Synthesis and evaluation of the antidepressant activity of the enantiomers of bupropion

The synthesis of the enantiomers of bupropion, (rac)-2-tert-butylamino-3′-chloropropiophenone 1 (Wellbutrin®) is described. The enantiomers were compared with the racemate in both the tetrabenazine-induced sedation model and the inhibition of uptake of biogenic amine assay. No significant differences were found in their potencies to reverse tetrabenazine-induced sedation in mice or in their IC₅₀ values as inhibitors of biogenic amine uptake into nerve endings obtained from mouse brain. © 1993 Wiley-Liss, Inc.     

Thermodynamic Parameters of Ligand-Receptor Interactions: Computation and Error Margins

Abstract

A semiempirical relationship describing the temperature function of ligand-receptor dissociation constants (K_d), derived from heat capacities of the system in equilibrium, is suggested for computation of the standard enthalpy (δH°) and standard entropy (δS°) changes in equilibrium. The use of the linear expression (called Gibbs-van't Hoff equation) may lead to inaccurate results when heat capacity C_p displays a considerable temperature dependence. The accuracy of K_d, δH° and δS° has been studied by simulation experiments. In the case of K_d, deviations of computed from “true” values are determined by both the accuracy of experimental data used for its estimation, and by the shape of the binding isotherm (for instance, by Hill coefficients or by the presence of low affinity sites). As a rule, if errors of bound ligand measurements are greater than 20 per cent, K_d estimates ought to be considered as less reliable. However, computations of δH° and δS° that use such K_d values, are more correct, probably due to an error compensation. The suggested nonlinear temperature function of K_d enables an estimate of the heat capacity of the system and its temperature dependence.     

The interaction of a range of serine proteinase inhibitors with bovine trypsin and Costelytra zealandica trypsin

Kinetic and other properties of the interaction between two serine proteinases, bovine trypsin and Costelytra zealandica (grass grub) larval trypsin and a range of proteinaceous serine proteinase inhibitors were investigated. Twenty-six inhibitors or isoinhibitors from 10 different inhibitor families were analysed. A 1700-fold range in equilibrium dissociation constant (K_d) values was obtained for bovine trypsin and a 10⁵-fold range for grass grub trypsin. The ratios of K_d (grass grub)/K_d (bovine) also spanned a range of 10⁵-fold. Qualitative observations indicated that the second order association rate constants were high for all except two inhibitors. Two classes of first order dissociation rate constant were determined from the dissociation of trypsin-inhibitor complexes induced by substrate. While most inhibitors were cleaved by grass grub trypsin, they still inhibited larval midgut crude extracts during long incubations. We suggest using the K_d value to assess the potential for any inhibitor to act as a grass grub larval resistance factor in plants, in preference to other parameters.     

Temperature dependence of acetylcholine receptor channels activated by different agonists

The temperature dependence of agonist binding and channel gating were measured for wild-type adult neuromuscular acetylcholine receptors activated by acetylcholine, carbamylcholine, or choline. With acetylcholine, temperature changed the gating rate constants (Q₁₀ ≈ 3.2) but had almost no effect on the equilibrium constant. The enthalpy change associated with gating was agonist-dependent, but for all three ligands it was approximately equal to the corresponding free-energy change. The equilibrium dissociation constant of the resting conformation (K_d), the slope of the rate-equilibrium free-energy relationship (Φ), and the acetylcholine association and dissociation rate constants were approximately temperature-independent. In the mutant αG153S, the choline association and dissociation rate constants were temperature-dependent (Q₁₀ ≈ 7.4) but K_d was not. By combining two independent mutations, we were able to compensate for the catalytic effect of temperature on the decay time constant of a synaptic current. At mouse body temperature, the channel-opening and -closing rate constants are ∼400 and 16 ms⁻¹. We hypothesize that the agonist dependence of the gating enthalpy change is associated with differences in ligand binding, specifically to the open-channel conformation of the protein.     

