Drug-protein System Research Articles

Effect of irradiated nonionic surfactant on drug-protein binding

In this work, the effect of γ-irradiated surfactants on drug–protein binding has been assessed. Irradiated aqueous solutions of Pluronic F-127, Pluronic L-35, Tween 20, and Tween 80 surfactants were used. Gamma irradiation was carried out for three different doses, to these four surfactants viz., 6, 30, and 36 kGy. Two drugs, Ornidazole (ONZ) and Telmisartan (TMS) were used for the binding study. The effect of four irradiated surfactants in the presence of drug – Bovine serum albumin (BSA) protein was analyzed. The drug solutions in methanol-aqueous media were combined with BSA in the initial step. In the next two succeeding steps, drug–BSA interaction in the presence of unirradiated and irradiated surfactants were carried out. The results of drug-BSA due to addition of irradiated and unirradiated surfactants were compared. The interaction processes were assessed through UV Spectroscopy, DLS, zeta potential, turbidity, and docking studies. Improved binding was observed for both the drugs and four surfactants for irradiated surfactants as determined from the binding constant values using UV spectroscopic studies. The DLS measurements demonstrated no general trend of increase or decrease in micellar size with absorbed dose. The binding and change in the size of micelles were observed to be highly drug and surfactant-specific. Among the four surfactants, irradiated Pluronic F-127 showed higher binding affinity. To the best of our knowledge, this is the first report on the effect of γ-irradiated surfactant on drug–BSA interaction. This can be applied to other drug–protein systems to tune their interaction to the required level. Communicated by Ramaswamy H. Sarma

Deciphering the molecular details of interactions between anti-COVID drugs and functional human proteins: in silico approach

The molecular docking calculations have been employed to investigate the interactions a set of proteins with the repurposed anti-COVID drugs. The position of the therapeutic agents within the protein structure was dependent on a particular drug-protein system and varied from the binding cleft to the periphery of the polypeptide chain. Interactions involved in the drug-protein complexation includes predominantly hydrogen bonding and hydrophobic contacts. The obtained results may be of particular importance while developing the anti-COVID strategies as well as for deeper understanding of the drug pharmacodynamics and pharmacokinetics.

Insights on the in-vitro binding interaction between donepezil and bovine serum albumin

In this work, the binding mechanism between donepezil (DNP) and bovine serum albumin (BSA) was established using several techniques, including fluorimetry, UV- spectrophotometry, synchronous fluorimetry (SF), fourier transform infrared (FTIR), fluorescence resonance energy transfer (FRET) besides molecular docking study. The fluorescence quenching mechanism of DNP-BSA binding was a combined dynamic and static quenching. The thermodynamic parameters, binding forces, binding constant, and the number of binding sites were determined using a different range of temperature settings. Van't Hoff's equation was used to calculate the reaction parameters, including enthalpy change (ΔHο) and entropy change (ΔSο). The results pointed out that the DNP-BSA binding was endothermic. It was shown that the stability of the drug-protein system was predominantly due to the intermolecular hydrophobic forces. Additionally, the site probing method revealed that subdomain IIA (Site I) is where DNP and BSA's binding occurs. This was validated using a molecular docking study with the most stable DNP configuration. This study might help to understand DNP's pharmacokinetics profile and toxicity as well as provides crucial information for its safe use and avoiding its toxicity.

Exploring the molecular interaction of mebendazole with bovine serum albumin using multi-spectroscopic approaches and molecular docking

This article presents the binding interaction between mebendazole (MBZ) and bovine serum albumin. The interaction has been studied using different techniques, such as fluorescence quenching spectroscopy, UV–visible spectroscopy, synchronous fluorescence spectroscopy, fourier transform infrared, and fluorescence resonance energy transfer in addition to molecular docking. Results from Stern Volmer equation stated that the quenching for MBZ-BSA binding was static. The fluorescence quenching spectroscopic study was performed at three temperature settings. The binding constant (kq), the number of binding sites (n), thermodynamic parameters (ΔHο, ΔSο and ΔGο), and binding forces were determined. The results exhibited that the interaction was endothermic. It was revealed that intermolecular hydrophobic forces led to the stabilization of the drug-protein system. Using the site marker technique, the binding between MBZ and BSA was found to be located at subdomain IIA (site I). This was furtherly approved using the molecular docking technique with the most stable MBZ configuration. This research may aid in understanding the pharmacokinetics and toxicity of MBZ and give fundamental data for its safe usage to avoid its toxicity.

