to the sidedness of application of indomethacin. In MDCK and HCT-8 epithelial layers, transepithelial vinblastine secretion mediated by P-glycoprotein was not inhibited by indomethacin. The data are consistent with the hypothesis that BCECF efflux is a manifestation of a novel ATP-dependent xenobiotic secretory efflux mechanism in renal and gastrointestinal epithelia. The factors regulating the polarity of BCECF efflux, both the indomethacin-sensitive and -insensitive components, have yet to be elucidated.     

Antiallergic agents. 2. N-(1H-tetrazol-5-yl)-6-phenyl-2-pyridinecarboxamides

A new series of N-(1H-tetrazol-5-yl)-6-phenyl-2-pyridinecarboxamides was prepared to determine the effects of substituents on the benzene and pyridine rings on antiallergic activity in the rat passive cutaneous anaphylaxis (PCA) assay after oral administration. One member of this series, N-(1H-tetrazol-5-yl)-4-methyl-6-[4-(methylamino)-phenyl]-2- pyridinecarboxamide (231), has an ED50 value of 0.8 mg/kg po and is 85 times more potent than disodium cromoglycate (DSCG) on intravenous administration. Further evaluation of 231 as a clinically useful antiallergic agent is in progress.     

Chemical reactivity of ethyl (6R)-6-[N-(2-chloro-4-fluorophenyl)sulfamoyl]cyclohex-1-ene-1-carboxylate (TAK-242) in vitro

Ethyl (6R)‐6‐[N‐(2‐chloro‐4‐fluorophenyl)sulfamoyl]cyclohex‐1‐ene‐1‐carboxylate (TAK‐242) was metabolized to cyclohexene and phenyl ring moieties in non‐clinical pharmacokinetic studies and it was suggested that the cyclohexene ring moiety of TAK‐242 is tightly bound to endogenous macromolecules. After incubation of TAK‐242 and glutathione (GSH) in phosphate buffer (pH 7.4) at 37 °C, TAK‐242 reacted with GSH to produce a glutathione conjugate of the cyclohexene ring moiety of TAK‐242, which had been observed as a metabolite (M‐SG) in non‐clinical pharmacokinetic studies. Formation of M‐SG was time dependent with a first order reaction and M‐I, a metabolite from the phenyl ring moiety of TAK‐242, was also produced in parallel. The formation of M‐SG was accelerated with increasing pH, therefore it was indicated that TAK‐242 reacted with GSH by a nucleophilic substitution reaction. Because glutathione transferase (GST) enhanced M‐SG formation in vitro, it is expected that the conjugation of TAK‐242 with GSH is also facilitated by GST in vivo in addition to a spontaneous chemical reaction. When radio‐labeled TAK‐242 ([cyclohexene ring‐U‐¹⁴ C]TAK‐242) was incubated with rat serum albumin (RSA) or human serum albumin (HSA) in vitro, the radioactive material was covalently bound to RSA and HSA, and M‐I was generated simultaneously in the reaction mixture. The chemical structure of the TAK‐242 adduct covalently bound to HSA was characterized by the accurate mass spectra that cyclohexene ring moiety of TAK‐242 was covalently bound to the lysine residue in HSA. The adduct was also detected in the plasma of rats and humans after single i.v. dosing of TAK‐242 (in vivo). Copyright © 2011 John Wiley & Sons, Ltd.     

Anilides related to substituted benzamides. Potential antipsychotic activity of N-(4-amino-5-chloro-2-methoxyphenyl)-1-(phenylmethyl)-4-piperidinecarboxamide

The substituted benzamides are used clinically both as antipsychotics and as stimulants of gastric motility. The antipsychotic effects are considered to be a consequence of their central dopamine antagonist properties, but there is evidence that the gastric stimulatory effects may be mediated by other mechanisms. Clebopride (3) is a substituted benzamide that although marketed for its stimulatory effects on gastric motility, is also a potent central dopamine antagonist. The corresponding anilide, BRL 20596 (4a), where the amide bond has been reversed, has been synthesized and found to lack gastric stimulatory activity. However, the potent central dopamine antagonist activity is retained, suggesting that benzamides and anilides have similar affinities for central dopamine receptors. The implications of the conformations adopted by benzamides and anilides at such receptors are discussed. Evidence is also presented that there is a further lipophilic binding site on such receptors for which the N-benzyl group is an optimal fit.     

