Binding Constants Of Drugs Research Articles

Vinca alkaloid binding to P-glycoprotein occurs in a processive manner

A mechanistic understanding of how P-glycoprotein (Pgp) is able to bind and transport its astonishing range of substrates remains elusive. Pharmacological data demonstrated the presence of at least four distinct binding sites, but their locations have not been fully elucidated. The combination of biochemical and structural data suggests that initial binding may occur in the central cavity or at the lipid-protein interface. Our objective was to define the binding sites for two transported substrates of Pgp; the anticancer drug vinblastine and the fluorescent probe rhodamine 123. A series of mutations was generated in positions proximal to previously defined drug-interacting residues on Pgp. The protein was purified and reconstituted into styrene-maleic acid lipid particles (SMALPs) to measure the apparent drug binding constant or into liposomes for assessment of drug-stimulated ATP hydrolysis. The biochemical data were reconciled with structural models of Pgp using molecular docking. The data indicated that the binding of rhodamine 123 occurred predominantly within the central cavity of Pgp. In contrast, the significantly more hydrophobic vinblastine bound to both the lipid-protein interface and within the central cavity. The data suggest that the initial interaction of vinca alkaloids with Pgp occurs at the lipid interface followed by internalisation into the central cavity, which also provides the transport conduit. This model is supported by recent structural observations with Pgp and early biophysical and cross-linking approaches. Moreover, the proposed model illustrates that the broad substrate profile for Pgp is underpinned by a combination of multiple initial interaction sites and an accommodating transport conduit.

Binding Constants of Clinical Drugs and Other Organic Ligands with Human and Mammalian Serum Albumins

A dataset containing the experimental values of the equilibrium binding constants of clinical drugs, and some other organic ligands with human and mammalian (predominantly bovine) serum albumins, is assembled. The affinity of drugs to albumin governs their pharmacokinetic properties, related to permeability through physiological barriers and distribution within the organism. The dataset contains 1755 records gathered from 346 original literature sources describing the albumin affinity of 324 different substances. The data were extracted from both articles and existing protein-binding databases applied strict data selection rules in order to exclude the values influenced by the third-party compounds. The dataset provides the details on the experimental conditions of the measurements, such as temperature; protein and ligand concentrations; buffer pH, composition and concentration; and the method and model used for the binding constant calculations. Analysis of the data reveals discrepancies between the values from different studies, as well as the significant influence of the measurement method. Averaging the values from multiple independent measurements from the dataset may help to determine the reliable values of the binding constants. The dataset can be used as the reference dataset for the development of predictive models to calculate binding constants, and as the choice for the experimental setup in the future albumin-binding studies.

Development of an on-line immunoextraction/entrapment system for protein capture and use in drug binding studies by high-performance affinity chromatography

An on-line purification and entrapment system was developed that could extract a protein from a sample such as serum and entrap this protein within a small column for use in high-performance affinity chromatography. Human serum albumin (HSA) was employed as a model protein for this work. Immunoextraction columns containing polyclonal anti-HSA antibodies were developed to capture and isolate HSA from applied samples. This was followed by the use of a strong cation-exchange column to recapture and focus HSA as it eluted from the immunoextraction columns. The recaptured HSA was entrapped within 1.0 cm × 2.1 mm I.D. columns containing hydrazide-activated silica and in the presence of oxidized glycogen as a capping agent. The binding and elution properties of HSA on the various components of this system were examined and optimized. The entrapped columns produced by this system were then evaluated for their use in binding studies with several sulfonylurea drugs. The HSA columns created by this approach typically contained 0.3–0.6 nmol HSA and were stable over several weeks and more than 50–60 sample injections. Drug binding constants could be determined with these columns in 8 min or less by zonal elution and gave good agreement with literature values. The same system could be used for the capture and entrapment of other proteins by utilizing antibodies against the given target for immunoextraction.

Study on the interaction of anti-inflammatory drugs with human serum albumin using molecular docking, quantitative structure-activity relationship, and fluorescence spectroscopy.

