Investigation on the Binding of Terazosin Hydrochloride and Naftopidil to an Immobilized α 1-Adrenoceptor by Zonal Elution

The interaction between drugs and receptors is particularly important in revealing the drug acting mechanism and developing new leads. In this work, α ₁-Adrenoceptor (α ₁-AR) from HEK293 cell line is purified and immobilized on the surface of macro-pore silica gel to prepare an high-performance affinity chromatography stationary phase for the pursuit of drug–receptor interactions by competition zonal elution. Naftopidil is found to have only one type of binding site to α ₁-AR with an association constant of 1.45 × 10⁶ M^?1 and a concentration of binding sites of 1.56 × 10^?6 M, while terazosin hydrochloride proves to present two kinds of binding site on the receptor at which the association constants are determined to be 1.61 × 10⁵ M^?1 and 2.06 × 10³ M^?1, and the corresponding concentrations of the binding sites are 1.56 × 10^?6 M and 1.11 × 10^?3 M, respectively. It is concluded that the stationary phase containing attached α ₁-AR can be used to realize the binding of a drug to the receptor.     

Investigation on the Binding of Terazosin Hydrochloride and Naftopidil to an Immobilized α 1-Adrenoceptor by Zonal Elution

The interaction between drugs and receptors is particularly important in revealing the drug acting mechanism and developing new leads. In this work, α ₁-Adrenoceptor (α ₁-AR) from HEK293 cell line is purified and immobilized on the surface of macro-pore silica gel to prepare an high-performance affinity chromatography stationary phase for the pursuit of drug–receptor interactions by competition zonal elution. Naftopidil is found to have only one type of binding site to α ₁-AR with an association constant of 1.45 × 10⁶ M⁻¹ and a concentration of binding sites of 1.56 × 10⁻⁶ M, while terazosin hydrochloride proves to present two kinds of binding site on the receptor at which the association constants are determined to be 1.61 × 10⁵ M⁻¹ and 2.06 × 10³ M⁻¹, and the corresponding concentrations of the binding sites are 1.56 × 10⁻⁶ M and 1.11 × 10⁻³ M, respectively. It is concluded that the stationary phase containing attached α ₁-AR can be used to realize the binding of a drug to the receptor.

     

INPHARMA-Based Determination of Ligand Binding Modes at α1-Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity

The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α_1A-AR and α_1B-AR, which belong to the G protein-coupled receptor (GPCR) superfamily, by employing the solution-based ligand-observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype-selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α_1A-AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α_1A-AR and α_1B-AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back-calculated spectra based on ligand poses obtained from all-atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of (R)-A-61603 towards α_1A-AR, thus demonstrating an approach for targeting subtype selectivity in ARs.     

Biointeraction analysis of carbamazepine binding to α1‐acid glycoprotein by high‐performance affinity chromatography

Interactions of the drug carbamazepine with the serum protein α₁‐acid glycoprotein (AGP) were examined by high‐performance affinity chromatography. Frontal analysis studies with an immobilized AGP column and control column indicated carbamazepine had both low‐affinity interactions with the support and high‐affinity interactions with AGP. When a correction was made for binding to the support, the association equilibrium constant measured at pH 7.4 and 37°C for carbamazepine with AGP was 1.0 (±0.1)×10⁵ M^?1, with values that ranged from 5.1 to 0.58×10⁵ M^?1 in going from 5 to 45°C. It was found in competition studies that these interactions were occurring at the same site that binds propranolol on AGP. Temperature studies indicated that the change in enthalpy was the main driving force for the binding of carbamazepine to AGP. These results provide a more complete picture of how carbamazepine binds to AGP in serum. This report also illustrates how high‐performance affinity chromatography can be used to examine biological interactions and drug–protein binding in situations in which significant interactions for an analyte are present with both the chromatographic support and an immobilized ligand.     

Spectroscopic investigations of hematoxylin–Eu(III) complex interacting with Herring-sperm DNA

The interaction of HE–Eu(III) complex (HE?=?hematoxylin) with Herring-sperm DNA (hsDNA) has been studied by absorption spectra, fluorescence, and viscosity measurements in physiological buffer (pH?=?7.40). The binding constant of HE–Eu(III) complex to hsDNA was obtained by double reciprocal method at 298 and 310?K and the corresponding thermodynamic parameters (Δ_r Hm??=?8.55?×?10⁴?J?mol^?1, Δ_r Gm??=??3.01?×?10⁴?J?mol^?1, Δ_r Sm??=?387.95?J?mol^?1?K^?1) were calculated, showing that the interaction between HE–Eu(III) complex and hsDNA was driven mainly by entropy. The value of K indicated that the binding mode of HE–Eu(III) complex with DNA was not classical intercalation. These results were further supported by viscosity method and competitive binding experiment. Scatchard analysis suggests that the interaction mode was a mixed binding, which contains partial intercalation and groove binding.     

Rapid analysis of interaction between six drugs and β2‐adrenergic receptor by injection amount‐dependent method

Drug–protein interaction analysis has become a considerable topic in life science which includes clarifying protein functions, explaining drug action mechanisms and uncovering novel drug candidates. This work was to determine the association constants (K _A ) of six drugs to β ₂‐adrenergic receptor by injection amount‐dependent method using stationary phase containing the immobilized receptor. The values of K _A were calculated to be (25.85 ± 0.035) × 10⁴ m ⁻¹ for clorprenaline, (42.51 ± 0.054) × 10⁴ m ⁻¹ for clenbuterol, (6.67 ± 0.008) × 10⁴ m ⁻¹ for terbutaline, (33.99 ± 0.025) × 10⁴ m ⁻¹ for tulobuterol, (7.59 ± 0.011) × 10⁴ m ⁻¹ for salbutamol and (78.52 ± 0.087) × 10⁴ m ⁻¹ for bambuterol. This rank order agreed well with the data determined by zonal elution, frontal analysis and nonlinear chromatography, even using different batches of β ₂‐AR column. A good correlation was found between the association constants by the current method and radio‐ligand binding assay. Our data indicates that the injection amount‐dependent method is a powerful alternative for rapid analysis of ligand–receptor interactions.     

DNA Binding Studies and Cytotoxicity of a Dinuclear PtII Diazapyrenium‐Based Metallo‐supramolecular Rectangular Box

The interaction with native DNA of a 2,7‐diazapyrenium‐based ligand 1 and its Pt^II rectangular metallacycle 2 is explored through circular and linear dichroism and fluorescence spectroscopies. The metal‐free ligand 1 binds through intercalation, with a binding constant of approximately 5×10⁵ M ^?1, whereas the metallacycle 2 binds and bends the DNA with a binding constant of 7×10⁶ M ^?1. PCR assays show that metallo‐supramolecular box 2 interferes with DNA transactions in vitro whereas the intercalator 1 does not. The metallacycle is active against four human cancer cell lines, with IC₅₀ values ranging between 3.1 and 19.2 μM and shows similar levels of efficacy, but a different spectrum of activity, to cisplatin.     

Study of Binding of Benzyl-Thiouracil to Human Serum Albumin by Gel Filtration Chromatography

Abstract

The binding of benzyl-thiouracil to human serum albumin was studied at 37°C and pH 7.4 by Sephadex filtration chromatography based upon Hummel and Dreyer's method. As the benzyl-thiouracil (ligand) was adsorbed on to the gel matrix, the free ligand concentrations had to be corrected through the ligand distribution between the stationary and mobile phases.

A good agreement was found between binding parameters—calculated by this method and by the classical method (equilibrium dialysis). Binding is characterized by one binding site with a moderate association constant (K₁ = 5.7 × 10⁴ M^-1) and two binding sites with a low association constant (K₂ = 7.8 × 10³ M^-1).     

Extraction and Immobilization of SA-α-2,6-Gal Receptors on Magnetic Nanoparticles to Study Receptor Stability and Interaction with Sambucus nigra Lectin

The interaction between influenza virus hemagglutinins and host cell with terminal sialic acid linked receptors, SA-α-2,6-Gal for human strains is important to obtain insights into this infectious disease. Sambucus nigra lectin has high affinity for SA-α-2,6-Gal receptors. The goals of this work were: to extract the SA-α-2,6-Gal receptors from porcine airways; to perform receptors immobilization and study their storage stability; and to determine some parameters of interaction between the receptor and S. nigra lectin. The receptor isolation was monitored by means of bound sialic acid (BSAc) detection. A major band of protein at 66.7 kDa was clearly visible in SDS-PAGE assay. Eighty-one percent of isolated glycoproteins were immobilized on magnetic nanoparticles. The kinetics of BSAc storage stability at 4 °C was approximated as the first order reaction with kinetic constant and half-life estimated as 0.062 day^?1 and 11.2 days, respectively. The dissociation constant (K _d) calculated from Scatchard's plot was 2.47?×?10^?7 M, and the receptor concentration was equal to 7.92?×?10^?5 M. Procedure for N-SA-α-2,6-Gal -receptors extraction based on their affinity to S. nigra lectin with magnetic nanoparticles, and their immobilization in active form, was not described previously, and may have wide application in designing biosensors or virus removal from areas or contaminated samples.     

