Zsolt Bikádi

Probing the binding of the flavonoid, quercetin to human serum albumin by circular dichroism, electronic absorption spectroscopy and molecular modelling methods.

The plant derived flavonoid compound quercetin, possesses wide range of biological activities in the human body by interacting with nucleic acids, enzymes and other proteins. As has recently been shown this molecule of polyphenolic type extensively binds to human serum albumin (HSA), the most abundant carrier protein in the blood. Electronic absorption, circular dichroism (CD) spectroscopy and molecular modelling methods were used to characterize optical properties of the quercetin-HSA complex, and to gain information on the binding mechanism at molecular level. The red shift and hypochromism of the longest-wavelength absorption band of quercetin relative to the spectral properties in ethanol suggests that one or more phenolic OH groups of the bound ligand is ionized and that the exocyclic phenyl ring is not coplanar with the benzopyrone moiety. It was found that quercetin shows extrinsic optical activity on interaction with HSA. The induced CD spectra were utilized to calculate the association constant at 37 degrees (1.46+/-0.21 x 10(4)M(-1)) and to probe the ligand binding site. Results of the CD displacement experiments performed with palmitic acid and salicylate were interpreted together with the findings of molecular modelling calculation performed on the quercetin-HSA complex. Computational mapping of possible binding sites of quercetin revealed the molecule to be bound in the large hydrophobic cavity of subdomain IIA. The protein microenvironment of this site was found to be rich in polar (basic) amino acid residues which are able to help to stabilize the negatively charged ligand bound in non-planar conformation. Additionally, the position of quercetin within the binding pocket allows simultaneous binding of other ligands such as warfarin, or sodium salycilate.

Application of the PM6 semi-empirical method to modeling proteins enhances docking accuracy of AutoDock.

BackgroundMolecular docking methods are commonly used for predicting binding modes and energies of ligands to proteins. For accurate complex geometry and binding energy estimation, an appropriate method for calculating partial charges is essential. AutoDockTools software, the interface for preparing input files for one of the most widely used docking programs AutoDock 4, utilizes the Gasteiger partial charge calculation method for both protein and ligand charge calculation. However, it has already been shown that more accurate partial charge calculation - and as a consequence, more accurate docking- can be achieved by using quantum chemical methods. For docking calculations quantum chemical partial charge calculation as a routine was only used for ligands so far. The newly developed Mozyme function of MOPAC2009 allows fast partial charge calculation of proteins by quantum mechanical semi-empirical methods. Thus, in the current study, the effect of semi-empirical quantum-mechanical partial charge calculation on docking accuracy could be investigated.ResultsThe docking accuracy of AutoDock 4 using the original AutoDock scoring function was investigated on a set of 53 protein ligand complexes using Gasteiger and PM6 partial charge calculation methods. This has enabled us to compare the effect of the partial charge calculation method on docking accuracy utilizing AutoDock 4 software. Our results showed that the docking accuracy in regard to complex geometry (docking result defined as accurate when the RMSD of the first rank docking result complex is within 2 Å of the experimentally determined X-ray structure) significantly increased when partial charges of the ligands and proteins were calculated with the semi-empirical PM6 method.Out of the 53 complexes analyzed in the course of our study, the geometry of 42 complexes were accurately calculated using PM6 partial charges, while the use of Gasteiger charges resulted in only 28 accurate geometries. The binding affinity estimation was not influenced by the partial charge calculation method - for more accurate binding affinity prediction development of a new scoring function for AutoDock is needed.ConclusionOur results demonstrate that the accuracy of determination of complex geometry using AutoDock 4 for docking calculation greatly increases with the use of quantum chemical partial charge calculation on both the ligands and proteins.

Structure and Function of the Human Breast Cancer Resistance Protein (BCRP/ABCG2)

The human breast cancer resistance protein (BCRP/ABCG2) is the second member of the G subfamily of the large ATP-binding cassette (ABC) transporter superfamily. BCRP was initially discovered in multidrug resistant breast cancer cell lines where it confers resistance to chemotherapeutic agents such as mitoxantrone, topotecan and methotrexate by extruding these compounds out of the cell. BCRP is capable of transporting non-chemotherapy drugs and xenobiotiocs as well, including nitrofurantoin, prazosin, glyburide, and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. BCRP is frequently detected at high levels in stem cells, likely providing xenobiotic protection. BCRP is also highly expressed in normal human tissues including the small intestine, liver, brain endothelium, and placenta. Therefore, BCRP has been increasingly recognized for its important role in the absorption, elimination, and tissue distribution of drugs and xenobiotics. At present, little is known about the transport mechanism of BCRP, particularly how it recognizes and transports a large number of structurally and chemically unrelated drugs and xenobiotics. Here, we review current knowledge of structure and function of this medically important ABC efflux drug transporter.