The interaction of 14 anti-inflammatory drugs with human serum albumin (HSA) was investigated using fluorescence quenching, molecular docking studies, and quantitative structure-activity relationship (QSAR) methodology. Binding of anti-inflammatory drugs to HSA plays a fundamental role in their transport, distribution, delivery, and elimination. Binding constants of these drugs to HSA, obtained using the fluorescence quenching method, were within the range 0.01×104 M-1 (acetaminophen) to 1881.05×104 M-1 (meloxicam). Binding sites and binding constants of these anti-inflammatory drugs were estimated using molecular docking. Inspection of the obtained values for docking score, logKb and Kb , showed that the drugs in this data set have a relatively strong binding constant for HSA. QSAR modelling was applied for binding constants obtained from fluorescence quenching and theoretical molecular descriptors. This modelling led to a linear two-parameter model with a correlation coefficient of 0.95 and adequate robustness. The descriptor results showed the importance of a bonding network and electronegativity as the discriminative structural factors in binding affinity for the HSA molecule.

Nanohydrogels Based on Self-Assembly of Cationic Pullulan and Anionic Dextran Derivatives for Efficient Delivery of Piroxicam.

A cationic derivative of pullulan was obtained by grafting reaction and used together with dextran sulfate to form polysaccharide-based nanohydrogel cross-linked via electrostatic interactions between polyions. Due to the polycation-polyanion interactions nanohydrogel particles were formed instantly and spontaneously in water. The nanoparticles were colloidally stable and their size and surface charge could be controlled by the polycation/polyanion ratio. The morphology of the obtained particles was visualized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The resulting structures were spherical, with hydrodynamic diameters in the range of 100–150 nm. The binding constant (Ka) of a model drug, piroxicam, to the cationic pullulan (C-PUL) was determined by spectrophotometric measurements. The value of Ka was calculated according to the Benesi—Hildebrand equation to be (3.6 ± 0.2) × 103 M−1. After binding to cationic pullulan, piroxicam was effectively entrapped inside the nanohydrogel particles and released in a controlled way. The obtained system was efficiently taken up by cells and was shown to be biocompatible.

Karvedilolun Çözünürlüğünü Arttırıcı Olarak Jeffamine® Çekirdekli PAMAM Dendrimerler

The aim of this study was to investigate the effect of Jeffamine® core poly(amidoamine) PAMAM dendrimers (JCPDs) on the aqueous solubility of carvedilol (CAR), a Biopharmaceutical Classification System (BCS) Class II drug, and a nonselective beta-adrenegenic blocking agent with alpha 1-blocking activity. The aqueous solubility of CAR was measured in the presence of JCPDs at room temperature in phosphate-buffered saline using traditional rotating bottle technique. Results obtained from the phase solubility studies revealed that the molar aqueous solubility of CAR increased significantly with a proportional increase in the concentration of fourth-generation JCPD, P4.NH2. Likewise, the encapsulation efficiency of JCPD, P4.NH2 improved as its concentration increased and the highest capacity was observed to be 60.75%. Furthermore, the drug binding constant of P4.NH2 (11177.31 ± 0.15 M-1) was found to be fifty times higher than that of β-cyclodextrin (227 M-1), which is the most common studied solubility enhancer excipient for CAR drug. Overall, it can be concluded that PAMAMs, used for the first time in this study as the successful solubility enhancer of CAR, might be helpful and good candidates for the development of various formulations in the future studies.

Chiral probes for α1-AGP reporting by species-specific induced circularly polarised luminescence.

