Ferenc Zsila

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.

Subdomain IB is the third major drug binding region of human serum albumin: toward the three-sites model.

According to the conventional view, noncovalent association of small molecules with human serum albumin (HSA) occurs principally at the so-called Sudlows sites located in subdomain IIA and IIIA. By employing a circular dichroism (CD) spectroscopic approach, it is shown that biliverdin is the specific CD label of an additional drug binding area in subdomain IB. CD competition experiments disclosed the entrapment of a diverse assortment of acidic, neutral, and basic molecules within subdomain IB including anticancer agents (camptothecin, doxorubicin, daunorubicin, teniposide, suramin, tyrosine kinase inhibitors), anticoagulants (dicoumarol), various steroids (bile acids, carbenoxolone), nonsteroidal antiinflammatory drugs, natural substances (aristolochic acid, glycyrrhetinic acid), and synthetic dyes (methyl orange, azocarmine B). These finding imply that subdomain IB can be considered as the third major drug binding region of HSA featured with promiscuous ligand recognition ability. Additionally, subdomain IB is allosterically coupled with the Sudlows sites, the ligand binding of which is shown to alter the HSA binding mode and affinity of biliverdin and hemin. Brief case studies are presented to illustrate how the evaluation of spectral changes of tetrapyrrole CD probes gains new insight into the HSA binding properties of endogenous as well as pharmaceutical compounds.

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.

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.

Determination of Human Serum α1-Acid Glycoprotein and Albumin Binding of Various Marketed and Preclinical Kinase Inhibitors

There are about 380 protein kinase inhibitors in drug development as of today and 15 drugs have been marketed already for the treatment of cancer. This time 139 validated kinase targets are in the focus of drug research of pharmaceutical companies and big efforts are made for the development of new, druglike kinase inhibitors. Plasma protein binding is an important factor of the ADME profiling of a drug compound. Human serum albumin (HSA) and alpha(1)-acid glycoprotein (AAG) are the most relevant drug carriers in blood plasma. Since previous literature data indicated that AAG is the principal plasma binding component of some kinase inhibitors the present work focuses on the comprehensive evaluation of AAG binding of a series of marketed and experimental kinase inhibitors by using circular dichroism (CD) spectroscopy approach. HSA binding was also evaluated by affinity chromatography. Protein binding interactions of twenty-six kinase inhibitors are characterized. The contribution of AAG and HSA binding data to the pharmacokinetic profiles of the investigated therapeutic agents is discussed. Structural, biological and drug binding properties of AAG as well as the applicability of the CD method in studying drug-protein binding interactions are also briefly reviewed.

Absolute Configuration of Two New 6-Alkylatedα-Pyrones (=2H-Pyran-2-ones) fromRavensara crassifolia

The stem bark CH2Cl2 extract of Ravensara crassifolia showed antifungal activity against the phytopathogenic fungus Cladosporium cucumerinum in a bioautographic TLC assay. Activity-guided fractionation afforded two new alpha -pyrones : (6S)-5,6-dihydro-6-[(2R)-2-hydroxy-6-phenylhexyl]-2H-pyran-2-one (1) and (6R)-6-[(4R,6R)-4,6-dihydroxy-10-phenyldec-1-enyl]-5,6-dihydro-2H-pyran-2-one (2). Their structures and absolute configurations were established by NMR spectroscopy, chemical methods, and CD spectroscopy. The antifungal activity against C. cucumerinum was determined for both compounds.

Induced chirality upon crocetin binding to human serum albumin: origin and nature

Abstract Binding to human serum albumin (HSA) of the natural, achiral carotenoid crocetin, having hypocholesterolemic and antitumour effects, was investigated in detail by circular dichroism (CD) and absorption spectroscopy. It has been shown that in the visible absorption region the crocetin–HSA complex exhibits a well-defined induced circular dichroic spectrum with two major bands of opposite sign, proving excitonic interaction between carotenoids bound in a left-handed chiral arrangement on the albumin molecule. In the course of CD titration experiments, palmitic acid gradually decreased the exciton band intensities indicating that crocetin and palmitic acid have common binding sites on HSA. To investigate potential sources of the intermolecular excitonic interaction, molecular modeling studies were performed fitting crocetin molecules to the long-chain fatty acid binding sites of HSA, determined recently by X-ray crystallographic measurements. The results suggest that binding of crocetin to domain III of the albumin might be responsible for the observed intermolecular exciton coupling. Crocetin binding was accompanied by a significant red shift in the visible absorption spectrum which has showed no excitonic contribution but rather indicates the higher polarizability of the protein environment.