Exploring drug–protein interactions using the relationship between injection volume and capacity factor

Yang

et al. 2015

J. Mol. Recognit.

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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) × 10(9) 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.

Section: Resultsmentioning

confidence: 99%

“…The binding mechanism of GFP–GFP antibody was also investigated by molecular docking. Our data suggest that the injection amount‐dependent analysis cannot only study the drug–protein interaction (Zhao et al ., , ), but also a promising method for realizing the protein–protein interaction.…”

Section: Introductionmentioning

confidence: 99%

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

Yang

et al. 2015

J. Mol. Recognit.

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“…During an affinity chromatographic process, the binding of a ligand to an immobilized protein is largely considered as an equilibrium between adsorption and desorption expressed by A + L = AL, where A, L, and AL represent the protein, ligand, and the ligand-protein complex, respectively. Under the conditions of (1) it is rapid to achieve the equilibrium, (2) the longitudinal diffusion is negligible, and (3) the adsorption sites are evenly distributed on the surface of the protein, Equation1 is usually used to describe the ligand-protein-binding interaction 29 :…”

Section: Injection Amount-dependent Methodsmentioning

confidence: 99%

“…During an affinity chromatographic process, the binding of a ligand to an immobilized protein is largely considered as an equilibrium between adsorption and desorption expressed by A + L = AL, where A, L, and AL represent the protein, ligand, and the ligand‐protein complex, respectively. Under the conditions of (1) it is rapid to achieve the equilibrium, (2) the longitudinal diffusion is negligible, and (3) the adsorption sites are evenly distributed on the surface of the protein, Equation is usually used to describe the ligand‐protein–binding interaction:

\frac{k' n_{L}}{1 + k'} = n_{t} - \frac{1}{K_{A}} \times k' V_{m},

where k ′ represents the capacity factor of a ligand, V m is the void volume of the chromatographic system, K A is the association constant of the binding interaction, n L is the molar amount of the injection solute, and n t is the total molar amount of adsorption sites. This equation predicts a linear relationship between k ′ n L /(1+ k ′) and k ′ in a certain range.…”

Section: Theorymentioning

confidence: 99%

Confirming therapeutic target of protopine using immobilized β₂‐adrenoceptor coupled with site‐directed molecular docking and the target‐drug interaction by frontal analysis and injection amount–dependent method

Liu

J of Molecular Recognition

et al. 2017

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Drug-protein interaction analysis is pregnant in designing new leads during drug discovery. We prepared the stationary phase containing immobilized β -adrenoceptor (β -AR) by linkage of the receptor on macroporous silica gel surface through N,N'-carbonyldiimidazole method. The stationary phase was applied in identifying antiasthmatic target of protopine guided by the prediction of site-directed molecular docking. Subsequent application of immobilized β -AR in exploring the binding of protopine to the receptor was realized by frontal analysis and injection amount-dependent method. The association constants of protopine to β -AR by the 2 methods were (1.00 ± 0.06) × 10 M and (1.52 ± 0.14) × 10 M . The numbers of binding sites were (1.23 ± 0.07) × 10 M and (9.09 ± 0.06) × 10 M, respectively. These results indicated that β -AR is the specific target for therapeutic action of protopine in vivo. The target-drug binding occurred on Ser in crystal structure of the receptor. Compared with frontal analysis, injection amount-dependent method is advantageous to drug saving, improvement of sampling efficiency, and performing speed. It has grave potential in high-throughput drug-receptor interaction analysis.

“…In our previous work (Zhao et al, ), we have developed a mathematical model based on the independent relationship between injection volume and capacity factor for analysis of interaction between β 2 ‐AR and its ligands. Although the model has the potential to address time‐consuming and labor‐intensive issues occurred in frontal analysis and zonal elution, the feasibility of this model in rapid drug‐receptor analysis needs further proof.…”

Section: Introductionmentioning

confidence: 99%

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

Zeng

Biomedical Chromatography

Sun

et al. 2017

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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 ) of six drugs to β -adrenergic receptor by injection amount-dependent method using stationary phase containing the immobilized receptor. The values of K were calculated to be (25.85 ± 0.035) × 10 m for clorprenaline, (42.51 ± 0.054) × 10 m for clenbuterol, (6.67 ± 0.008) × 10 m for terbutaline, (33.99 ± 0.025) × 10 m for tulobuterol, (7.59 ± 0.011) × 10 m for salbutamol and (78.52 ± 0.087) × 10 m 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 -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.