Luminescence spectroscopy has been used to monitor the selective and reversible binding of pH sensitive, macrocyclic lanthanide complexes, [LnL1], to the serum protein α1-AGP, whose concentration can vary significantly in response to inflammatory processes. On binding α1-AGP, a very strong induced circularly-polarised europium luminescence signal was observed that was of opposite sign for human and bovine variants of α1-AGP - reflecting the differences in the chiral environment of their drug-binding pockets. A mixture of [EuL1] and [TbL1] complexes allowed the ratiometric monitoring of α1-AGP levels in serum. Moreover, competitive displacement of [EuL1] from the protein by certain prescription drugs could be monitored, allowing the determination of drug binding constants. Reversible binding of the sulphonamide arm as a function of pH, led to a change of the coordination environment around the lanthanide ion, from twisted square antiprism (TSAP) to a square antiprismatic geometry (SAP), signalled by emission spectral changes and verified by detailed computations and the fitting of NMR pseudocontact shift data in the sulphonamide bound TSAP structure for the Dy and Eu examples. Such analyses allowed a full definition of the magnetic susceptibility tensor for [DyL1].

Adsorptive and affinity linear sweep voltammetry for the determination of singulair in tablets and its interaction with human serum albumin

A new simple, sensitive and reliable adsorptive and affinity linear sweep voltammetric method at in situ mercury film electrode for the determination of singulair in tablets and its interaction with human serum albumin (HSA) was investigated. The solubility of singulair in aqueous media was enhanced by inclusion with hydroxypropyl-β-cyclodextrin (HPβCD). The nature of the electrochemical process of singulair was studied by affinity cyclic voltammetry (ACV). Reproducibility of the proposed method was checked giving interday precision of 0.073 standard deviation. The limit of detection and limit of quantification were 0.05 and 0.16 fg/L, respectively. In the present work, singulair was interacted with HSA by 1: 1 stoichiometry to form electroinactive supramolecular complex. The binding constant was precisely estimated by coupling affinity linear sweep voltammetry (ALSV) and ACV with non-linear regression analysis based on the shifting of analyte peak potentials. The proposed experiments and data analysis could be used to investigate the binding constant of drug with protein within one hour.

Polymeric nanomicelles for sustained delivery of anti-cancer drugs

In the first section of this paper, the existing and emerging nanotechnology-based cancer therapies – nanoparticles, drug conjugates, nanomicelles – are reviewed. In a second part, we present our original and unpublished findings on the sustained release of anti-cancer drugs such as paclitaxel, doxorubicin and camptothecin using block copolymer micelles [PEG-b-poly(dioxanone-co-methyl dioxanone)]. Copolymers with variable lengths of hydrophobic and hydrophilic blocks have been synthesized and successfully loaded with paclitaxel, doxorubicin and camptothecin anti-cancer drugs, with micelles size in the range 130–300nm. Drug encapsulation efficiencies varied between 15% and 70% depending on drug and copolymer composition. The drug binding constants, which give a good insight into drug encapsulation and release, were evaluated from UV spectroscopy as we reported previously for anti-TB drugs. Through variation of the methyl dioxanone content of the copolymer, our systems can be tailored for sustained release of the different drugs.

Binding of benzodiazepine drugs to bovine serum albumin: A second derivative spectrophotometric study

The binding constants ( K values) of three benzodiazepine drugs to bovine serum albumin were determined by a second derivative spectrophotometric method. Despite the sample and reference samples were prepared in the same way to maintain the same albumin content in each sample and reference pair, the absorption spectra show that the baseline compensation was incomplete because of the strong background signals caused by bovine serum albumin. Accordingly, further quantitative spectral information could not be obtained from these absorption spectra. On the other hand, the calculated second derivative spectra clearly show isosbestic points indicating the complete removal of the residual background signal effects. Using the derivative intensity differences (Δ D values) of the studied benzodiazepine drugs before and after the addition of albumin, the binding constants were calculated and obtained with R.S.D. of less than 8%. The interactions of drugs with bovine serum albumin were investigated using Scatchard's plot. In addition, the consistency between the fractions of bound benzodiazepine calculated from the obtained K values and the experimental values were established. The results indicate that the second derivative method can be advantageously applicable to the determination of binding constants of drugs to serum albumin without prior separation. Moreover, the validity of the proposed method was confirmed.

