Use of 19F NMR spectroscopy to screen chemical libraries for ligands that bind to proteins

Identification of compounds from chemical libraries that bind to macromolecules by use of NMR spectroscopy has gained increasing importance during recent years. A simple methodology based on (19)F NMR spectroscopy for the screening of ligands that bind to proteins, which also provides qualitative information about relative binding strengths and the presence of multiple binding sites, is presented here. A library of fluorinated compounds was assembled and investigated for binding to the two bacterial chaperones PapD and FimC, and also to human serum albumin (HSA). It was found that library members which are bound to a target protein could be identified directly from line broadening and/or induced chemical shifts in a single, one-dimensional (19)F NMR spectrum. The results obtained for binding to PapD using (19)F NMR spectroscopy agreed well with independent studies based on surface plasmon resonance, providing support for the versatility and accuracy of the technique. When the library was titrated to a solution of PapD chemical shift and linewidth changes were observed with increasing ligand concentration, which indicated the presence of several binding sites on PapD and enabled the assessment of relative binding strengths for the different ligands. Screening by (19)F NMR spectroscopy should thus be a valuable addition to existing NMR techniques for evaluation of chemical libraries in bioorganic and medicinal chemistry.     

Determination of drug–serum protein interactions via fluorescence polarization measurements

New fast methods for the determination of pharmacokinetic behaviour of potential drug candidates are receiving increasing interest. We present a new homogeneous method for the determination of drug binding and drug competition for human serum albumin and α₁-acid glycoprotein that is amenable to high-throughput-screening. It is based on selective fluorescent probes and the measurement of fluorescence polarization. This leads to decreased interference with fluorescent drugs as compared with previously published methods based on similar probes and the measurement of fluorescence intensity. The binding of highly fluorescent drugs that still interfere with the probes can be measured by simply titrating the drugs in a two-component system with the serum protein. The assay may also be used to discover strongly binding protein ligands that are interesting for drug-targeting strategies. Additionally, binding data could be obtained from larger libraries of compounds for in silico predictive pharmacokinetics. Figure Fluorescence polarization displacement titration of dansylsarcosine (3D-structure as insert) bound to human serum albumin (HSA) by naproxene Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Rational design of diflunisal analogues with reduced affinity for human serum albumin.

Many lead compounds bind to serum albumin and exhibit markedly reduced efficacy in vivo as compared to their potency in vitro. To aid in the design of compounds with reduced albumin binding, we performed nuclear magnetic resonance (NMR) structural and binding studies on the complex between domain III of human serum albumin (HSA-III) and diflunisal, a cyclooxygenase inhibitor with antiinflammatory activity. The structural studies indicate that the aromatic rings of diflunisal are involved in extensive and specific interactions with hydrophobic residues that comprise the binding pocket in subdomain IIIA. The carboxylic acid of diflunisal forms electrostatic interactions with the protein similar to those observed in the X-ray structure of HSA complexed to myristic acid. In addition to the structural studies, NMR-derived binding constants were obtained for diflunisal and closely related analogues to develop a structure-affinity relationship for binding to subdomain IIIA. On the basis of the structural and binding data, compounds were synthesized that exhibit more than a 100-fold reduction in binding to domain III of HSA, and nearly a 10-fold reduction in affinity for full length albumin. Significantly, several of these compounds maintain activity against cyclooxygenase-2. These results suggest a rational strategy for designing out albumin binding in potential drug molecules by using structure-based design in conjunction with NMR-based screening.     

Characterization of interaction property of multicomponents in Chinese Herb with protein by microdialysis combined with HPLC

Interaction of traditional Chinese Herb Rhizoma Chuanxiong and protein was studied by microdialysis coupled with high performance liquid chromatography. Compounds in Rhizoma Chuanxiong, such as ferulic acid, senkyunolide A and 3-butylphthalide, were identified by HPLC, HPLC-MS and UV-vis. Microdialysis recoveries and binding degrees of compounds in Rhizoma Chuanxiong with human serum albumin (HSA) and other human plasma protein were determined: recoveries of microdialysis sampling ranged from 36.7 to 98.4% with R.S.D. below 3.1%; while binding to HSA ranged from 0 to 91.5% (0.3 mM HSA) and from 0 to 93.5% (0.6 mM HSA), respectively. Compared with HSA, most of compounds bound to human blood serum more extensively and the results showed that binding of these compounds in Rhizoma Chuanxiong was influenced by pH. Two compounds were found to bind to HSA and human blood serum, their binding degrees were consistent with ferulic acid and 3-butylphthalide, the active compounds in Rhizoma Chuangxiong.     

