The interplay of electrostatic and binding interactions determining active centre chemistry and catalytic activity in actinidin and papain.
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Catalysis↗
Biomedical subjects
E W Thomas
Publications and source records attributed to E W Thomas.
Synthesis of acylguanidine analogues: inhibitors of ADP-induced platelet aggregation.
Routine screening of compounds for inhibition of ADP-induced platelet aggregation in vitro revealed that 1,1'-hexamethylenebis[3-cyclohexyl-3-[(cyclohexylimino) (4-morpholinyl) methyl]urea] (1) was active and represented the first example of a bis(acylguanidine) with possible antithrombotic activity. In order to develop a structure-activity relationship for this class of compounds, we synthesized a number of new bis(acylguanidines). These were tested in vitro, and several analogues were also active. Ex vivo testing revealed that compounds 22, 41, 58, and 70-73 were orally active in rats or guinea pigs.
Adenosine Diphosphate↗
Radiosensitization of E. coli B/r by arylhydrazonopropanedinitriles.
Several arylhydrazonopropanedinitriles and an arylhydrazonopropane-diethyl ester (derivatives of well-known uncouplers of oxidative phosphorylation) have been studied with respect to their ability to radiosensitize E. coli B/r under oxic and hypoxic conditions. Of the compounds studied, 2-carboxyphenylhydrazono-propanedinitrile and 2-carboxyphenylhydrazonopropanediethylester were found to be the most efficient radiosensitizers under hypoxia, whilst the former compound was also found to provide radiosensitization under oxic conditions. Increased radiosensitization by 4-carboxyphenylhydrazonopropanedinitrile was observed on decreasing the pH of the irradiation incubation medium. The results are discussed with respect to the physicochemical properties of these compounds and their reactivity with thiols, for which data are presented.
Acetylcysteine↗
Consequences of molecular recognition in the S1-S2 intersubsite region of papain for catalytic-site chemistry. Change in pH-dependence characteristics and generation of an inverse solvent kinetic isotope effect by introduction of a P1-P2 amide bond into a two-protonic-state reactivity probe.
1. The pH-dependences of the second-order rate constant (k) for the reactions of papain (EC 3.4.22.2) with 2-(acetamido)ethyl 2'-pyridyl disulphide and with ethyl 2-pyridyl disulphide and of k for the reaction of benzimidazol-2-ylmethanethiol (as a minimal model of cysteine proteinase catalytic sites) with the former disulphide were determined in aqueous buffers at 25 degrees C at I 0.1. 2. Of these three pH-k profiles only that for the reaction of papain with 2-(acetamido)ethyl 2'-pyridyl disulphide has a rate maximum at pH approx. 6; the others each have a rate minimum in this pH region and a rate maximum at pH 4, which is characteristic of reactions of papain with other 2-pyridyl disulphides that do not contain a P1-P2 amide bond in the non-pyridyl part of the molecule. 3. The marked change in the form of the pH-k profile consequent upon introduction of a P1-P2 amide bond into the probe molecule for the reaction with papain but not for that with the minimal catalytic-site model is interpreted in terms of the induction by binding of the probe in the S1-S2 intersubsite region of the enzyme of a transition-state geometry in which nucleophilic attack by the -S- component of the catalytic site is assisted by association of the imidazolium ion component with the leaving group. 4. The greater definition of the rate maximum in the pH-k profile for the reaction of papain with an analogous 2-pyridyl disulphide reactivity probe containing both a P1-P2 amide bond and a potential occupant for the S2 subsite [2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [Brocklehurst, Kowlessur, O'Driscoll, Patel, Quenby, Salih, Templeton, Thomas & Willenbrock (1987) Biochem. J. 244, 173-181]) suggests that a P2-S2 interaction substantially increases the population of transition states for the imidazolium ion-assisted reaction. 5. The overall kinetic solvent 2H-isotope effect at pL 6.0 was determined to be: for the reaction of papain with 2,2'-dipyridyl disulphide, 0.96 (i.e. no kinetic isotope effect), for its reaction with the probe containing only the P1-P2 amide bond, 0.75, for its reaction with the probe containing both the P1-P2 amide bond and the occupant for the S2 subsite, 0.61, and for kcat./Km for its catalysis of the hydrolysis of N-methoxycarbonylglycine 4-nitrophenyl ester, 0.67.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetamides↗
Pharmacologic effects of A-56234, a new high-ceiling diuretic.