Predicting P-Glycoprotein-Mediated Drug Transport Based On Support Vector Machine and Three-Dimensional Crystal Structure of P-glycoprotein

Human P-glycoprotein (P-gp) is an ATP-binding cassette multidrug transporter that confers resistance to a wide range of chemotherapeutic agents in cancer cells by active efflux of the drugs from cells. P-gp also plays a key role in limiting oral absorption and brain penetration and in facilitating biliary and renal elimination of structurally diverse drugs. Thus, identification of drugs or new molecular entities to be P-gp substrates is of vital importance for predicting the pharmacokinetics, efficacy, safety, or tissue levels of drugs or drug candidates. At present, publicly available, reliable in silico models predicting P-gp substrates are scarce. In this study, a support vector machine (SVM) method was developed to predict P-gp substrates and P-gp-substrate interactions, based on a training data set of 197 known P-gp substrates and non-substrates collected from the literature. We showed that the SVM method had a prediction accuracy of approximately 80% on an independent external validation data set of 32 compounds. A homology model of human P-gp based on the X-ray structure of mouse P-gp as a template has been constructed. We showed that molecular docking to the P-gp structures successfully predicted the geometry of P-gp-ligand complexes. Our SVM prediction and the molecular docking methods have been integrated into a free web server (http://pgp.althotas.com), which allows the users to predict whether a given compound is a P-gp substrate and how it binds to and interacts with P-gp. Utilization of such a web server may prove valuable for both rational drug design and screening.

Circular dichroism spectroscopic studies reveal pH dependent binding of curcumin in the minor groove of natural and synthetic nucleic acids

For the first time, an interaction between the non-toxic, cancer chemopreventive agent curcumin and both natural and synthetic DNA duplexes has been demonstrated by using circular dichroism (CD) and absorption spectroscopy techniques. Upon addition of curcumin to calf thymus DNA, poly(dG-dC).poly(dG-dC) and poly(dA-dT).poly(dA-dT) solutions, an intense positive induced CD band centered around 460-470 nm was observed depending on the actual pH and Na+ ion concentration of the medium; no CD signal was obtained, however, with single stranded poly(dC). Interaction of curcumin with calf thymus DNA was observed already at pH 6.5 in contrast with poly(dG-dC).poly(dG-dC) which induces no extrinsic Cotton effect above a pH value of 5. The protonated, Hoogsteen base-paired structure of poly(dG-dC).poly(dG-dC) is necessary for curcumin binding while the alternating AT-rich polymer formed complexes with curcumin only at certain Na+ concentrations. Evaluation of the spectral data and molecular modeling calculations suggested that curcumin, this dietary polyphenolic compound binds in the minor groove of the double helix. The mechanism of the induced CD activity, the effects of the pH and Na+ ions on the ligand binding and conformation of the double helix are discussed in detail. As well as being an essentially new phenolic minor groove binder agent curcumin is also a promising molecular probe to study biologically important, pH and cation induced conformational polymorphisms of nucleic acids.

Molecular basis of the Cotton effects induced by the binding of curcumin to human serum albumin

Abstract Curcumin binding to human serum albumin (HSA) has been found recently to induce bisignate CD curves due to intramolecular exciton coupling between the two feruloyl chromophoric parts. The present study reports further results on this interaction. UV–vis and chiroptical properties of HSA-bound curcumin were analyzed in detail by comparison with bilirubin–albumin complexes. Data obtained by UV–vis and fluorescence spectroscopy, CD displacement experiments and molecular modelling methods suggested the primary binding site of curcumin to be located in site I of HSA. Since acid–base dissociation of the polyphenol type curcumin molecule plays a fundamental role in albumin binding, light absorption spectra of curcumin and half-curcumin (dehydrozingerone) were studied in ethanol and in water at different pH values. It is established that the phenolic OH group of curcumin is the most acidic and that its dissociation is responsible for both the large red-shift of the main absorption band and the binding of curcumin to HSA in a right-handed chiral conformation. Additionally, it is demonstrated that pH dependent induced CD spectra can be utilized to determine the acid–base dissociation constant; from chiroptical data the first pKa value of curcumin was calculated (8.28).

