Affinity capillary electrophoresis (ACE) has been applied to estimation of apparent binding constant of complexes of (R,S)-enantiomers of selected acyclic nucleoside phosphonates (ANPs) with chiral selector β-cyclodextrin (βCD) in aqueous alkaline medium. The noncovalent interactions of five pairs of (R,S)-enantiomers of ANPs-based antiviral drugs and their derivatives with βCD were investigated in the background electrolyte (BGE) composed of 35 or 50 mM sodium tetraborate, pH 10.0, and containing variable concentration (0-25 mM) of βCD. The apparent binding constants of the complexes of (R,S)-enantiomers of ANPs with βCD were estimated from the dependence of effective electrophoretic mobilities of (R,S)-enantiomers of ANPs (measured simultaneously by ACE at constant reference temperature 25°C inside the capillary) on the concentration of βCD in the BGE using different nonlinear and linear calculation methodologies. Nonlinear regression analysis provided more precise and accurate values of the binding constants and a higher correlation coefficient as compared to the regression analysis of the three linearized plots of the effective mobility dependence on βCD concentration in the BGE. The complexes of (R,S)-enantiomers of ANPs with βCD have been found to be relatively weak - their apparent binding constants determined by the nonlinear regression analysis were in the range 13.3-46.4 L/mol whereas the values from the linearized plots spanned the interval 12.3-55.2 L/mol.

• MeSH
• beta-cyklodextriny chemie   MeSH
• elektroforéza kapilární metody   MeSH
• nukleosidy chemie   izolace a purifikace   MeSH
• organofosfonáty chemie   izolace a purifikace   MeSH
• stereoizomerie MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this study, two capillary electrophoresis-based ligand binding assays, namely, mobility shift affinity capillary electrophoresis (ms-ACE) and capillary electrophoresis-frontal analysis (CE-FA), were applied to determine binding parameters of human serum albumin toward small drugs under similar experimental conditions. The substances S-amlodipine (S-AML), lidocaine (LDC), l-tryptophan (l-TRP), carbamazepine (CBZ), ibuprofen (IBU), and R-verapamil (R-VPM) were used as the main binding partners. The scope of this comparative study was to estimate and compare both the assays in terms of their primary measure's precision and the reproducibility of the derived binding parameters. The effective mobility could be measured with pooled CV values between 0.55% and 7.6%. The precision of the r values was found in the range between 1.5% and 10%. Both assays were not universally applicable. The CE-FA assay could successfully be applied to measure the drugs IBU, CBZ, and LDC, and the interaction toward CBZ, S-AML, l-TRP, and R-VPM could be determined using ms-ACE. The average variabilities of the estimated binding constants were 64% and 67% for CE-FA and ms-ACE, respectively.

• MeSH
• akutní myeloidní leukemie * MeSH
• elektroforéza kapilární metody   MeSH
• ibuprofen MeSH
• izotachoforéza * MeSH
• lidé MeSH
• lidský sérový albumin metabolismus   MeSH
• reprodukovatelnost výsledků MeSH
• tryptofan MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Affinity capillary electrophoresis (ACE) and quantum mechanical density functional theory (DFT) calculations have been employed for the investigation of noncovalent interactions between hexaarylbenzene-based receptor (R) and ammonium cation NH(4)(+). Firstly, by means of ACE, the binding constant of the NH(4)R(+) complex in methanol was estimated from the dependence of the effective electrophoretic mobility of the receptor R (in advance corrected by our earlier developed procedure to a reference temperature of 25°C) on the concentration of ammonium ion in the background electrolyte using non-linear regression analysis. The logarithmic form of the apparent binding (stability) constant of NH(4)R(+) complex in the methanolic background electrolyte (25 mM Tris, 50 mM chloroacetate, pH(MeOH) 7.8) was evaluated as log K(NH(4)R) = 4.03 ± 0.15. Secondly, the structural characteristics of NH(4)R(+) complex were determined by DFT calculations.