Fluorescence Spectroscopic Studies on the Interaction of Oleanolic Acid and its Triterpenoid Saponins Derivatives with Two Serum Albumins

The interaction of oleanolic acid (OA) and its glycosylated derivatives (LL-2 and LL-4) with human and bovine serum albumins were investigated using the methods of fluorescence spectroscopy. The spectroscopic analysis of the fluorescence quenching that occurs when OA and its derivatives interact with serum albumin indicates that these quenching constants are inversely correlated with temperature and the quenching process involves static interactions. The binding affinity of OA and OA-derived compounds to bovine serum albumin (BSA) and human serum albumin (HSA) follow the trend LL-4 > LL-2 > OA, suggesting that glycosylation of OA can facilitate its binding to serum albumins. Additionally, the binding affinity of these compounds to HSA is stronger than it is to BSA. The calculated thermodynamic parameters suggest that hydrophobic interactions dominate these interaction processes. We also found that only a single type of binding site exists for OA and its derivatives to HSA and BSA. Synchronous fluorescence results indicate that the binding of OA, LL-2 and LL-4 to BSA and HSA can lead to the conformational changes around the tryptophan residues of the two serum albumins. These results provided valuable clues to the pharmacokinetics and the pharmacologic activities of OA and its types of triterpenoid saponins derivatives.     

Evalution of capillary electrophoresis-frontal analysis for the study of low molecular weight drug-human serum albumin interactions

Capillary electrophoresis frontal analysis was applied to 12 low molecular weight compounds including 8 drug substances displaying a range of different properties with respect to binding affinity, binding location, structure, lipophilicity, charge at physiological pH, and electrophoretic mobility. It was found that capillary electrophoresis frontal analysis can be used as a general method to study and quantify drug-human serum albumin interactions. The binding parameters obtained were consistent with literature values. Dextran was in some cases added to the run buffer to improve separation of the drug and human serum albumin plateau peaks. Results indicate that mobility differences between free and complexed human serum albumin give rise to only minor errors. Capillary electrophoresis frontal analysis was also found applicable to the study of human serum albumin drug displacement reactions. Low sensitivity of the UV-detection system was found to be the major limitation of capillary electrophoresis frontal analysis. The method is simple, and minimal effort has to be put into method development, which makes it well suited for screening in early drug development.     

Characterization of interactions between polyphenolic compounds and human serum proteins by capillary electrophoresis

The interaction of ten natural polyphenolic compounds (chlorogenic acid, apigenin, catechin, epicatechin, flavanone, flavone, quercetin, rutin, vicenin-2 and vitexin) with human serum albumin and mixtures of human serum albumin and α₁-acid glycoprotein under near physiological conditions is studied by capillary electrophoresis–frontal analysis. Furthermore, the binding of these polyphenolic compounds to total plasmatic proteins is evaluated using ultrafiltration and capillary electrophoresis. In spite of the relatively small differences in the chemical structures of the compounds studied, large differences were observed in their binding behaviours to plasmatic proteins. The hydrophobicity, the presence/absence of some functional groups, steric hindrance and spatial arrangement seem to be key factors in the affinity of natural polyphenols towards plasmatic proteins.     

Binding of glycyrrhizin to human serum and human serum albumin

The binding of glycyrrhizin (GLZ) to human serum and human serum albumin (HSA) was examined by an ultrafiltration technique. Specific and nonspecific bindings were observed in both human serum and HSA. The association constants (K) for the specific bindings were very similar: 1.31 x 10(5) M-1 in human serum and 3.87 x 10(5) M-1 in HSA. The number of binding sites (n) and the linear binding coefficient (phi) in HSA were 1.95 and 3.09 x 10(3) M-1, respectively. When the human serum protein concentration was assumed to be 4.2% (equal to the measured serum albumin concentration), n in human serum was 3.09, which is similar to the n value in HSA, and phi in human serum was 0.71 x 10(3) M-1, which is reasonably close to that for HSA. The binding pattern of GLZ with human serum protein on Sephadex G-200 column chromatography showed that GLZ binds to only the albumin fraction. It was concluded that the GLZ-binding sites in human serum exist mainly on albumin and GLZ binds to specific and nonspecific binding sites at lower and higher concentrations than approximately 2 mM, respectively.     