Cationic Polyelectrolytes as Drug Delivery Vectors: Calorimetric and Fluorescence Study of Rutin Partitioning

The interaction between hydrophobically modified cationic polysaccharides based on dextran and a flavonoid drug (Rutin) was studied by isothermal titration calorimetry (ITC) and fluorescence spectroscopy, in order to assess the factors responsible for the interaction and characterize its energetics, as well as for evaluating their encapsulation capacity, for possible applicability of these polymers as drug delivery vectors. To address the importance of the hydrophobic pendant groups in the solution behavior of these polymer/drug systems, we also studied the interaction of Rutin with a cationic surfactant, cetyltrimethylammonium chloride (CTAC). The interaction enthalpies and drug binding constants for D40R30/Rutin systems were derived from ITC through a simple binding model. The binding constants were independently derived from fluorescence results, with fair agreement between the parameters obtained from both methods. By changing the Rutin concentration, we were able to get evidence for a solubility enhancement induced by the presence of the polymers, a promising effect regarding its use to improve bioavailability.

Chloramphenicol binding to human serum albumin: Determination of binding constants and binding sites by steady-state fluorescence

The interaction between chloramphenicol and human serum albumin (HSA) was studied by fluorescence, UV/vis, circular dichroism (CD) and three-dimensional fluorescence spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by chloramphenicol was the result of the formation of drug–HSA complex, and the effective quenching constants ( K a) were 2.852 × 10 4, 2.765 × 10 4, 2.638 × 10 4 and 2.542 × 10 4 M −1 at 287, 295, 303 and 311 K, respectively. The thermodynamic parameters, enthalpy change (Δ H) and entropy change (Δ S) for the reaction were calculated to be −3.634 kJ mol −1 and 72.66 J mol −1 K −1 according to van’t Hoff equation. The results indicated that the hydrophobic and electrostatic interactions played a major role in the binding of drug to HSA. The distance r between donor and acceptor was obtained to be 3.63 nm according to Förster’s theory. Site marker competitive experiments indicated that the binding of drug to HSA primarily took place in subdomain IIA. The alterations of HSA secondary structure in the presence of chloramphenicol were confirmed by the evidences from synchronous fluorescence, CD and three-dimensional fluorescence spectra. In addition, the effect of common ions on the binding constants of drug–HSA complex was also discussed.

Derivative Spectrophotometric and NMR Spectroscopic Study in Pharmaceutical Science

This review starts with an introduction of derivative spectrophotometry followed by a description on the construction of a personal computer-assisted derivative spectrophotometric (DS) system. An acquisition system for inputting digitalized absorption spectra into personal computers and a BASIC program for calculating derivative spectra were developed. Then, applications of the system to drug analyses that are difficult with traditional absorption methods are described. Following this, studies on the interactions of drugs with biological macromolecules by the DS and NMR methods were discussed. An (1)H NMR study elucidated that the small unilamellar vesicle (SUV) has a single membrane made of a phosphatidylcholine bilayer, and that chlorpromazine interacts with both the outer and inner layers. (13)C NMR revealed a reduction of the dissociation constants of phenothiazine drugs due to their interaction with SUV. The partition coefficients of phenothiazine, benzodiazepine and steroid drugs in an SUV-water system and the effects of cholesterol or amino lipids content on these partition coefficients were examined by the DS method. The binding constants of phenothiazine drugs to bovine serum albumin (BSA) and the influence of Na(+), K(+), Cl(-), Br(-), and I(-) on these binding constants were determined by DS. It was found that I(-), Br(-), Cl(-) reduce the binding constants in this order, and that Na(+) and K(+) have no effect. A (19)F NMR study revealed that triflupromazine binds to BSA and human serum albumin in two regions including Site II with different populations, and that a nonsteroidal anti-inflammatory drug, niflumic acid, binds Sites Ia and Ib.