Unique, pH-dependent biphasic band shape of the visible circular dichroism of curcumin-serum albumin complex

Interaction between the plant derived polyphenolic type curcumin molecule having anticarcinogenic, antiinflammatory, and antioxidant activities, and human serum albumin was studied at different pH values by circular dichroism (CD) and electronic absorption spectroscopy. The weak, induced CD spectrum of curcumin-HSA complex measured at pH 7.4 in the visible spectral region shows striking changes upon alkalization; CD spectra collected between pH 7.7 and 9.3 exhibit characteristic, oppositely signed CD band pair according to the visible absorption band of HSA-bound curcumin. At 0.3 curcumin/HSA molar ratio, typical molar CD values are Delta epsilon (496.6nm)+40M(-1)cm(-1) and Delta epsilon (426.8nm)-40M(-1)cm(-1), respectively (pH 9.0, t=37 degrees C). The induced optical activity is attributed to a bent, right-handed chiral conformation of the HSA-bound curcumin molecule within which intramolecular exciton coupling occurs between the electric dipole transition moments of the dissymmetrically juxtaposed feruloyl chromophores. Deprotonation of phenolic OH group(s) of curcumin seems to be the reason leading to the conformational alteration of HSA-bound curcumin.

Homology modeling of breast cancer resistance protein (ABCG2)

BCRP (also known as ABCG2, MXR, and ABC-P) is a member of the ABC family that transports a wide variety of substrates. BCRP is known to play a key role as a xenobiotic transporter. Since discovering its role in multidrug resistance, considerable efforts have been made in order to gain deeper understanding of BCRP structure and function. The recent study was aimed at predicting BCRP structure by creating a homology model. Based on sequence similarity with known structures of full-length, NB and TM domain of ABC transporters, TM, NB, and linker regions of BCRP were defined. The NB domain of BCRP was modeled using MalK as a template. Based on secondary structure prediction of BCRP and comparison of the transmembrane connecting regions of known structures of ABC transporters, the TM domain arrangement of BCRP was established and was found to resemble to that of the recently published crystal structure of Sav1866. Thus, an initial alignment of TM domain of BCRP was established using Sav1866 as a template. This alignment was subsequently refined using constrains derived from secondary structure and TM predictions and the final model was built. Finally, the complete homodimer ABCG2 model was generated using Sav1866 as template. Furthermore, known ligands of BCRP were docked to our model in order to define possible binding sites. The results of molecular dockings of known BCRP substrates to the BCRP model were in agreement with recently published experimental data indicating multiple binding sites in BCRP.

Probing Protein Binding Sites by Circular Dichroism Spectroscopy

Pharmacological and pharmacodynamic properties of biologically active natural and synthetic compounds are crucially determined via their binding to proteins of the human body. Several spectroscopic techniques are available to study these mainly non-covalent interactions. Circular dichroism (CD) spectroscopy, being sensitive to the chirality of ligand molecules induced by the asymmetric protein environment, has widely and successfully been applied for many decades. Chiral conformation of the ligand due to conformational adaptation to its binding site, or interaction between ligand molecules held in chiral arrangement relative to each other by the protein sites, results in one or more induced CD bands with different shape, sign and intensity. These extrinsic Cotton effects present in light absorbing region of the optically active or inactive ligand molecules give qualitative and quantitative information of the binding process. It can provide valuable data on the stereochemistry, number, location and nature of the binding sites. This paper is aimed to survey briefly the literature and the results of recent investigations undertaken in this field.

Retinoic acid binding properties of the lipocalin member β-lactoglobulin studied by circular dichroism, electronic absorption spectroscopy and molecular modeling methods

Interaction between the Vitamin A derivative all-trans retinoic acid and the lipocalin member bovine beta-lactoglobulin (BLG) was studied by circular dichroism (CD) and electronic absorption spectroscopy at different pH values. In neutral and alkaline solutions achiral retinoic acid forms a non-covalent complex with the protein as indicated by the appearance of a negative Cotton effect around 347 nm associated to the narrowed and red shifted pi-pi(*) absorption band of the ligand. The induced optical activity is attributed to the helical distortion of the conjugated chain caused by the chiral protein binding environment. As the disappearing CD activity showed in the course of CD-pH titration experiment, retinoic acid molecules dissociate from BLG upon acidification but this release is completely reversible as proved by the reconstitution of the CD and absorption spectra after setting the pH back to neutral. This unique behavior of the complex is explained by the conformational change of BLG (Tanford transition) which involves a movement of the EF loop at the entrance of the central cavity from open to closed conformation in the course of pH lowering. From these results it was inferred that retinoic acid binds within the hydrophobic calyx of the beta-barrel.