Inhibition of Ligand Binding Ability of Three Porphyrins by an Organic Effector

A stimulus‐responsive receptor 1 was designed and prepared to control the ligand‐binding ability of three active sites, two zinc tetraphenylporphyrin units (P1) and one zinc diethynyldiphenylporphyrin unit (P2), with one effector molecule 2 . Bulky hexarylbenzene units were incorporated as shielding panels in the middle of the flexible side arms of 1 . Spectroscopic titrations indicated that a stable supramolecular complex 1 ? 2 (K _{1 ? 2} =6.7×10⁶ m ^?1) was produced by the cooperative formation of multiple hydrogen and coordination bonds. As a result, the binding of a ligand to P1 was inhibited by 2 in a competitive manner. Additionally, the formation of 1 ? 2 brought about conformational restriction of the side arms to cover both faces of P2 with the shielding panels. The binding constant of 4‐phenylpyridine with P2 in 1 ? 2 decreased to 8.9 % of that in 1 . Namely, the ligand‐binding ability of P2 was inhibited according to an allosteric mechanism.     

Antibacterial,nuclease, and SOD-mimic behaviors of copper(II) complexes of norfloxacin and phenanthrolines

Square-pyramidal complexes [Cu(NFL)(A ⁿ )Cl]?·?5H₂O (A ^{n ?}=?phenanthroline derivatives and NFL?=?deprotonated norfloxacin) have been synthesized and characterized. Interactions with Herring Sperm DNA and pUC19 DNA have been investigated. Mode and extent of interaction was measured by the perturbation in absorbance of complexes in the absence and presence of DNA. Hydrodynamic volume change and gel electrophoretic results were also kept under consideration. Synthesized complexes bind to DNA via intercalation with binding constant 0.875–1.446?×?10⁴?(mol?L^?1)^?1 based on bathochromism and hypochromism observed. Intercalative binding of complexes with DNA was further supported by relative viscosity, where 5 intercalates more strongly with most increase in relative viscosity, and K _b value of 1.446?×?10⁴?(mol?L^?1)^?1. Evaluation of electrophoretic separation of plasmid on agarose gel reveals that 5 cleaves more efficiently. Square-pyramidal geometry at the metal center supports superoxide-dismutase (SOD)-mimic behavior in addition to an electron-withdrawing group on the ancillary ligand stabilizing Cu–O bonding.     

Electrocatalytic studies of covalently immobilized metal tetra-amino phthalocyanines onto derivatized screen-printed gold electrodes

Metal tetra-amino phthalocyanine complexes (MTAPc; where M is Co or Mn) were immobilized on screen-printed gold electrodes pre-modified with monolayers of benzylamino groups. The functionalized electrodes were then activated using benzene-1,4-dicarbaldehyde as a linker before MTAPc complexes were immobilized. The surface coverages for the modified electrodes confirmed the perpendicular orientation of the MTAPcs. The apparent electron transfer constant (k_app) for the electrodes is 2.2?×?10^?5 cm.s^?1 for both CoTAPc and MnTAPc modified electrodes as calculated with data from impedance measurements. The k_app values for the bare and benzylamino modified electrodes were found to be 1.2?×?10^?4 cm.s^?1 and 4.9?×?10^?6 cm.s^?1, respectively. The electrocatalysis of the modified electrodes towards detection of H₂O₂ gave significant peak current densities and electrocatalytic potentials at ?0.28 V and ?0.31 V for the MnTAPc and CoTAPc modified electrodes, respectively.     

Use of Frontal Chromatography to Measure the Binding Interaction of Berberine Chloride with Bovine Serum Albumin

There is much interest in the interactions between the active constituents of traditional Chinese medicine and biomolecules. By use of frontal analysis on an affinity column we have examined the binding interaction of berberine chloride (BC), a major active constituent of coptis, with bovine serum albumin (BSA) in 40 mM phosphate buffer, pH 7.0. Adsorption of BC on immobilized BSA was in accordance with the Langmuir isotherm, suggesting BC is binding to a single type of site on the immobilized BSA. The binding constant was 4.79 × 10⁴ L mol^?1 at 30 °C, less than the value of 6.61 × 10⁴ L mol^?1 obtained by fluorescence spectroscopy under the same buffer and temperature conditions. The effects of temperature on the retention, binding constant, and active binding sites, and on the percentage protein binding of BC, were also investigated. Thermodynamic measurements indicated that the increase in entropy was an important process promoting the interaction between BC and BSA.     

