Synthesis,characterization, HSA interaction,and antibacterial activity of a new water-soluble Pt(II) complex containing the drug cephalexin

Abstract

A new water-soluble platinum(II) complex, [Pt(CEX)Cl(DMSO)]Cl (CEX is cephalexin), was synthesized and characterized by physicochemical, spectroscopic, and computational methods. Multispectroscopic techniques were used to investigate the interaction of Pt(II) complex with human serum albumin (HSA) under the physiological conditions. The results of fluorescence titration indicated that the binding of the Pt(II) complex to HSA induced fluorescence quenching through static quenching mechanism with binding constant of 1.24?×?10⁴?M^?1 at 298?K. The thermodynamic parameters at different temperatures indicated that van der Waals forces, hydrogen bonds, and electrostatic forces play major roles in the stability of Pt(II) complex–HSA association. The displacement experiments using the site probes warfarin and ibuprofen substantiated that Pt(II) complex could bind to both site I and II of HSA. Furthermore, UV–Vis and fluorescence spectra were used to investigate the conformational changes of HSA molecule with the addition of Pt(II) complex. The binding constant of Pt(II) complex is more than two orders of magnitude higher than the corresponding value of cephalexin. These results indicate that the binding affinity of Pt(II) complex is stronger than the free drug. In addition, the antibacterial study showed that the MIC of platinum complex of cephalexin for variety of organisms was lower than free cephalexin.     

Binding Studies of AICAR and Human Serum Albumin by Spectroscopic,Theoretical, and Computational Methodologies

The interactions of small molecule drugs with plasma serum albumin are important because of the influence of such interactions on the pharmacokinetics of these therapeutic agents. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) is one such drug candidate that has recently gained attention for its promising clinical applications as an anti-cancer agent. This study sheds light upon key aspects of AICAR’s pharmacokinetics, which are not well understood. We performed in-depth experimental and computational binding analyses of AICAR with human serum albumin (HSA) under simulated biochemical conditions, using ligand-dependent fluorescence sensitivity of HSA. This allowed us to characterize the strength and modes of binding, mechanism of fluorescence quenching, validation of FRET, and intermolecular interactions for the AICAR–HSA complexes. We determined that AICAR and HSA form two stable low-energy complexes, leading to conformational changes and quenching of protein fluorescence. Stern–Volmer analysis of the fluorescence data also revealed a collision-independent static mechanism for fluorescence quenching upon formation of the AICAR–HSA complex. Ligand-competitive displacement experiments, using known site-specific ligands for HSA’s binding sites (I, II, and III) suggest that AICAR is capable of binding to both HSA site I (warfarin binding site, subdomain IIA) and site II (flufenamic acid binding site, subdomain IIIA). Computational molecular docking experiments corroborated these site-competitive experiments, revealing key hydrogen bonding interactions involved in stabilization of both AICAR–HSA complexes, reaffirming that AICAR binds to both site I and site II.     

Synthesis,structural determination and HSA interaction studies of a new water-soluble Cu(II) complex derived from 1,10-phenanthroline and ranitidine drug

A new water-soluble Cu(II) complex containing ranitidine drug and 1,10-phenanthroline was synthesized and characterized by elemental analysis, molar conductivity, spectroscopic and computational methods. In vitro human serum albumin (HSA)-interaction studies of Cu(II) complex were performed by employing fluorescence spectroscopy in combination with UV–vis absorption and circular dichroism (CD) spectroscopies. The results of fluorescence titration showed that Cu(II) complex strongly quenched the intrinsic fluorescence of HSA through a static quenching mechanism with an intrinsic binding constant (6.05 × 10⁴ M^?1) at 286 K. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated and suggested that the hydrophobic and hydrogen bonding interactions play major roles in Cu(II) complex-HSA association. The displacement experiments using warfarin and ibuprofen as site I and II probes proved that the Cu(II) complex could bind to site I (subdomain IIA) of HSA. Finally, CD spectra indicated that the interaction of the Cu(II) complex with HSA leads to an increase in the α-helical content. The main result of this study was the finding that the binding affinity of the Cu(II) complex to HSA is three orders of magnitude stronger than that of ranitidine drug.     

Spectrofluorimetric studies on the binding of salicylic acid to bovine serum albumin using warfarin and ibuprofen as site markers with the aid of parallel factor analysis

The interactions of salicylic acid (SL) and two different site markers (warfarin for site I and ibuprofen for site II) with bovine serum albumin (BSA) in pH 7.4 Tris–HCl buffer have been investigated with the use of spectrofluorimetry. An equilibrium solution of BSA and SA was titrated separately with the two markers. This initial work showed that the binding of SL with BSA could be quite complex, and that there was probably a competitive interaction occurring between ibuprofen and SL. However, the spectral results were difficult to interpret clearly for the interaction of warfarin and SL in similar circumstances.