A combinatorial approach to obtain affinity media with binding properties towards the aflatoxins

In our work we performed a combinatorial solid-phase synthesis in aqueous medium to prepare peptide libraries from which to select an amino acid sequence with binding properties towards aflatoxins.We used polystyrene beads, functionalised with carboxylic groups as solid support and eight amino acids as monomers. During the first step 64 different sequences of two amino acids were prepared by exploiting the principles of combinatorial chemistry; then the binding properties of all sequences towards aflatoxin B(1 )were checked. We determined binding constants towards aflatoxin B(1) and towards aflatoxins B(2), G(1) and G(2). Results were promising, so we prepared a new library by using the selected dipeptide as the starting solid phase. After selecting the best tetrapeptide sequences, we determined binding constants towards the quoted aflatoxins. We obtained binding constants ( K>10(4) M(-1)) similar to those shown by human serum albumin for similar compounds. Preliminary studies on an extraction column were promising for the development of an SPE system and for its application in food matrices.     

Bilirubin/rat serum albumin interaction

Essential differences are demonstrated between bilirubin binding to rat serum proteins and to albumin in human serum. Acidimetric titration of rat serum with and without added bilirubin shows that binding of bilirubin acid in the range of pH from 6.8 to 8.8 takes place with release of less than one hydrogen ion per molecule of bound bilirubin. With human serum, two hydrogen ions are released, indicating binding of bilirubin dianion. The binding equilibrium of N-[4-[(4-aminophenyl)-sulfonyl]phenyl]-acetamide (MADDS) to rat serum albumin is influenced slightly by cobinding of bilirubin whereas MADDS and bilirubin bind competitively to human serum albumin. Finally, the rate of oxidation of bilirubin with hydrogen peroxide and peroxidase is decreased moderately by addition of rat serum albumin and strongly by the human protein, indicating that biliribin in its complex with rat serum albumin is subject to oxidation while the complex with human serum albumin is protected. These differences should be considered when rats are used as a model in experimental studies aiming at prevention of bilirubin encephalopathy in human neonates.     

Competition binding experiments for rapidly ranking lead molecules for their binding affinity to human serum albumin

Many lead molecules that have high affinity for a therapeutic target in vitro exhibit a reduced efficacy in vivo. Drug binding to human serum albumin is a major contributor to this reduction in potency, and many drug discovery programs expand significant resources preparing compounds that have decreased albumin binding. As rational and structure-based approaches have already been demonstrated to design compounds that have reduced affinity for albumin, the ability to rapidly and accurately assess protein binding will be valuable in lead optimization. This review will summarize some of the NMR-based efforts towards developing universal, rapid, accurate, and site-specific assays for estimating protein binding.     

表面等离子体共振生物传感器研究人参皂甙与血清白蛋白的相互作用

To use a newly developed wavelength modulation surface plasmon resonance （SPR） biosensor, an experimental protocol was developed to investigate the interaction of ginsenosides with serum albumin. With a known concentration of the ginsenosides, bound percentages of the ginsenosides with human serum albumin （HSA） or bovine serum albumin （BSA） were obtained. SPR technique could require no labeling and this method provided the detailed information on association and disassociation of molecules in real time. The results indicate that the sensitivity of wavelength modulation SPR biosensor is sufficient for detection and characterization of binding events involving low-molecular weight compounds and their immobilized protein targets.     