• MeSH
• benzenové deriváty chemie   MeSH
• chromatografie afinitní metody   MeSH
• elektroforéza kapilární metody   MeSH
• kationty chemie   MeSH
• kvartérní amoniové sloučeniny chemie   MeSH
• methanol MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• teplota MeSH
• vodíková vazba MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• bronchiální astma krev   MeSH
• dialýza MeSH
• dítě MeSH
• dospělí MeSH
• krevní proteiny metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• senioři MeSH
• sérový albumin metabolismus   MeSH
• theofylin krev   MeSH
• vazba proteinů MeSH
• Check Tag
• dítě MeSH
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH

An improved approach to the evaluation of the rate constants of drugs binding to the open channel underlying the transient outward potassium current I(t0) is described. It is based on an analysis of a quantitative model formulated by a set of twelve differential equations. The rate constants are calculated from the time constants resulting from an approximation of the time course of apparent inactivation of the recorded I(t0) by two exponentials in the absence and by three exponentials in the presence of a blocking agent. The model study confirmed significantly higher accuracy in comparison with the existing electrophysiological method.

• MeSH
• blokátory draslíkových kanálů farmakologie   MeSH
• buněčná membrána účinky léků   fyziologie   MeSH
• draslík metabolismus   MeSH
• draslíkové kanály účinky léků   fyziologie   MeSH
• gating iontového kanálu účinky léků   fyziologie   MeSH
• kardiomyocyty účinky léků   fyziologie   MeSH
• kinetika MeSH
• membránové potenciály fyziologie   MeSH
• modely kardiovaskulární MeSH
• počítačová simulace MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

TRPV1 is a nonselective cation channel that integrates wide range of painful stimuli. It has been shown that its activity could be modulated by intracellular ligands PIP2 or calmodulin (CaM). The detailed localization and description of PIP2 interaction sites remain unclear. Here, we used synthesized peptides and purified fusion proteins of intracellular regions of TRPV1 expressed in E.coli in combination with fluorescence anisotropy and surface plasmon resonance measurements to characterize the PIP2 binding to TRPV1. We characterized one PIP2 binding site in TRPV1 N-terminal region, residues F189-V221, and two independent PIP2 binding sites in C-terminus: residues K688-K718 and L777-S820. Moreover we show that two regions, namely F189-V221 and L777-S820, overlap with previously localized CaM binding sites. For all the interactions the equilibrium dissociation constants were estimated. As the structural data regarding C-terminus of TRPV1 are lacking, restraint-based molecular modeling combined with ligand docking was performed providing us with structural insight to the TRPV1/PIP2 binding. Our experimental results are in excellent agreement with our in silico predictions.

• MeSH
• ankyriny chemie   MeSH
• fosfatidylinositolfosfáty metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• kalmodulin chemie   metabolismus   MeSH
• kationtové kanály TRPV chemie   genetika   metabolismus   MeSH
• konformace proteinů MeSH
• krysa rodu rattus MeSH
• ligandy MeSH
• liposomy metabolismus   MeSH
• mutace MeSH
• rekombinantní fúzní proteiny chemie   genetika   metabolismus   MeSH
• simulace molekulového dockingu MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Neurons lose intrinsic axon regenerative ability with maturation, but the mechanism remains unclear. Using an in-vitro laser axotomy model, we show a progressive decline in the ability of cut CNS axons to form a new growth cone and then elongate. Failure of regeneration was associated with increased retraction after axotomy. Transportation into axons becomes selective with maturation; we hypothesized that selective exclusion of molecules needed for growth may contribute to regeneration decline. With neuronal maturity rab11 vesicles (which carry many molecules involved in axon growth) became selectively targeted to the somatodendritic compartment and excluded from axons by predominant retrograde transport However, on overexpression rab11 was mistrafficked into proximal axons, and these axons showed less retraction and enhanced regeneration after axotomy. These results suggest that the decline of intrinsic axon regenerative ability is associated with selective exclusion of key molecules, and that manipulation of transport can enhance regeneration.

• MeSH
• axony fyziologie   MeSH
• biologický transport MeSH
• buněčná diferenciace MeSH
• cytoplazmatické vezikuly metabolismus   MeSH
• potkani Sprague-Dawley MeSH
• rab proteiny vázající GTP metabolismus   MeSH
• regenerace * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The Na+/K+-ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na+/K+-ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na+/K+-ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP.