The pharmacokinetic characteristics, the diuretic, saluretic, and uricosuric properties, and the safety of single, rising, oral doses of A-56234, a new high-ceiling diuretic, were evaluated in this double-blind, placebo-controlled, cross-over study. Each of three groups of eight subjects received placebo and three different single doses of the diuretic at 1-week intervals. Doses ranged from 0.5 to 80 mg. Significant, dose-related increases in urine volume and in urinary excretion of sodium and chloride were produced during the 24 hours after administration of 20, 40, 60, and 80 mg of the drug. Uricosuria was not observed at any dose. The drug was rapidly absorbed and displayed linear pharmacokinetics within the dose range studied. The elimination-phase plasma half-life was approximately 6 hours. Hepatic clearance was the main route of excretion in humans; only 2 to 10% of the parent drug was excreted in the urine. The drug was well tolerated and no clinically important adverse events were noted.
Adolescent↗
Light-induced fusion of liposomes with release of trapped marker dye is sensitised by photochromic phospholipid.
Liposomes have been prepared from dipalmitoylphosphatidylcholine containing small amounts of a synthetic photochromic phospholipid, 'Bis-Azo PC'. In the dark, these are stable at room temperature, and contents do not significantly leak over weeks. Photoisomerisation results in immediate release of trapped marker, and in liposome fusion to form larger structures. Fusion has been detected using a fluorescence polarisation assay, and confirmed by electron microscopy. In mixtures, fusion occurs between 'photochromic' liposomes and those of pure lipid. Bis-Azo PC contains two photochromic acyl chains; analogues bearing a single photochromic chain appear to have little effect on bilayer permeability after isomerisation. Photo-induced leakage and liposome fusion suggest possible applications for localised drug delivery as an adjunct to phototherapy. The ability to non-invasively trigger fusion processes should be useful in fundamental studies of membrane interactions. We believe this to be the first report of photo-induced fusion to date.
1,2-Dipalmitoylphosphatidylcholine↗
Resonance Raman study of reconstituted carotenoproteins incorporating astaxanthin and 15,15'-didehydroastaxanthin.
Two reconstituted carotenoproteins have been studied by resonance Raman spectroscopy. They were prepared from the apoprotein of the Asterias rubens carotenoprotein, asteriarubin and either astaxanthin or 15,15'-didehydroastaxanthin. Spectral properties of dehydrocarotenoids are first discussed. The spectral properties of the complexes are compared to those of the free carotenoids and of other carotenoproteins containing astaxanthin, and possible protein-carotenoid interactions are discussed. Greater delocalisation of the pi-electron system in the central part of the polyene chain, and the role of lateral methyl groups in binding is emphasised.
Animals↗
Substrate-derived two-protonic-state electrophiles as sensitive kinetic specificity probes for cysteine proteinases. Activation of 2-pyridyl disulphides by hydrogen-bonding.
1. 2-(N'-Acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide [compound (III)] and 2-(acetamido)ethyl 2'-pyridyl disulphide [compound (IV)] were synthesized by acylation of the common intermediate, 2-aminoethyl 2'-pyridyl disulphide, to provide examples of chromogenic thiol-specific substrate-derived two-protonic-state electrophilic probe reagents. These two reagents, together with n-propyl 2-pyridyl disulphide [compound (II)], provide structural variation in the non-pyridyl part of the molecule from a simple hydrocarbon side chain in compound (II) to a P1-P2 amide bond in compound (IV) and further to both a P1-P2 amide bond and a hydrophobic side chain (of phenylalanine) at P2 as a potential occupant of S2 subsites. 2. These disulphides were used as reactivity probes to investigate specificity and binding-site-catalytic-site signalling in a number of cysteine proteinases by determining (a) the reactivity at pH 6.0 at 25 degrees C at I 0.1 of compound (III) (a close analogue of a good papain substrate) towards 2-mercaptoethanol, benzimidazol-2-ylmethanethiol [compound (V), as a minimal catalytic-site model], chymopapains B1-B3, chymopapain A, papaya proteinase omega, actinidin, cathepsin B and papain, (b) the effect of changing the structure of the probe as indicated above on the reactivities of compound (V) and of the last five of these enzymes, and (c) the forms of pH-dependence of the reactivities of papain and actinidin towards compound (III). 3. The kinetic data suggest that reagents of the type investigated may be sensitive probes of molecular recognition features in this family of enzymes and are capable not only of detecting differences in binding ability of the various enzymes but also of identifying enzyme-ligand contacts that provide for binding-site-catalytic-site signalling mechanisms. 4. The particular value of this class of probe appears to derive from the possibility of activating the 2-mercaptopyridine leaving group not only by formal protonation, as was recognized previously [see Brocklehurst (1982) Methods Enzymol. 87C, 427-469], but also by hydrogen-bonding to the pyridyl nitrogen atom when the appropriate geometry in the catalytic site is provided by enzyme-ligand contacts involving the non-pyridyl part of the molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
2,2'-Dipyridyl↗
Free radical reactions with alpha-tocopherol and N-stearoyl tryptophan methyl ester in micellar solutions.