Inefficient nucleotide excision repair in human cell extracts of the N-(deoxyguanosin-8-yl)-6-aminochrysene and 5-(deoxyguanosin-N(2)-yl)-6-aminochrysene adducts derived from 6-nitrochrysene

Ubiquitous environmental agents [e.g., polynuclear aromatic hydrocarbons (PAHs) and their nitrated derivatives (NO(2)-PAHs)] that are known to induce mammary cancer in rodents are regarded as potential human risk factors for inducing analogous human cancers. Although 6-nitrochrysene (6-NC) is less abundant than other NO(2)-PAHs in the environment, it is the most potent mammary carcinogen in the rat; its carcinogenic potency is not only higher than that of the carcinogenic PAH, benzo[a]pyrene (B[a]P), but also of the well-known carcinogenic heterocylic aromatic amine, 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP). Studies in rats and in vitro assays have indicated that 6-NC can be activated by simple nitroreduction leading to the formation of 6-hydroxylaminochrysene (N-OH-6-AC); this metabolite yielded N-(deoxyguanosin-8-yl)-6-aminochrysene (N-[dG-8-yl]-6-AC) and 5-(deoxyguanosin-N(2)-yl)-6-aminochrysene (5-[dG-N(2)-yl]-6-AC. These lesions are likely to cause mutations if they are not removed by cellular defense mechanisms before DNA replication occurs. However, nothing is known about the susceptibility of these adducts to nucleotide excision repair (NER), the major cellular repair system that removes bulky adducts. In order to address this issue, we synthesized the N-(dG-8-yl)-6-AC and 5-(dG- N(2)-yl)-6-AC lesions and site-specifically inserted these lesions into 135-mer DNA duplexes. These constructs were incubated with NER-competent nuclear extracts from human HeLa cells. The efficiency of repair of these lesions was ～ 8 times less efficient than that in the case of the well-known and excellent substrate of NER, the intrastrand cross-linked cis-diaminodichloroplatinum II adduct in double-stranded DNA (cis-Pt), but similar to N(2)-dG adducts derived from the (+)-bay region diol epoxide of B[a]P [(+)-trans-B[a]P-N(2)-dG]. The results support the hypothesis that the N-(dG-8-yl)-6-AC and 5-(dG-N(2)-yl)-6-AC lesions may be slowly repaired and thus persistent in mammalian tissue which could, in part, account for the potent tumorigenic activity of 6-NC in the rat mammary gland.     

In vitro andin vivo antifungal activities of 6-[(N-4-bromophenyl) amino]-7-chloro-5,8-quinolinediones

Antifungal activities of 6-[(N-4-bromophenyl)amino]-7-chloro-5,8-quinolinedione (RCK7) were tested. The MIC values of RCK7 were determined for antifungal suceptibility,in vitro againstAspergillus niger, Cryptococcus neoformans andTrichophyton mentagrophyte by standard agar streak method.In vitro, RCK7 showed more potent antifungal activity than fluconazole and ketoconazole. Also, RCK7 was tested forin vivo antifungal activity in the treatment of systemic infection withCandida albicans in normal mice. The therapeutic potential of RCK7 had been assessed by evaluating their survival rate against systemic infections compared with that of ketoconazole. ED₅₀ of intraperitoneally administered RCK7 was 2.05±0.30 mg/kg but that of ketoconazole was 8.00±0.73 mg/kg, respectively. When RCK7 was administered intravenously at the ED₅₀ (2.05 mg/kg), the colony counts ofCandida albicans in the liver after 7 days and 14 days were reduced as likely as ketoconazole at the ED₅₀ (8.00 mg/kg), and the better survival rates than ketoconazole’s were achieved after 14 days. The results suggest that RCK7 may be a potent antifungal agent.     

Transcellular transport of creatinine in renal tubular epithelial cell line LLC-PK1

BACKGROUND/AIM: Creatinine is excreted into urine via tubular secretion in addition to glomerular filtration. In the present study, characteristics of the creatinine transport in renal epithelial cells were investigated. METHODS: The transcellular transport and accumulation of [14C]creatinine and [14C]tetraethylammonium (TEA) were assessed using LLC-PK1 cell monolayers cultured on porous membrane filters. RESULTS: [14C]Creatinine was transported directionally from the basolateral to apical side of LLC-PK1 cell monolayers. Basolateral uptake of [14C]creatinine was dependent on membrane potential, and was saturable with apparent K(m) and V(max) values of 13.2+/-2.8 mM and 13.1+/-3.1 nmol/mg protein/5 min, respectively. Concomitant administration of organic cations (1 mM) such as cimetidine, quinidine and trimethoprim inhibited both the transcellular transport and accumulation of [14C]creatinine. Furthermore, apical excretion of [14C]creatinine was not dependent on acidification of the apical medium. CONCLUSIONS: Creatinine was subjected to directional transport across renal epithelial cells from the basolateral to apical side. The organic cation transporter should be involved in the basolateral uptake of creatinine.