• MeSH
• adenosintrifosfát metabolismus   MeSH
• buněčná membrána metabolismus   MeSH
• cytoplazma metabolismus   MeSH
• fluorescenční barviva metabolismus   MeSH
• kinetika MeSH
• mutageneze cílená metody   MeSH
• simulace molekulového dockingu MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• tryptofan metabolismus   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In this study, two complementary approaches, affinity capillary electrophoresis (ACE) and quantum mechanical density functional theory (DFT) calculations, have been employed for quantitative characterization and structure elucidation of the complex between hexaarylbenzene (HAB)-based receptor R and lithium ion Li(+) . First, by means of ACE, the apparent binding constant of LiR(+) complex (K LiR +) in methanol was determined from the dependence of the effective electrophoretic mobilities of LiR(+) complex on the concentration of lithium ions in the 25 mM Tris/50 mM chloroacetate background electrolyte (BGE) using non-linear regression analysis. Prior to regression analysis, the effective electrophoretic mobilities of the LiR(+) complex were corrected to reference temperature 25 °C and constant ionic strength 25 mM. The apparent binding constant of the LiR(+) complex in the above methanolic BGE was evaluated as logK LiR + = 1.15±0.09. Second, the most probable structures of nonhydrated LiR(+) and hydrated LiR(+)·3H(2)O complexes were derived by DFT calculations. The optimized structure of the hydrated LiR(+)·3H(2)O complex was found to be more realistic than the nonhydrated LiR(+) complex because of the considerably higher binding energy of LiR(+)·3H(2)O complex (500.4 kJ/mol) as compared with LiR(+) complex (427.5 kJ/mol).

• MeSH
• benzenové deriváty analýza   MeSH
• chromatografie afinitní MeSH
• elektroforéza kapilární MeSH
• ionty analýza   MeSH
• kvantová teorie MeSH
• lithium analýza   MeSH
• molekulární struktura MeSH
• osmolární koncentrace MeSH
• teplota MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

INTRODUCTION: Competitive binding assays can be used to decipher not only the binding kinetics of studied ligands but also the binding site preference. Such assays are an essential step in the characterization of radioligands. However, the currently used competition assays require high concentrations of usually expensive ligands and still provide only binding site preference. By employing the time-resolved competition assay presented in this paper, binding characteristics including binding site preference can be obtained using less ligand. METHODS: To demonstrate the appropriateness of the time-resolved competition assay, we developed an assay in which the ligand binding was interrupted with a competitor. Experiments were performed on human carcinoma cell lines expressing epidermal growth factor receptor (EGFR). The targeting of the receptor was performed with radio-iodinated epidermal growth factor (EGF). The employed competitors involved either natural ligand transforming growth factor alpha (TGF-α) or anti-EGFR antibodies cetuximab and panitumumab targeting the same EGFR domain. RESULTS: Radio-iodinated EGF bound to EGFR was displaced with either low concentrations of cetuximab or high concentrations of panitumumab. In the case of TGF-α, we observed no competitive displacement of bound EGF at either high or low concentrations. When comparing the time-resolved competition assay with a manual competition assay, the resulting data of measured inhibition constants were in agreement. DISCUSSION: The results summarised in this study confirm the appropriateness of the time-resolved competition assay for assessing ligand binding properties. The assay has the potential to complement or replace conventional competition assays for determining binding site preference in the future.

• MeSH
• časové faktory MeSH
• epidermální růstový faktor chemie   metabolismus   MeSH
• erbB receptory antagonisté a inhibitory   chemie   metabolismus   MeSH
• humanizované monoklonální protilátky chemie   farmakologie   MeSH
• kompetitivní vazba účinky léků   MeSH
• lidé MeSH
• ligandy MeSH
• monoklonální protilátky chemie   farmakologie   MeSH
• nádorové buňky kultivované MeSH
• substrátová specifita MeSH
• transformující růstový faktor alfa chemie   metabolismus   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH