Liujiao Bian

Thermodynamic study of the interaction between terbutaline and salbutamol with an immobilized β2-adrenoceptor by high-performance liquid chromatography

Investigation of the interaction between drugs and receptors is very important in revealing the biologic basis and mechanism of the drug, and designing new bioactive compounds. The beta(2)-adrenoceptor (beta(2)-AR) was purified from rabbit lung tissues and immobilized on the surface of macro-pore silica gel through covalent bonds to prepare the stationary phase. Binding isotherms of terbutaline and salbutamol were determined by frontal analysis and the perturbation method, respectively. On the basis of the model of binding isotherm assumed, zonal elution was used to investigate the binding interaction of the receptor with terbutaline and salbutamol. The two drugs had one type of common binding site on immobilized beta(2)-AR. Salbutamol had at least one other major binding region. The association constant for terbutaline was (9.76+/-0.67)x10(4)/M, and the concentration of the binding sites was (9.37+/-1.32)x10(-6)M. Under identical conditions, association constants for salbutamol at the two types of binding site were (1.11+/-0.08)x10(4)/M and (1.34+/-0.13)x10(3)/M, and the concentration of the binding sites was (5.46+/-0.35)x10(-6)M. Entropy increase was the main driving force for terbutaline and salbutamol to bind with beta(2)-AR. The associating reaction of terbutaline and beta(2)-AR was an exothermal process primarily from electrostatic interactions; hydrophobic force was the major factor contributing to the process for salbutamol.

Immobilised Histidine Tagged β2-Adrenoceptor Oriented by a Diazonium Salt Reaction and Its Application in Exploring Drug-Protein Interaction Using Ephedrine and Pseudoephedrine as Probes

A new oriented method using a diazonium salt reaction was developed for linking β 2-adrenoceptor (β 2-AR) on the surface of macroporous silica gel. Stationary phase containing the immobilised receptor was used to investigate the interaction between β 2-AR and ephedrine plus pseudoephedrine by zonal elution. The isotherms of the two drugs best fit the Langmuir model. Only one type of binding site was found for ephedrine and pseudoephedrine targeting β 2-AR. At 37 °C, the association constants during the binding were (5.94±0.05)×103/M for ephedrine and (3.80±0.02) ×103/M for pseudoephedrine, with the binding sites of (8.92±0.06) ×10−4 M. Thermodynamic studies showed that the binding of the two compounds to β 2-AR was a spontaneous reaction with exothermal processes. The ΔGθ, ΔHθ and ΔSθ for the interaction between ephedrine and β 2-AR were −(22.33±0.04) kJ/mol, −(6.51±0.69) kJ/mol and 50.94±0.31 J/mol·K, respectively. For the binding of pseudoephedrine to the receptor, these values were −(21.17±0.02) kJ/mol, −(7.48±0.56) kJ/mol and 44.13±0.01 J/mol·K. Electrostatic interaction proved to be the driving force during the binding of the two drugs to β 2-AR. The proposed immobilised method will have great potential for attaching protein to solid substrates and realizing the interactions between proteins and drugs.

Recognition and binding of β-lactam antibiotics to bovine serum albumin by frontal affinity chromatography in combination with spectroscopy and molecular docking

Serum albumins are the most abundant carrier proteins in blood plasma and participate in the binding and transportation of various exogenous and endogenous compounds in the body. This work was designed to investigate the recognition and binding of three typical β-lactam antibiotics including penicillin G (Pen G), penicillin V (Pen V) and cefalexin (Cef) with bovine serum albumin (BSA) by frontal affinity chromatography in combination with UV-vis absorption spectra, fluorescence emission spectra, binding site marker competitive experiment and molecular docking under simulated physiological conditions. The results showed that a BSA only bound with one antibiotic molecule in the binding process, and the binding constants for Pen G-BSA, Pen V-BSA and Cef-BSA complexes were 4.22×10(1), 4.86×10(2) and 3.32×10(3) (L/mol), respectively. All the three β-lactam antibiotics were mainly inserted into the subdomain IIA (binding site 1) of BSA by hydrogen bonds and Van der Waals forces. The binding capacity between the antibiotics and BSA was closely related to the functional groups and flexibility of side chains in antibiotics. This study provided an important insight into the molecular recognition and binding interaction of BSA with β-lactam antibiotics, which may be a useful guideline for the innovative clinical medications and new antibiotic designs with effective pharmacological properties.

Binding of angiogenesis inhibitor kringle 5 to its specific ligands by frontal affinity chromatography.

The interactions between angiogenesis inhibitor Kringle 5 and its five specific ligands were investigated by frontal affinity chromatography in combination with fluorescence spectra and site-directed molecular docking. The binding constants of trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), epsilon-aminocaproic acid (EACA), benzylamine, 7-aminoheptanoic acid (7-AHA) and L-lysine to Kringle 5 were 19.0×10(3), 7.97×10(3), 6.45×10(3), 6.07×10(3) and 4.04×10(3) L/mol, respectively. The five ligands bound to Kringle 5 on the lysine binding site in equimolar amounts, which was pushed mainly by hydrogen bond and Van der Waals force. This binding affinity was believed to be dependent on the functional group and flexible feature in ligands. This study will provide an important insight into the binding mechanism of angiogenesis inhibitor Kringle 5 to its specific ligands.

Distribution, Transition and Thermodynamic Stability of Protein Conformations in the Denaturant-Induced Unfolding of Proteins

Background Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. Methodology/Principal Findings In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters k i and Δm i in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. Conclusions/Significance This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.

Liquid Chromatography/Quadrupole Time-of-Flight Mass Spectrometry for Identification of In Vitro and In Vivo Metabolites of Bornyl Gallate in Rats

Bornyl gallate (BG) is a potential drug candidate synthesized by the reaction of two natural products, gallic acid and borneol. Previous studies have strongly suggested that BG is worthy of further investigation due to antioxidant, antiatherosclerosis activities, and obvious activity of stimulating intersegmental vessel growth in zebrafish. This work was designed to elucidate the metabolic profile of BG through analyzing its metabolites in vitro and in vivo by a chromatographic separation coupled with a mass spectrometry. The metabolites of BG were characterized from the rat liver microsome incubation solution, as well as rat urine and plasma after oral administration. Chromatographic separation was performed on an Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm) with gradient elution using methanol and water containing 0.2% (V : V) formic acid as the mobile phase. Metabolites identification involved analyzing the retention behaviors, changes of molecular weights and MS/MS fragment patterns of BG and the metabolites. Five compounds were identified as isomers of hydroxylated BG metabolites in vitro. The major metabolites of BG in rat urine and plasma proved to be BG-O-glucuronide and O-methyl BG-O-glucuronide. The proposed method confirmed to be a reliable and sensitive alternative for characterizing metabolic pathways of BG.

Screening bioactive compounds with multi-targets from Rhodiola crenulata by a single column containing co-immobilized beta2-adrenergic receptor and voltage dependent anion channel isoform 1

The pursuit of drugs having improved therapeutic efficacy necessitates increasing research on new assays for screening bioactive compounds with multi-targets. This work synthesized a chromatographic stationary phase containing co-immobilized beta2-adrenergic receptor (β2-AR) and voltage dependent anion channel isoform 1 (VDAC-1) to achieve such purpose. Specific ligands of the two receptors (e.g. salbutamol, methoxyphenamine, ATP and NADH) were utilized to characterize the specificity and bioactivity of the column. Validated application of the stationary phase was performed by screening multi-target compounds of Rhodiola crenulata using high performance affinity chromatography coupled with ESI-Q-TOF-MS. By zonal elution, we identified salidroside as a bioactive compound simultaneously binding to β2-AR and VDAC-1. The compound exhibited the binding sites of 1.0 × 10-7 and 4.0 × 10-7 M on the β2-AR and VDAC-1. On these sites, the association constants were calculated to be 3.3 × 104 and 1.0 × 104 M-1. Molecular docking indicated that the binding of salidroside to the two receptors occurred on Ser169 and Phe255of β2-AR, and the channel wall of VDAC-1. Taking together, we concluded that the column containing co-immobilized receptors has potential for screening bioactive compounds with multi-targets from complex matrices including traditional Chinese medicines.

Binding of TEM-1 beta-lactamase to beta-lactam antibiotics by frontal affinity chromatography

TEM-1 beta-lactamases can accurately catalyze the hydrolysis of the beta-lactam rings in beta-lactam antibiotics, which make beta-lactam antibiotics lose its activity, and the prerequisite for the hydrolysis procedure in the binding interaction of TEM-1 beta-lactamases with beta-lactam antibiotics is the beta-lactam rings in beta-lactam antibiotics. Therefore, the binding of TEM-1 beta-lactamase to three beta-lactam antibiotics including penicillin G, cefalexin as well as cefoxitin was explored here by frontal affinity chromatography in combination with fluorescence spectra, adsorption and thermodynamic data in the temperature range of 278-288K under simulated physiological conditions. The results showed that all the binding of TEM-1 beta-lactamase to the three antibiotics were spontaneously exothermic processes with the binding constants of 8.718×103, 6.624×103 and 2.244×103 (mol/L), respectively at 288K. All the TEM-1 beta-lactamases were immobilized on the surface of the stationary phase in the mode of monolayer and there existed only one type of binding sites on them. Each TEM-1 beta-lactamase bound with only one beta-lactam antibiotic and hydrogen bond interaction and Van der Waals force were the main forces between them. This work provided an insight into the binding interactions between TEM-1 beta-lactamases and beta-lactam antibiotics, which may be beneficial for the designing and developing of new substrates resistant to TEM-1 beta-lactamases.

Estimation of interaction between oriented immobilized green fluorescent protein and its antibody by high performance affinity chromatography and molecular docking.

Although green fluorescence protein (GFP) and its antibody are widely used to track a protein or a cell in life sciences, the binding behavior between them remains unclear. In this work, diazo coupling method that synthesized a new stationary GFP was oriented immobilized on the surface of macro‐porous silica gel by a phase. The stationary phase was utilized to confirm the validation of injection amount‐dependent analysis in exploring protein–protein interaction that use GFP antibody as a probe. GFP antibody was proved to have one type of binding site on immobilized GFP. The number of binding site and association constant were calculated to be (6.41 ± 0.76) × 10‐10 M and (1.39 ± 0.12) × 109 M‐1. Further analysis by molecular docking showed that the binding of GFP to its antibody is mainly driven by hydrogen bonds and salt bridges. These results indicated that injection amount‐dependent analysis is capable of exploring the protein–protein interactions with the advantages of ligand and time saving. It is a valuable methodology for the ligands, which are expensive or difficult to obtain. Copyright