Protein interaction affinity determination by quantitative FRET technology

The dissociation constant, K_d, is an important parameter for characterizing protein–protein interaction affinities. SUMOylation is one of the important protein post‐translational modifications and it involves a multi‐step enzymatic cascade reaction, resulting in peptide activation and substrate conjugation. Multiple covalent and non‐covalent protein–protein interactions are involved in this cascade. Techniques involving Förster resonance energy transfer (FRET) have been widely used in biological studies in vitro and in vivo, and they are very powerful tools for elucidating protein interactions in many regulatory cascades. In our previous studies, we reported the attempt to develop a new method for the determination of the K_d by FRET assay using the interaction of SUMO1 and its E2 ligase, Ubc9 as a test system. However, the generality and specifications of this new method have not been fully determined. Here we report a systematic approach for determining the dissociation constant (K_d) in the SUMOylation cascade and for further sensitivity and accuracy testing by the FRET technology. From a FRET donor to acceptor concentration ratio range of 4–40, the K_ds of SUMO1 and Ubc9 consistently agree well with values from surface plasmon resonance and isothermal titration calorimetry. These results demonstrate the high sensitivity and accuracy of the FRET‐based K_d determination approach. This technology, therefore, can be used in general for protein–protein interaction dissociation constant determination. Biotechnol. Bioeng. 2012; 109: 2875–2883. © 2012 Wiley Periodicals, Inc.     

Analysis of the interaction between human interleukin-5 and the soluble domain of its receptor using a surface plasmon resonance biosensor

A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin-5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophilis. The receptor for IL5 is composed of two subunits, α and β. The α subunit provides the specificity for IL5 and consist of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor α subunit (shIL5Rα) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5Rα were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore? (Pharmacia) SPR biosensor after N-hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5Rα or hIL5, in solution. Kinetics of binding of soluble analyst to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (K_d) were derived. With immobilized shIL5Rα and soluble hIL5, the measured K_d was 2 nM . A similar value was obtained by titration calorimetry. The K_d for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used for differences in protein glycosylation. Receptor-IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range the dissociation rate increased with compressibility little increased in association rate. The values obtained for the interaction of hIL5 and shIL5Rα were found to depend on which component was immobilized; the K_d was 5.5 nM with immobilized hIL5 and soluble shIL5Rα. The SPR biosensor provides a unified methodology to measure the interaction properties of shIL5Rα and hIL5 derivatives, mutants and mimetic as well as to evaluate potential antagonists of the receptor-cytokine interaction.     

Modulation of the serotonin S2-receptor in brain after chronic lithium

In vivo effects of chronic lithium administration on dopaminergic and serotonergic receptor binding were studied in the striatum and cerebral cortex of the rat. [³H]Domperidone was used as the ligand for the dopaminergic receptor, and [³H]ketanserin for the serotonergic system. Long-term ingestion of lithium (2–3 months) resulted in high levels of lithium in the cerebral cortex and significantly higher potassium levels; the sodium content remained at normal levels. The kinetic constants (K _d andB _max) of [³H]domperidone binding sites measured in the striatum did not show any deviation from control values, but the receptor concentration (B _max) of [³H]ketanserin binding sites was significantly reduced in the cerebral cortex of lithium-treated rats. The apparent dissociation constant (K _d) was not changed. The results indicate that the serotonergic component of the [³H]spiperone binding site, which we had previously found to be affected by chronic lithium treatment and which was shown by Peroutka and Snyder (1) to be the 5-HT₂ receptor, is selectively affected by lithium.Special Issue dedicated to Prof. Eduardo De Robertis.     

Identification of b,c, and d cytochromes in the membrane of Vitreoscilla

Cytochromes b, c, d, and o were identified by spectroscopic analysis of respiratory membrane fragments from Vitreoscilla sp., strain C1. Carbon monoxide difference spectra of the reduced membranes had absorption maxima at 416, 534, and 571 nm (ascribed to cytochrome o) and 632 nm (cytochrome d). Derivative spectra of the pyridine hemochromogen spectra of the membranes identified the presence of b- and c-type cytochromes in Vitreoscilla. The cyanide binding curve of the membranes was biphasic with dissociation constants of 2.14 mM and 10.7 mM which were assigned to cytochrome o and cytochrome d, respectively. Membranes bound carbon monoxide with dissociation constant 3.9 M, which was assigned to cytochrome o. Cytochrome c ₅₅₆ and a NADH-p-iodonitrotetrazolium violet reductase component were partially purified from Vitreoscilla membranes.Abbreviations INT p-iodonitrotetrazolium violet - RMF respiratory membrane fragments - K _d dissociation constant - CHAPS 3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate - DOC sodium deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Limitations of the Eadie-Hofstee plot to estimate kinetic parameters of intestinal transport in the presence of an unstirred water layer