Multiresolution non-covalent interaction analysis for ligand–protein promolecular electron density distributions

Nowadays, the topology of electron density (ED) distributions as well as the non-covalent interaction (NCI) analysis of reduced density gradient (RDG) distributions, are extensively used to characterize intermolecular contacts. Here, topological and NCI-based analyses are combined for the multiresolution study of the intermolecular interactions occurring in a drug-protein system (PDB access code: 3UNK). The method involves the search for the critical points (CP) of the well-known PASA model developed by Carbo-Dorca and co-workers, and the minima in the corresponding RDG grids. The CP-based representation of the intermolecular contacts enriches the geometry-based interactions found using a Web server. The stability of the intermolecular interactions is studied by following their corresponding CP trajectory in space at several degrees of the ED smoothing, and by the evaluation of the local potential energy density (LPDE). It is observed that several RDG minima are located at the meeting point of CP trajectories initiated in the unsmoothed ED distribution. At high smoothing levels, the accumulation of ED charges at the ligand–protein interface progressively emerges and is detected through the study of the Laplacian values at the CPs and RDG minima. In parallel, the distance-dependency of the LPED values is less and less clear while the density-dependency is favored. The CP networks and their descriptors are seen as a signature of the ligand–protein arrangement, which is proposed to be further used in the characterization of ligand–protein stackings obtained from, e.g., crystal structures, docking calculations, Molecular Dynamics simulations, and pharmacophore designs.

Multi-spectroscopic characterization of bovine serum albumin upon interaction with atomoxetine

The quenching interaction of atomoxetine (ATX) with bovine serum albumin (BSA) was studied in vitro under optimal physiological condition (pH=7.4) by multi-spectroscopic techniques. The mechanism of ATX-BSA system was a dynamic quenching process and was confirmed by the fluorescence spectra and lifetime measurements. The number of binding sites, binding constants and other binding characteristics were computed. Thermodynamic parameters ∆H° and ∆S° indicated that intermolecular hydrophobic forces predominantly stabilized the drug-protein system. The average binding distance between BSA and ATX was studied by Försters theory. UV-absorption, Fourier transform infrared spectroscopy (FT-IR), circular dichroism (CD), synchronous spectra and three-dimensional (3D) fluorescence spectral results revealed the changes in micro-environment of secondary structure of protein upon the interaction with ATX. Displacement of site probes and the effects of some common metal ions on the binding of ATX with BSA interaction were also studied.

On-column entrapment of alpha1-acid glycoprotein for studies of drug-protein binding by high-performance affinity chromatography.

An on-column approach for protein entrapment was developed to immobilize alpha1-acid glycoprotein (AGP) for drug-protein binding studies based on high-performance affinity chromatography. Soluble AGP was physically entrapped by using microcolumns that contained hydrazide-activated porous silica and by employing mildly oxidized glycogen as a capping agent. Three on-column entrapment methods were evaluated and compared to a previous slurry-based entrapment method. The final selected method was used to prepare 1.0cm × 2.1mm I.D. affinity microcolumns that contained up to 21 (±4) μg AGP and that could be used over the course of more than 150 sample applications. Frontal analysis and zonal elution studies were performed on these affinity microcolumns to examine the binding of various drugs with the entrapped AGP. Site-selective competition studies were also conducted for these drugs. The results showed good agreement with previous observations for these drug-protein systems and with binding constants that have been reported in the literature. The entrapment method developed in this study should be useful for future work in the area of personalized medicine and in the high-throughput screening of drug interactions with AGP or other proteins. Graphical abstract On-column protein entrapment using a hydrazide-activated support and oxidized glycogen as a capping agent.

Elucidation of the binding mechanism of renin using a wide array of computational techniques and biological assays

We investigate the binding mechanism in renin complexes, involving three drugs (remikiren, zankiren and enalkiren) and one lead compound, which was selected after screening the ZINC database. For this purpose, we used ab initio methods (the effective fragment potential, the variational perturbation theory, the energy decomposition analysis, the atoms-in-molecules), docking, molecular dynamics, and the MM-PBSA method. A biological assay for the lead compound has been performed to validate the theoretical findings. Importantly, binding free energy calculations for the three drug complexes are within 3kcal/mol of the experimental values, thus further justifying our computational protocol, which has been validated through previous studies on 11 drug-protein systems. The main elements of the discovered mechanism are: (i) minor changes are induced to renin upon drug binding, (ii) the three drugs form an extensive network of hydrogen bonds with renin, whilst the lead compound presented diminished interactions, (iii) ligand binding in all complexes is driven by favorable van der Waals interactions and the nonpolar contribution to solvation, while the lead compound is associated with diminished van der Waals interactions compared to the drug-bound forms of renin, and (iv) the environment (H2O/Na+) has a small effect on the renin–remikiren interaction.