The rate constants have been measured for one-electron oxidation by N3. and Br2-. of N-stearoyl tryptophan methyl ester (STME) and alpha-tocopherol (alpha-T) in micelles of sodium dodecyl sulphate (SDS) or tetradecyl trimethyl ammonium bromide (TTAB). Compared with analogous reactions of tryptophan and Trolox C in aqueous solution, the rate constants for oxidation in micellar solution by N3. are reduced by 30-70%. The micellar charge increased the rate of oxidation of STME by Br2-. in TTAB micelles by almost an order of magnitude, compared with the reaction of Br2-. with tryptophan in aqueous solution. In SDS micelles the rate of oxidation of STME by Br2-. was reduced more than 30-fold. Quenching of fluorescence from STME in micelles by acrylamide confirmed the accessibility of the indole ring to the aqueous solvent. The rate of repair of the neutral STME radical by alpha-T in TTAB micelles was found to be accelerated by a factor of at least 27, compared with the similar reaction between Trolox C and tryptophan radicals in aqueous solution.
Free Radicals↗
Inhibition of (Ca2+ + Mg2+)-ATPase by carbodiimides. A structure-activity study.
Nine hydrophobic carbodiimides were synthesized and their chemical reactivities (towards acetic acid) and inhibitory capacities (towards the (Ca2+ + Mg2+)-ATPase were measured. No correlation between chemical reactivity and inhibitory efficacy emerges, but a significant effect of molecular bulk on reactivity towards the calcium-protectable carboxyl groups of the ATPase is noted: methyl-substituted compounds inhibit the enzyme in the presence of Ca2+, while aryl- or cyclohexyl-substituted compounds do not inactivate in the presence of Ca2+.
Acetates↗
Synthesis and platelet aggregation inhibitory activity of 4,5-bis(substituted)-1,2,3-thiadiazoles.
Routine screening of compounds for inhibition of collagen-induced platelet aggregation in vitro revealed 4,5-bis-(4-methoxyphenyl)-1,2,3-thiadiazole (2) was active and it represents the first example of a 1,2,3-thiadiazole with possible antithrombotic activity. In order to develop a structure-activity relationship for this heterocycle, a number of new 4(5)-mono- and -disubstituted 1,2,3-thiadiazoles were synthesized. These were tested in our screen and a number of additional active compounds were found. The most active compounds (2, 5a, 5b, and 6c) were those in which the heterocycle was substituted with benzene rings possessing para electron-donating groups.
Collagen↗
Ligand binding properties of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase labelled with N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide.
The (Ca2+ + Mg2+)-ATPase of rabbit sarcoplasmic reticulum, when labelled at two Ca2+-protected sites with N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide (NCD-4) retains Ca2+ binding capacity at the sites with Kd values of approx. 3 microM and 0.12 mM as assessed by fluorescence titration. The sites correspond to the two high-affinity Ca2+ binding sites present in the native ATPase. The NCD-4 labelled ATPase exhibits slow conformational changes at each site on addition of Ca2+. It retains the ability to form phosphoenzyme, and can most likely translocate Ca2+.
Adenosine Triphosphate↗
High-performance liquid chromatographic determination of a new calcium antagonist, fostedil, in plasma and urine using fluorescence detection.