Summary In the presence of an intestinal unstirred water layer, the relationship between substrate concentration (C ₁) and unidirectional flux (J _d) is not described by the equation for a rectangular hyperbole. Accordingly, transformations of the Michaelis-Menten equation may not necessarily be linear and may lead to serious errors in the estimation of the affinity constant (K _m) and maximal transport rate (J _d ^m ) of carrier-mediated processes. An equation has previously been derived which describedJ _d under conditions of varying effective thickness or surface area of the unstirred water layer, the free diffusion coefficient of the probe molecule, and the distribution of transport sites along the villus. These theoretical curves have been analyzed by using the Eadie-Hofstee transformation (J _d vs. J_d/C₁) of the Michaelis-Menten equation. Use of this plot leads to serious discrepancies between the true and apparent affinity constants and between true and apparent maximal transport rates. These differences are further magnified by failure to correct for the contribution of passive permeation. The Eadie-Hofstee plot is of use, however, to infer certain qualitative characteristics of active transport processes, such as the variation in affinity constants and overlying resistance of the unstirred water layer at different sites along the villus and to predict the adequacy of the correction for the contribution of passive permeation.Abbreviations Used in the Text C ₁ Concentration of the probe molecule in the bulk phase - C ₂ Concentration of the probe molecule at the aqueous-membrane interface - d Effective thickness of UWL - D Free diffusion coefficient - d ⁿ d atn th segment of the villus - f _n Proportion of total carrier transport sites present on each segment of villus - J Unidirectional flux of probe molecule, uncorrected for surface area - J _d Unidirectional flux of probe molecule determined experimentally, corrected for surface area - J _d ^m Maximal transport rate, corrected for surface area - J _d ^m* Apparent maximal transport rate - J ^m Maximal transport rate, uncorrected for surface area - K _m Michaelis constant (true affinity constant) - K _m ^* Apparent affinity constant - K _m ⁿ K _m atnth segment of the villus - n The perpendicular height of the villus was divided into ten equal segments numberedn ₁ ton ₁₀ - p Passive permeability coefficient - S _m Functional surface area of the membrane - S _w Effective surface of UWL - S _w ⁿ S _w atnth segment of the villus - UWL Intestinal unstirred water layer     

Nucleotide binding affinities of the intact proton-translocating transhydrogenase from Escherichia coli

Transhydrogenase (E.C. 1.6.1.1) couples the redox reaction between NAD(H) and NADP(H) to the transport of protons across a membrane. The enzyme is composed of three components. The dI and dIII components, which house the binding site for NAD(H) and NADP(H), respectively, are peripheral to the membrane, and dII spans the membrane. We have estimated dissociation constants (K_d values) for NADPH (0.87 μM), NADP⁺ (16 μM), NADH (50 μM), and NAD⁺ (100-500 μM) for intact, detergent-dispersed transhydrogenase from Escherichia coli using micro-calorimetry. This is the first complete set of dissociation constants of the physiological nucleotides for any intact transhydrogenase. The K_d values for NAD⁺ and NADH are similar to those previously reported with isolated dI, but the K_d values for NADP⁺ and NADPH are much larger than those previously reported with isolated dIII. There is negative co-operativity between the binding sites of the intact, detergent-dispersed transhydrogenase when both nucleotides are reduced or both are oxidised.     

Stereoselective pharmacokinetics of ibuprofen in rats: effect of enantiomer-enantiomer interaction in plasma protein binding

Stereoselective pharmacokinetics of ibuprofen (IB) enantiomers were studied in rats. Unidirectional conversion from R-ibuprofen (R-IB) to S-ibuprofen (S-IB) was observed following intravenous administration. S-IB concentrations in plasma following racemate administration were simulated according to a conventional compartmental model using the parameters obtained after the administration of individual enantiomers, and resulted in overestimation of S-IB concentrations. Binding of IB enantiomers measured in rat plasma was stereoselective, the binding of R-IB being more favorable than that of S-IB. Moreover, there are interactions between IB enantiomers in binding, which may cause the increase of distribution volumes of IB enantiomers in the presence of their antipodes. Hence simulated S-IB concentrations according to a conventional compartment model were significantly greater than those observed. Indeed, when the enantiomer-enantiomer interactions were taken into account, simulation of S-IB concentrations in plasma following racemate administration was in good agreement with observed values. Therefore, interactions between stereoisomers as well as dispositional stereoselectivity have to be considered when pharmacokinetics of stereoisomers after administration of the racemate are compared to those after administration of individual isomers. Chirality 9:354-361, 1997. © 1997 Wiley-Liss, Inc.     

Enantioselectivity of aromatase inhibitors: Substituted 3-(4-aminophenyl)pyrrolidine-2,5-diones

The (+)-, (?)-, and (±)-forms of 1- and 1,3-substituted 3-(4-aminophenyl)pyrrolidine-2,5-dione have been examined as inhibitors of P450_AROM and P450_CSCC. The inhibitory potency for P450_AROM resided in the (+)-enantiomers of ( 1 ), ( 2 ), and ( 4 ) and the (?)-enantiomers of ( 3 ) and ( 5 ). These findings have been accommodated within a molecular graphics-derived model for binding of P450_AROM inhibitors to the substrate binding site. Crystallography showed that (+)-( 2 ) has the (R)-configuration. Spectral binding studies with human placental P450_AROM showed type II binding but although the K_S values were in line with the IC₅₀ values for individual compounds there was no overall correlation between K_S and IC₅₀ within the series. There was little difference in the inhibitory potency of the enantiomers and racemate of individual compounds toward P450_CSCC. © 1995 Wiley-Liss, Inc.     

Quantitative In Vivo Fluorescence Cross-Correlation Analyses Highlight the Importance of Competitive Effects in the Regulation of Protein-Protein Interactions

Computer-assisted simulation is a promising approach for clarifying complicated signaling networks. However, this approach is currently limited by a deficiency of kinetic parameters determined in living cells. To overcome this problem, we applied fluorescence cross-correlation spectrometry (FCCS) to measure dissociation constant (K_d) values of signaling molecule complexes in living cells (in vivo K_d). Among the pairs of fluorescent molecules tested, that of monomerized enhanced green fluorescent protein (mEGFP) and HaloTag-tetramethylrhodamine was most suitable for the measurement of in vivo K_d by FCCS. Using this pair, we determined 22 in vivo K_d values of signaling molecule complexes comprising the epidermal growth factor receptor (EGFR)–Ras–extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway. With these parameters, we developed a kinetic simulation model of the EGFR-Ras-ERK MAP kinase pathway and uncovered a potential role played by stoichiometry in Shc binding to EGFR during the peak activations of Ras, MEK, and ERK. Intriguingly, most of the in vivo K_d values determined in this study were higher than the in vitro K_d values reported previously, suggesting the significance of competitive bindings inside cells. These in vivo K_d values will provide a sound basis for the quantitative understanding of signal transduction.     

Day–night specific binding of 2-[125I]Iodomelatonin and melatonin content in gill, small intestine and kidney of three fish species

Some of melatonin’s (Mel) well-established physiological effects are mediated via high-affinity cell-membrane receptors belonging to the superfamily of G-protein-coupled receptors. Specific binding of ligand 2-[¹²⁵I]iodomelatonin, using membrane preparations from osmoregulatory tissues of flounder, rainbow trout and sea bream, together with Mel concentrations in the tissues and plasma were studied. The kidney, gill and small intestine samples were collected during the day and at night. The dissociation constants (K _d) and maximal binding densities (B _max) were calculated for each tissue at 11:00 and 23:00 h. The binding sites with K _d values in the tissues in the picomolar range indicated the high affinity. K _d and B _max values were tissue- and species-dependent. The GTP analogue [Guanosine 5′-O-(3-thiotriphosphate)] treatment significantly reduced the B _max value, indicating that the 2-[¹²⁵I]iodomelatonin-binding sites are probably coupled to a G-protein. No daily variations in K _d and B _max values were observed. These are the first studies of the presence of 2-[¹²⁵I]iodomelatonin-binding sites in the small intestine, kidney tubule and gill of fish. The data strongly suggest new potential targets for Mel action and the influence of Mel on water/ion balance in fish. The intestine seems to be a site of Mel synthesis and/or an active accumulation of the hormone.     

Kinetic constants for the inhibition of camel retinal acetylcholinesterase by the carbamate insecticide lannate

We have designed this study to determine various kinetic parameters of camel retinal membrane‐bound acetylcholinesterase (AChE; EC 3.1.1.7) inhibition by carbamate insecticide lannate [methyl N‐{{(methylamino)carbonyl}oxy} ethanimidothioate]. All these kinetic constants were derived by simple graphical methods. The value of kinetic parameters was estimated as follows: 0.061 (μM)⁻¹, 1.14 (μM)⁻¹, 0.216 μM, 0.016 min⁻¹, 0.0741 (μM min)⁻¹, 0.746 μM, and 4.42 μM for velocity constant (K_v), new inhibition constant (K_nic), dissociation constant (K_d), carbamylation rate constant (k_2c), overall carbamylation rate constant (k′2 ), 50% inhibition constant (K_I50), and 99% inhibition constant (K_I99), respectively. These unique methods may be used to estimate such kinetic parameters for time‐dependent inhibition of enzymes by variety of chemicals, insecticides, herbicides, and drugs. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 13: 41–46, 1999     