Species-dependent binding of tocainide analogues to albumin: Affinity chromatography and circular dichroism study

A series of novel tocainide analogues were characterized for their HSA and RSA binding, by using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). In this HPLAC study, HSA and RSA were covalently immobilized to the silica matrix of HPLC columns, with a procedure that maintained unaltered the binding properties of the proteins. The tocainide analogues were ranked for their affinity to HSA and RSA on the basis of their bound fractions measured by the two albumin-based columns. This technique was also applied to characterize the high affinity binding sites of these tocainide analogues to the protein. For this purpose displacement experiments were carried out by means of increasing concentrations in the mobile phase of competitors known to bind selectively to the main binding sites of HSA. The results obtained with the immobilized proteins were confirmed by investigating the same drug-protein systems in solution by circular dichroism. The comparison of the data collected with both methodologies highlighted the dramatic effect of small differences in the amino acidic sequences of the two proteins. In fact, despite their similar primary and secondary structures, a small difference in the amino acidic sequence leads to significant differences in their three-dimensional structure reflecting their different binding capacity and their stereoselectivity. Therefore, this study confirms how it is crucial to consider the significant differences among the animal models when performing pharmacokinetic studies. It is also clear that the knowledge of serum carrier binding parameters at an early stage of drug discovery represents a great advantage that may help to save time and efforts.

5-Imino-1,2-4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors: Determination of blood–brain barrier penetration and binding to human serum albumin

5-Imino-1,2,4-thiadiazoles and quinazolines derivatives as glycogen synthase kinase 3β (GSK-3β) and phosphodiesterase 7 (PDE7) inhibitors were characterized for their ability to pass the blood–brain barrier (BBB) together with their human serum albumin (HSA) binding using high-performance liquid affinity chromatography (HPLAC) and circular dichroism (CD). To study the blood–brain barrier penetration, a parallel artificial membrane permeability assay (PAMPA) using a porcine brain lipid was employed. For the HPLAC investigation, HSA was previously covalently immobilized to the silica matrix of the HPLC column. This HSA-based column was used to characterize the high affinity binding sites of 5-imino-1,2,4-thiadiazoles and quinazolines derivatives to HSA. Displacement experiments in the presence of increasing concentrations of competitors known to bind selectively to the main binding sites of HSA were carried out to determine their possible binding site. The same drug–protein system was studied by CD.The analysed compounds were able to pass BBB, they present good drug-like properties and they showed a high affinity to HSA. Competition experiments showed an anticooperative interaction at sites I and II, and an independent binding at bilirubin binding site on HSA.

Investigating the Antagonistic Action Between Aspirin and Tamoxifen with HSA: Identification of Binding Sites in Binary and Ternary Drug-Protein Systems by Spectroscopic and Molecular Modeling Approaches

The combination of several drugs is often necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease of the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. The aim of this study has been to analyze the interactions of tamoxifen (TMX) and aspirin (ASA) with human serum albumin (HSA) and to evaluate the mechanism of a simultaneous binding of TMX and ASA to the protein. Fluorescence analysis was used to estimate the effect of the drugs on the protein fluorescence and to define the binding and quenching properties of drug-HSA complexes. The binding sites for TMX and ASA were identified in ternary structures of HSA by means of spectrofluroscence. The analysis of the fluorescence quenching of HSA in binary and ternary systems pointed at TMX and ASA having an effect on the HSA-ASA and HSA-TMX complexes. Furthermore, the results of synchronous fluorescence, resonance light scattering and circular dichroism of the binary and ternary systems showed that the binding of TMX and ASA to HSA could induce conformational changes in HSA. Moreover, the simultaneous presence of TMX and ASA during binding to HSA should be taken into account in multi-drug therapy, as it induces the necessity of a monitoring therapy owing to the possible increase of uncontrolled toxic effects. Competitive site marker experiments demonstrated that the binding site of ASA and TMX to HSA differed in the binary system as opposed to in its ternary counterpart. Finally, molecular modeling of the possible binding sites of TMX and ASA in binary and ternary systems to HSA confirmed the experimental results.

CHROMATOGRAPHIC ANALYSIS OF DRUG INTERACTIONS IN THE SERUM PROTEOME.

The binding of drugs with serum proteins and binding agents such as human serum albumin, α1-acid glycoprotein, and lipoproteins is an important process in determining the activity and fate of many pharmaceuticals in the body. A variety of techniques have been used to study drug interactions with serum proteins, but there is still a need for faster or better methods for such work. High-performance liquid chromatography (HPLC) is one tool that has been utilized in many formats for these types of measurements. Advantages of using HPLC for this application include its speed and precision, its ability to be automated, its good limits of detection, and its compatibility with a wide range of assay formats and detectors. This review will discuss various approaches in which HPLC can be employed for the study of drug-protein interactions. These techniques include the use of soluble proteins in zonal elution and frontal analysis methods or vacancy techniques such as the Hummel-Dreyer method. Zonal elution and frontal analysis methods that make use of immobilized proteins and high-performance affinity chromatography will also be presented. A variety of applications will be examined, ranging from the determination of free drug fractions to the measurement of the strength or rate of a drug-protein interaction. Newer developments that will be discussed include recent work in the creation of novel mathematical approaches for HPLC studies of drug-protein binding, the use of HPLC methods for the high-throughput screening of drug-protein binding, and the creation and use of affinity monoliths or affinity microcolumns for examining drug-protein systems.