Thiophilic interaction chromatography of serum albumins

An investigation of the binding of native and recombinant human serum albumin and bovine serum albumin on three thiophilic gels, PyS, 2S, and 3S was performed. In addition to these proteins, we studied serum albumins from several species such as goat, rabbit, guinea pig, rat, hamster, baboon, and pig. Our results reveal that recombinant human serum albumin (rHSA) binds completely to PyS whereas native human serum albumin and bovine serum albumin bind only partially to PyS. The binding affinities of rHSA, human serum albumin and bovine serum albumin to 2S and 3S gels are less than their binding to PyS. Serum albumins from goat, rabbit, guinea pig, rat, hamster, baboon, and pig bind much stronger to 3S gel than human and bovine serum albumins. The binding of pig and hamster serum albumins is stronger than that of rat, goat, baboon, and rabbit.     

Studies on Interactions of Antibiotics with Serum Albumin by Surface Plasmon Resonance Biosensor

Introduction Humanserumalbumin(HSA)isawell known transportproteinforavarietyofmoleculesandions[1].Thebindingofadrugtoserumalbuminhasimportant pharmacokineticconsequencesbecauseitinfluences distribution,excretionandpharmacologicaleffectsof thedruginthebody…     

Isolation of clobazam-orosomucoid complexes from patients' sera.

Orosomucoid, a member of the lipocalin family, may function in the in vivo transport of lipophilic compounds such as basic and neutral drugs. We describe the identification of 7-chloro-1-methyl-1,5-benzodiazepine-2,4-dione (clobazam) bound to the serum orosomucoid from individuals actively taking this tranquillizer. This suggests not only that other endogenous factors limit access to the benzodiazepine binding site on human serum albumin, but also that the differential binding of benzodiazepines and their metabolites by orosomucoid should be considered in determining therapeutic doses, particularly in the acute phase response.     

Droplet microfluidics with magnetic beads: a new tool to investigate drug–protein interactions

In this study, we give the proof of concept for a method to determine binding constants of compounds in solution. By implementing a technique based on magnetic beads with a microfluidic device for segmented flow generation, we demonstrate, for individual droplets, fast, robust and complete separation of the magnetic beads. The beads are used as a carrier for one binding partner and hence, any bound molecule is separated likewise, while the segmentation into small microdroplets ensures fast mixing, and opens future prospects for droplet-wise analysis of drug candidate libraries. We employ the method for characterization of drug–protein binding, here warfarin to human serum albumin. The approach lays the basis for a microfluidic droplet-based screening device aimed at investigating the interactions of drugs with specific targets including enzymes and cells. Furthermore, the continuous method could be employed for various applications, such as binding assays, kinetic studies, and single cell analysis, in which rapid removal of a reactive component is required.     

Characterization of the Interaction of 6-Thioguanine with Human Serum Albumin by Surface-Enhanced Raman Scattering and Molecular Modeling

The interaction of 6-thioguanine and human serum albumin was investigated by fluorescence, ultraviolet-visible absorption, and surface-enhanced Raman scattering. The fluorescence of human serum albumin decreased with the concentration of 6-thioguanine, and the fluorescence quenching of human serum albumin by 6-thioguanine was static. Molecular modeling showed that 6-thioguanine was located in the hydrophobic cavity in subdomain IIA of human serum albumin. Surface-enhanced Raman scattering was combined with density function theory to characterize the orientation of 6-thioguanine on gold and the 6-thioguanine functional groups bonded to human serum albumin. The 6-thioguanine was shown to be tilted on the gold surface by a N-C?S moiety. The binding sites of 6-thioguanine to human serum albumin were the NH and amino groups of the pyrimidine ring of 6-thioguanine. This study may provide information regarding the metabolism of anticancer pharmaceuticals in the human body and assist in the development of effective compounds.     

The TcO4-binding to human serum albumin --the stoichiometry and characteristics of the binding equilibrium.

The stoichiometry and the characteristics of the TcO4-binding equilibrium to human serum albumin were investigated by the use of 99TcO4-. Based on the Scatchard plots, the number of binding sites and the association constants were obtained at pH 7.38,6.04, 5.10, 4.18, 2.80 and 0.65, respectively. From the parameters at pH 7.38, it was estimated that 64% of TcO4- added was bounded to human serum albumin under physiological condition. Variance in the values at pH 7.38, 6.04 and 5.10 shows that these bindings are stabilized by electrostatic forces. Below pH 4.18, the number of binding sites increased and the association constants constants diminished. These phenomena may be attributed to the conformation change of human serum albumin.