Stereodynamics of Small 1,2-Dialkyldiaziridines

Diaziridines are very interesting representatives of organic compounds containing stereogenic nitrogen atoms. In particular, 1,2-dialkyldiaziridines show extraordinarily high stereointegrity. The lone electron pairs of the nitrogen atoms are in trans configuration, avoiding a four-electron repulsive interaction. Furthermore, the trans configuration of the substituents at the nitrogen atoms is energetically favored because of reduced steric interactions. Therefore only two stereoisomers (enantiomers) are observed. At elevated temperatures the enantiomers are interconverting because of the limited stereointegrity of the chirotopic nitrogen atoms. The enantiomerization rate constants and the activation parameters of interconversion are of great interest. Here, we investigated the stereodynamics of a set of small 1,2-dialkyldiaziridines bearing short substituents (Me, Et, iPr, tBu), using enantioselective dynamic gas chromatography (DGC). Separation of enantiomers of all compounds, including the highly volatile 1,2-dimethyldiaziridine, was achieved using heptakis(2,3-di-O-ethyl-6-O-tert-butyldimethylsilyl)-β-cyclodextrin in 50% PS086 (w/w) as chiral stationary phase in fused silica capillaries with a length of up to 50 m. Measurements at variable temperatures were performed and reaction rate constants were determined using the unified equation of chromatography implemented in the software DCXplorer. The activation barriers at room temperature for 1-(tert-butyl)-2-ethyldiaziridine, ΔG^╪_298K = 123.8 kJ mol^–1 (ΔH^╪ = 115.5 ± 2.9 kJ mol^–1, ΔS^╪ = –28 ± 1 J mol^–1 K^–1), and 1-ethyl-2-isopropyldiaziridine, ΔG^╪_298K = 124.2 kJ mol^–1 (ΔH^╪ = 113.1 ± 2.4 kJ mol^–1, ΔS^╪ = –37 ± 2 J mol^–1 K^–1), were determined, representing some of the highest values observed for nitrogen inversion in diaziridines. Chirality 00:000–000, 2013. © 2013 Wiley Periodicals, Inc.     

Selection of aptamers for aflatoxin M1 and their characterization

In the present work, aptamers against aflatoxin M1 and aflatoxin B1 were generated and tested for creating proof of principle of recognition of aflatoxin M1 by generated aptamers. The aptamers were selected through the process referred as systematic evolution of ligands by exponential enrichment. A total of 41 different aptamer (36 aptamers for aflatoxin M1 and 5 for aflatoxin B1) sequences were obtained. The determination of dissociation constant (K_d) values revealed that aptamers generated against aflatoxin M1 exhibited K_d values in the range of 35–1515 nM. Selected aptamers were grouped on the basis of the presence of common motifs or G‐quadruplex. We find it interesting that one aptamer with no conserved motif or G‐quadruplex had lowest K_d value (K_d = 35 nM). This structural motif is very distinct from motifs present in other aptamers. The K_d values of selected aptamers for aflatoxin B1 were in the range of 96–221 nM. One aptamer from each group was further tested for its ability to be used in aptasensor. The aptamer recognized aflatoxin M1 as indicated by color change (red to purple or blue) of aptamer‐coated gold nanoparticles in the presence of 250–500 nM aflatoxin M1. The aptamers can be used in developing methods for detection/estimation/separation of aflatoxin or antidote for aflatoxin toxicity. Copyright © 2014 John Wiley & Sons, Ltd.     

Computational and experimental investigation of DNA repair protein photolyase interactions with low molecular weight drugs

This paper reports the previously unknown interactions between eight low molecular weight commercially available drugs (130–800 Da) and DNA repair protein photolyase using computational docking simulations and surface plasmon resonance (SPR) experiments. Theoretical dissociation constants, K_d, obtained from molecular docking simulations were compared with the values found from SPR experiments. Among the eight drugs analyzed, computational and experimental values showed similar binding affinities between selected drug and protein pairs. We found no significant differences in binding interactions between pure and commercial forms of the drug lornoxicam and DNA photolyase. Among the eight drugs studied, prednisone, desloratadine, and azelastine exhibited the highest binding affinity (K_d = 1.65, 2.05, and 8.47 μM, respectively) toward DNA photolyase. Results obtained in this study are promising for use in the prediction of unknown interactions of common drugs with specific proteins such as human clock protein cryptochrome. Copyright © 2013 John Wiley & Sons, Ltd.