Electrochemistry of complex formation of carbaryl with ds-DNA using [Ru(bpy)2dppz]2+ as probe

An electrochemical competition method was used to study the interaction of carbaryl with natural double-stranded DNA (ds-DNA). Layer-by-layer films of negatively charged natural ds-DNA and polycationic poly (diallyldimethylammonium chloride) were assembled on the surface of a glassy carbon electrode. The DNA intercalator [Ru(bpy)₂(dppz)]²⁺ (bpy?=?2, 2′-bipyridine, dppz?=?dipyrido [3, 2-a: 2′,3′-c] phenazine) was chosen as an electrochemical probe. Tripropylamine was used as an electron donor to chemically amplify the oxidation current of the probe. In order to examine the effects of substituting group on the binding interaction of carbaryl with DNA, the interaction of naphthalene or α-naphthol with DNA was also studied by square wave voltammetry (SWV). The values of binding constant K _b of the three compounds to DNA are determined, which fall in the range of (0.2?×?10⁵) to (1.3?×?10⁵)?M^?1. The correlation suggests that the functional groups may play an important role in the DNA/analyte competition binding interaction. We demonstrated that it is conducive to the combination of small molecules and DNA when the functional groups are hydrophobic and have the lone-pair electrons as the electron donor. Furthermore, UV-absorption and fluorescence intensity of Ru-dppz decreases in the presence of carbaryl. These characteristics strongly support the intercalation of carbaryl into double-stranded DNA.     

Study on the Interaction of Raloxifene and Bovine Serum Albumin by the Tachiya Model

The interaction of bovine serum albumin (BSA) with raloxifene was assessed via fluorescence spectroscopy. The number of binding sites and the apparent binding constants between raloxifene and BSA were analyzed using the Tachiya model and Stern-Volmer equation, respectively. The apparent binding constant and the number of binding sites at 298 K were 2.33×10⁵ L?mol^?1 and 1.0688 as obtained from the Stern-Volmer equation and 2.00×10⁵ L?mol^?1 and 2.6667 from the Tachiya model. The thermodynamic parameters ΔH and ΔS were calculated to be 69.46 kJ?mol^?1 and 121.12 J?K^?1?mol^?1, respectively, suggesting that the force acting between raloxifene and BSA was mainly a hydrophobic interaction. The binding distance between the donor (BSA) and acceptor (raloxifene) was 4.77 nm according to Förster’s nonradiational energy transfer theory. It was also found that common metal ions such as K⁺, Cu²⁺, Zn²⁺, Mg²⁺ and Ca²⁺ decreased the apparent association constant and the number of binding sites between raloxifene and BSA.     

Molecular dynamics and energetic perceptions of substrate recognition by thymidylate kinase

Plasmodium deoxyguanylate pathways are an attractive area of investigation for future metabolic and drug discovery studies due to their unusual substrate specificities. We investigated the energetic contribution to thymidylate kinase substrate binding, and the forces underlying ligand recognition. The binding constant varied from 8 × 10⁴ M^?1 at 290 K to 6 × 10⁴ M^?1 at 310 K for dGMP, and from 16 × 10⁴ M^?1 at 290 K to 4 × 10⁴ M^?1 at 310 K for TMP. ΔC _p was estimated as ?1.75 kJ mol^?1 K^?1 for TMP and +2 kJ mol^?1 K^?1 for dGMP. In comparison with TMP, the binding of dGMP to PfTMK produced less favorable enthalpy change, positive or favorable entropic contribution at lower temperature, positive heat capacity change, negative $ \Updelta S_{\text{HE}}^{^\circ } $ , positive ΔS _other, higher total solvent-exposed surface area and more or less rigid body binding. These changes indicate unfavorable conditions for proper binding and lower conformational changes, and suboptimal structural reordering during dGMP binding.     