Aspirin Interaction With Ribonuclease A

Aspirin is an anti-inflammatory drug and a main source of protein acetylation that can alter enzymatic activity and protein functions. Ribonuclease A (RNase A) with several high-affinity binding sites is a possible target for many organic and inorganic molecules (Leonidas at al., [2003] Protein Sci. 12, 2559-2574). This study was designed to examine the interaction of aspirin with RNase Aat physiologic conditions. Reaction mixtures of constant protein concentration (3 mM) and different aspirin contents (0.0002-2 mM) are studied by ultraviolet-visible, Fourier transform infrared, and circular dichroism spectroscopic methods to determine the drug binding mode, the drug-binding constant, and the effects of drug complexation on the protein conformation in aqueous solution. Spectroscopic results showed one major binding for the aspirin-RNase complexes with overall binding constant of K = 3.57 x 10(4) M-1. Minor reductions in the protein alpha-helix from 15.5 to 14.1% (circular dichroism) using CDPro program and 26 to 21% (infrared) were observed on aspirin interaction. The changes are indicative of some degree of protein unfolding on drug complexation.

Effects of Inorganic Ions on the Binding of Triflupromazine and Chlorpromazine to Bovine Serum Albumin Studied by Spectrometric Methods

The effects of inorganic salts, NaCl, NaBr, NaI, Na2SO4, KCl, KBr, KI, on the binding constants (Ks) of psychotropic phenothiazine drugs, triflupromazine (TFZ) and chlorpromazine, to bovine serum albumin (BSA) were examined by using second-derivative spectrophotometry. All of the salts examined, with the exception of Na2SO4, decreased the K values significantly, depending on the concentration of the salt, e.g., the decrease in the K values of both drugs were about 40% for 0.1 M NaCl. The results obtained with Na2SO4 indicated that neither Na+ nor SO4(2-) had any affect on the binding of the phenothiazines to BSA. Based on the Na2SO4 results and the finding that the effect of each potassium salt on binding was quite similar to that of the corresponding sodium salt, the effects of these halogen salts can be considered to be derived from their anions, although the phenothiazines are positively charged at pH 7.4. The effectiveness of the anions was determined to occur in the following order: I->>Br->Cl-; these results coincided with the published order of the binding affinity of these anions to albumin. The 19F-NMR spectra of TFZ in the presence of each of these halogen salts revealed a concentration-dependent decrease in the intensity of the signal at 13.8 ppm that had previously been assigned to the TFZ bound to Site II. Consequently, the effects of these anions on the binding of positively charged phenothiazine drugs are thought to be local steric effects caused by the binding of these anions to Site II.

AZT Binding to Na,K-ATPase

3'-azido-3'-deoxythymidine (AZT) is the first effective drug used clinically for the treatment of human immunodeficiency virus (HIV) infection. The drug interactions with DNA and protein are associated with its mechanism of action in vivo. This study was designed to examine the interaction of AZT with the Na,K-dependent adenosine triphosphatase (Na,K-ATPase) in H2O and D2O solutions at physiological pH using drug concentration of 0.1 microM to 1 mM and final protein concentration of 0.5 to 1 mg/mL. Ultraviolet absorption and Fourier transform infrared difference spectroscopy with its self-deconvolution, second-derivative resolution enhancement, and curve-fitting procedures were used to characterize the drug-binding mode, the drug-binding constant, and the effects of drug interaction on the protein secondary structure. Spectroscopic evidence showed that at low drug concentration (0.1 microM), AZT binds (H-bonding) mainly to the polypeptide C=O and C-N groups with two binding constants of K1 = 5.3 x 10(5) M(-1) and K2 = 9.8 x 10(3) M(-1). As drug content increased, AZT-lipid complex prevailed. At a high drug concentration (1 mM), drug binding resulted in minor protein secondary structural changes from that of the alpha-helix 19.8%; beta-pleated 25.6%; turn 9.1%; beta-antiparallel 7.5% and random 38%, in the free Na,K-ATPase to that of the alpha-helix 19%; beta-pleated 21.1%; turn 10.1%; beta-antiparallel 8.8% and random 41%, in the AZT-ATPase complexes.