Anomalous relationship between free drug fraction and its total concentration in drug–protein systems: II. Binding of different ligands to plasma proteins

As it was reported earlier, the free propofol percentage is dependent on its total concentration in human blood or plasma protein solution. At total propofol concentrations below 1 μg ml −1 the free fraction of the drug increases with the decrease of its total concentration. The results of the experiments performed in Part I indicate that the well-ordered water around a protein molecule (i.e. the hydration layer) is probably the main factor responsible for the mentioned anomalous free drug fraction changes. The experiments carried out in this study with different ligands (homologous saturated fatty acids, indomethacin and lidocaine) show that: 1. ligand hydrophobicity affects the run of the dependence between the free ligand fraction and its total concentration, which confirms the importance of the hydration layer in the drug binding process, 2. similar anomalous changes of the free drug fraction are observed not only for drugs interacting with different binding sites on human serum albumin (HSA), i.e. for propofol and indomethacin, but also for basic local anesthetic lidocaine which is known to bind to other plasma protein, alpha-1-acid glycoprotein (AGP). Therefore, the increase of the free drug fraction at its low total concentration does not appear to be an exclusive feature of propofol–HSA system. The results presented in this paper expand the present knowledge about drug–protein binding process and thereby encourage further research on this subject.

Anomalous relationship between free drug fraction and its total concentration in drug–protein systems: I. Investigation of propofol binding in model HSA solution

According to contemporary knowledge, only the free form of a drug exhibits pharmacological activity due to its ability to cross the membranes and to interact with receptors. For the majority of drugs their free fraction in human plasma is usually constant, hence the therapeutic effect of a drug is most often correlated with its total concentration. However, in the case of certain drugs of narrow therapeutic window, for which a slight elevation of a free drug fraction (e.g., occurring in some pathological states) may exert a toxic effect, the therapeutic effect should be correlated with free concentration. As we reported earlier, at total propofol concentrations below 1 μg ml −1 the free fraction of the drug increases with the decrease of its total concentration. The presented paper discusses the results of several experiments performed in order to elucidate this anomalous phenomenon. The study is mainly focused on the investigation of the relationship between free drug percentage and total drug concentration in human serum albumin (HSA) solution, performed at different temperatures and in the presence of the salting-out agent. It was observed that a temperature increase leads to an increased propofol binding, which confirms the importance of hydrophobic interactions for the binding process. The addition of the salting-out agent significantly changing water structure of the drug–protein system is followed by disappearance of the anomalous relationship and results in constant value of the free drug fraction in the whole range of the studied total drug concentration. The results of the performed experiments indicate that the well-ordered water around a protein molecule (i.e., the hydration layer) constitutes the main factor responsible for the discussed anomalous free drug fraction changes.

Ultraviolet Resonance Raman Study of Drug Binding in Dihydrofolate Reductase, Gyrase, and Catechol O-Methyltransferase

This paper presents a study of the use of ultraviolet resonance Raman (UVRR) spectroscopic methods as a means of elucidating aspects of drug-protein interactions. Some of the RR vibrational bands of the aromatic amino acids tyrosine and tryptophan are sensitive to the microenvironment, and the use of UV excitation radiation allows selective enhancement of the spectral features of the aromatic amino acids, enabling observation specifically of their change in microenvironment upon drug binding. The three drug-protein systems investigated in this study are dihydrofolate reductase with its inhibitor trimethoprim, gyrase with novobiocin, and catechol O-methyltransferase with dinitrocatechol. It is demonstrated that UVRR spectroscopy has adequate sensitivity to be a useful means of detecting drug-protein interactions in those systems for which the electronic absorption of the aromatic amino acids changes because of hydrogen bonding and/or possible dipole-dipole and dipole-polarizability interactions with the ligand.

Calorimetric study of complex formation in the drug-protein system. III. Association of phenoxymethylpenicillin with human serum albumin

Calorimetric study of complex formation in the drug-protein system. III. Association of phenoxymethylpenicillin with human serum albumin

Calorimetric study of complex formation of the drug-protein system II. Thermal stabilization of human serum albumin in its association with oxacillin and methicillin

Calorimetric study of complex formation of the drug-protein system II. Thermal stabilization of human serum albumin in its association with oxacillin and methicillin

Calorimetric study of complex formation in the drug-protein system. I. Binding of methicillin to human serum albumin

Calorimetric study of complex formation in the drug-protein system. I. Binding of methicillin to human serum albumin