Human Saliva-Based Quantitative Monitoring of Clarithromycin by Flow Injection Chemiluminescence Analysis: A Pharmacokinetic Study

Human saliva quantitative monitoring of clarithromycin (CLA) by chemiluminescence (CL) with flow injection analysis was proposed for the first time, which was based on the quenching effect of CLA on luminol–bovine serum albumin (BSA) CL system with a linear range from 7.5?×?10^?4 to 2.0 ng/ml. This proposed approach, offering a maximum sample throughput of 100 h^?1, was successfully applied to the quantitative monitoring of CLA levels in human saliva during 24 h after a single oral dose of 250 mg intake, with recoveries of 95.2～109.0 % and relative standard deviations lower than 6.5 % (N?=?7). Results showed that CLA reached maximum concentration of 2.28?±?0.02 μg/ml at approximately 3 h, and the total elimination ratio was 99.6 % in 24 h. The pharmacokinetic parameters including absorption rate constant (0.058?±?0.006 h^?1), elimination rate constant (0.149?±?0.009 h^?1) and elimination half-life time (4.66?±?0.08 h) were obtained. A comparison of human saliva and urine monitoring was also given. The mechanism study of BSA–CLA interaction revealed the binding of CLA to BSA is an entropy driven and spontaneous process through hydrophobic interaction, with binding constant K _BSA–CLA of 4.78?×?10⁶ l/mol and the number of binding sites n of 0.82 by flow injection–chemiluminescence model. Molecular docking analysis further showed CLA might be in subdomain IIA of BSA, with K _BSA–CLA of 6.82?×?10⁵ l/mol and ΔG of ?33.28 kJ/mol.     

Potentiometric Sensing of Aspirin Metabolite in Human Plasma and Pharmaceutical Preparations Using Co(III)‐complex Based Electrodes: Experimental and Quantum Chemical Calculations

Three novel poly vinyl chloride (PVC) ( A ), carbon paste (CP) ( B ), and coated glassy carbon‐MWCNT (CGC) ( C ) salicylate (sal^?) sensors based on new synthesized [Co(L^2Cl)Cl₃(H₂O)] ? H₂O complex (L^2Cl=(1H‐benzimidazol‐2‐ylmethyl)‐N‐(2‐chloro‐phenyl)‐ amine)), o‐nitrophenyloctyl ether as a mediator and tridodecylmethylammonium chloride as a cationic additive were successfully used for determination of sal^? in human plasma and pharmaceutical formulations. The sal^?‐sensors exhibited enhanced sensitivity with slope of ?63.5, ?60.5 and ?58.9 mV/decade and detection limit of 1.0×10^?5, 4.0×10^?7, and 1.0×10^?6 mol L^?1 for A – C sensors respectively. Quantum chemical calculations were carried out by HF and DFT/B3LYP methods to explore and investigate the interaction between the receptor and the different anions. The intermolecular H‐bond created between the uncoordinated C?O of salicylate group and the secondary amino group in the complex is the key factor of the selectivity of the proposed sensor. A linear relation is established between the natural charge on the Co center and the value of the binding energy, where the decrease in positive charge is associated by an increase in the anion binding energy.     

Lanthanide complex of 1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldehyde-(isonicotinoyl) hydrazone: crystal structure and DNA-binding properties

A Schiff-base ligand derived from 1-phenyl-3-methyl-4-formyl-2-pyrazolin-5-one (PMFP) and isoniazid was prepared and its La(III) complex was characterized by X-ray single crystal diffraction. The La(III) is nine-coordinate in a space group P2₁/n. DNA-binding was investigated by UV-Vis, fluorescence titration, ethidium bromide displacement experiments, and viscosity measurements, which indicated that the ligand and La(III) complex strongly binds to calf thymus DNA presumably via groove binding and intercalation. The intrinsic binding constants of the ligand and La(III) complex were 0.86?×?10⁵ and 2.46?×?10^5?mol?L^?1, respectively. Antioxidant data from hydroxyl radical scavenging experiments in vitro suggest that the La(III) complex possesses higher scavenging ratio than the free ligand, metallic salt, and some standard antioxidants like mannitol.     

Chemiluminescent and Colorimetric Aptamer-Based Assays of Human α-Thrombin

Abstract

A microplate-based sandwich assay for the determination of α-human thrombin (HTb) was developed. Fluorescein-modified 29-mer thrombin binding aptamer (FAM-TBA29) and biotinylated 15-mer thrombin binding aptamer (Bio-TBA15) reacting with different exosites of HTb were used as the biorecognition components in the assay. FAM-TBA29 (capture aptamer) was immobilized using its interaction with anti-fluorescein antibody adsorbed on the microplate surface. As a sensitive signaling system, a combination of Bio-TBA15 and streptavidin-polyHRP conjugate was used. Under the optimized conditions, the detection limit for HTb was 1.4?nM; this value was the same in both the colorimetric and the chemiluminescent assays. The replacement of colorimetry for HRP measurement with chemiluminescence increased the assay sensitivity from 0.06 to 1.7?×?10⁶?nM^?1 that clearly demonstrated advantage of the latter approach.