Atypical Antipsychotics: Mechanism of Action

Atypical Antipsychotics: Mechanism of Action

3'-azido-3'-deoxythymidine binding to ribonuclease A: model for drug-protein interaction.

Ribonuclease A (RNase A) with several high affinity binding sites is a possible target for many organic and inorganic molecules. 3'-Azido-3'-deoxythymidine (AZT) is the first clinically effective drug for the treatment of human immunodeficiency virus (HIV) infection. The drug interactions with protein and nucleic acids are associated with its mechanism of action in vivo. This study was designed to examine the interaction of AZT with RNase A under physiological conditions. Reaction mixtures of constant protein concentration (2%) and different drug contents (0.0001-0.1 mM) are studied by UV-visible, FTIR, and circular dichroism spectroscopic methods in order to determine the drug binding mode, the drug binding constant, and the effects of drug complexation on the protein and AZT conformations in aqueous solution. The spectroscopic results showed one major binding for the AZT-RNase complexes with an overall binding constant of 5.29 x 10(5) M(-1). An increase in the protein alpha helicity was observed upon AZT interaction, whereas drug sugar pucker remained in the C2'-endo/anti conformation in the AZT-RNase complexes.

Interaction of AZT with Human Serum Albumin Studied by Capillary Electrophoresis, FTIR and CD Spectroscopic Methods

The thymidine analoge 3′-azido-3′-deoxythymidine (AZT) is still one of the effective drugs against human immunodeficiency (HIV) infection. AZT has been used as inhibitor of HIV-1 reverse transcriptase, the virus encoded enzyme which catalyzes transcription of viral RNA to DNA. The drug interaction with protein has been included in its mechanism of action. Human serum albumin (HSA) is a carrier of many drugs in vivo and thus AZT-HSA complexation can serve as a model for drug-protein interaction. This study was designed to examine the interaction of AZT with human serum albumin at physiological conditions using constant protein concentration (0.2% or 2%) and different drug contents (5 to 1000 μM). Capillary electrophoresis, FTIR and CD spectroscopic methods were used to determine the drug binding mode, the drug binding constant and the effects of drug-HSA complexation on the protein and AZT conformations in aqueous solution. Capillary electrophoresis and spectroscopic results showed two major bindings for the AZT-HSA complexes with K1=1.9 x 106 M−1 and K2= 2.1 × 104 M−1. Minor alterations of the protein secondary structure from that of the a-helix to β-sheet were observed upon drug complexation, whereas the drug sugar pucker remained in the C2′-endo/anti conformation upon protein interaction.

Determination of protein–drug binding constants by pressure-assisted capillary electrophoresis (PACE)/frontal analysis (FA)

A simple method for the determination of binding constants of drugs to human serum albumin (HSA) and α 1-acid glycoprotein (AGP) was developed by pressured-assisted capillary electrophoresis (PACE) based on the principle of frontal analysis (FA). The free drug concentration was measured from the height of the frontal peak and calculated based on the external drug standard in the absence of protein. With a known concentration of total drug, the percentage of drug bound to HSA or AGP was then determined. The binding constants of drug to HSA or AGP were obtained from non-linear curve fitting of the percentage of bound drug as a function of total protein concentration or total drug concentration. The sample was prepared by mixing known concentrations of drug and protein in phosphate buffered saline (PBS) and equilibrated for 30 min. A large volume of sample solution (∼80 nl) was injected at 1.0 psi for 40 s into the fused silica capillary, which was filled with PBS buffer. Due to the difference in charge/size ratio, the free drug was separated from the protein/protein–drug complex when 15–25 kV voltage and 0.5–1.5 psi air pressure were applied. External air pressure was used to improve the throughput, prevent protein loss, and achieve a better drug plateau. By modifying experimental conditions, a wide range of binding constants could be measured. This PACE/FA method works well for basic, neutral, and weakly acidic compounds.