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Animals↗
Pharmacokinetics of fostedil, a new calcium antagonist, in beagle dogs following oral and intravenous administration.
Twelve adult beagle dogs received both an oral and intravenous dose (12 mg/kg) of fostedil in a cross-over design. The plasma levels and urinary excretion of intact fostedil were measured, and the pharmacokinetic parameters of the drug were defined. The results indicate that fostedil was at least 68% bioavailable after an oral dose given as a suspension or solution. The terminal half-life was about 7-8 h. The in vitro protein binding, at concentrations of 0.1-100 micrograms/mL, ranged from 95 to 97%. The binding was not concentration dependent, and saturation of the binding sites was not apparent at concentrations up to 100 micrograms/mL. Excretion of unchanged drug from the kidneys accounted for only a small percentage of drug clearance.
Administration, Oral↗
Inactivation of sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide.
The synthesis and characterisation of N-cyclohexyl-N'-(4-dimethylamino-alpha-naphthyl)carbodiimide (NCD-4) is described. Only the N-acetylurea and urea corresponding to NCD-4 are appreciably fluorescent: the O-phenylisourea and S-ethylisothiourea derivatives have negligible fluorescence. NCD-4 inhibits the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum irreversibly: Ca2+ protects against inhibition. Covalent incorporation of NCD-4 occurs into the Ca2+-protected sites, with a stoichiometry of approximately 1 mole/mole of ATPase. The modified enzyme has fluorescence emission properties similar to those of NCD-4 N-acetylurea in a relatively hydrophobic environment: it is concluded that NCD-4 has modified a carboxylate group (s) located in or near the Ca2+-binding sites of the ATPase.
Animals↗
The use of fluorescence energy transfer to distinguish between poly(ethylene glycol)-induced aggregation and fusion of phospholipid vesicles.
Two fluorescence energy transfer assays for phospholipid vesicle-vesicle fusion have been developed, one of which is also sensitive to vesicle aggregation. Using a combination of these assays it was possible to distinguish between vesicle aggregation and fusion as induced by poly(ethylene glycol) PEG 8000. The chromophores used were 1-(4'-carboxyethyl)-6-diphenyl-trans-1,3,5-hexatriene as fluorescent 'donor' and 1-(4'-carboxyethyl)-6-(4"-nitro)diphenyl-trans-1,3,5-hexatriene as 'acceptor'. These acids were appropriately esterified giving fluorescent phospholipid and triacylglycerol analogues. At 20 degrees C poly(ethylene glycol) 8000 (PEG 8000) caused aggregation of L-alpha-dipalmitoylphosphatidylcholine (DPPC) vesicles without extensive fusion up to a concentration of about 35% (w/w). Fusion occurred above this poly(ethylene glycol) concentration. The triacylglycerol probes showed different behaviour from the phospholipids: while not exchangeable through solution in the absence of fusogen, they appeared to redistribute between bilayers under aggregating conditions. DPPC vesicles aggregated with less than 35% poly(ethylene glycol) could not be disaggregated by dilution, as monitored by the phospholipid probes. However, DPPC vesicles containing approx. 5% phosphatidylserine which had been aggregated by poly(ethylene glycol) could be disaggregated by either dilution or sonication. Phospholipid vesicles aggregated by low concentrations of poly(ethylene glycol) appear to fuse to multilamellar structures on heating above the lipid phase transition temperature.
Cell Fusion↗
The use of a phospholipid analogue of diphenyl-1,3,5-hexatriene to study melittin-induced fusion of small unilamellar phospholipid vesicles.
A phospholipid analogue incorporating the diphenyl-1,3,5-hexatriene (DPH) chromophore has been synthesized. The compound has been shown to have similar fluorescence properties to DPH itself but, unlike DPH, is unable to exchange freely through solution when incorporated as probe in a subset of phospholipid vesicles of given composition. The non-exchangeability of this probe has been exploited to study the fusion of phospholipid vesicles to form larger structures. The peptide melittin was used to initiate fusion, and it was shown that vesicles which had been induced to fuse by heating in the presence of melittin would not fuse with subsequently added vesicles.
Bee Venoms↗
Analysis of buflomedil in mouse, rat and rabbit plasma by reversed-phase ion-pair high-performance liquid chromatography.
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Animals↗