To extract more information from the resolution of fluorescence excitation-emission spectra, the contour plots of the fluorescence spectra indicated that the optimal excitation wavelengths for BSA, SL, warfarin and ibuprofen were different, and were found to be at 278, 295, 306 and 218 nm, respectively. The spectral information was arranged into three-way excitation-emission fluorescence matrix (EEM) stack arrays, and was submitted for analysis by the parallel factor analysis (PARAFAC) algorithm. Firstly, it was demonstrated that the estimated excitation and emission spectral responses for SL, BSA and the site markers, warfarin and ibuprofen, agreed well with the measured spectra. Then, the interpretation of the plots of simultaneously extracted (by PARAFAC) equilibrium concentrations for the above four reactants, showed that: (i) the SL primarily appears to bind in site I but at a different location from the high-affinity binding site (HAS) for warfarin, and the interaction partially overlaps with the low-affinity binding site (LAS) for warfarin. (ii) The SL may have two LAS—one in site II where the HAS for ibuprofen is located, and the other in site I at the LAS for ibuprofen. Thus, application of the PARAFAC method for the study of competitive interaction of SL and BSA with the aid of two different site markers has extracted information unobtainable by traditional methods such as the Scatchard plot, and provided useful means of data visualization.     

Microdialysis-liquid chromatographic study on competitive binding of drugs to protein

A new method to determine the interaction between drug and protein has been developed by utilizing the technique of microdialysis sampling with the ketoprofen and the human serum albumin (HSA) as the model of drug and protein.Two kinds of binding sites of HSA to ketoprofen have been observed.The binding constants and number of binding sites obtained by the Scatchard equation are 0.799,3.18×106 mol-1 L and 2.15,2.01×105 mol-1 L,respectively The displacement binding of drugs to HSA has also been studied.The strong displacement of competitive binding of ibuprofen with ketoprofen to HSA was observed,which means that the primary binding site of HSA to ketoprofen and that to ibuprofen are the same.However,only a weaker displacement of warfarin for the association of ketoprofen with HSA was observed,which may suggest that the primary binding site of HSA to ketoprofen is different from that to warfarin.Such a displacement effect for competitive binding of drugs to HSA was explained by the displacement model i     

Exploring the binding mechanism of dihydropyrimidinones to human serum albumin: spectroscopic and molecular modeling techniques

The binding mechanism of molecular interaction between 5-(ethoxycarbonyl)-6-methyl-4-phenyl-3,4-dihydropyrimidin-2(1H)-one (a dihydropyrimidinones derivative, EMPD) and human serum albumin (HSA) was studied using spectroscopic methods and modeling technique. The quenching mechanism was investigated in terms of the binding constants and the basic thermodynamic parameters. The results of spectroscopic measurements suggested that EMPD have a strong ability to quench the intrinsic fluorescence of HSA through static quenching procedure. The drug-protein complex was stabilized by hydrophobic forces and hydrogen bonding as indicated from the thermodynamic parameters and synchronous fluorescence spectra, which was consistent with the results of molecular docking and accessible surface area calculation. Competitive experiments indicated that a displacement of warfarin by EMPD, which revealed that the binding site of EMPD to HSA was located at the subdomains IIA. The distance between the donor and the acceptor was 4.85nm as estimated according to F?rster's theory of non-radiation energy transfer. The effect of metal ions on the binding constants was also investigated. The results indicated that the binding constants between EMPD and HSA increased in the presence of common metal ions.     

具抗凝血作用的三种钕配合物与人血清白蛋白的相互作用

采用荧光光谱法,紫外光谱法以及圆二色谱法研究了具抗凝血作用的水杨酸钕((NdL′3.2H2O,L′=水杨酸离子))、华法灵钕(NdL3.2H2O,L=华法灵离子)和华法灵水杨酸钕(NdL2L′.2H2O)3种配合物与人血清白蛋白的相互作用。结果表明:配合物对人血清白蛋白(HSA)的荧光产生猝灭现象;配合物的存在使得HSA紫外吸收光谱的强度增加;配合物的存在也对HSA的构象产生影响。水杨酸钕的猝灭方式为动态与静态猝灭,而华法灵钕和华法灵水杨酸钕的猝灭方式属于两者之间生成了不发荧光的复合物而导致的静态猝灭。并分别确定了它们的结合力类型:华法灵钕与HSA之间主要作用力是静电作用力;水杨酸钕与HSA之间主要作用力为典型的疏水作用力;华法灵水杨酸钕与HSA之间为氢键和范德华力。计算了配合物与人血清白蛋白的结合常数K和结合位点数n。     

Steady state and time-resolved fluorescence investigation of the specific binding of two chlorin derivatives with human serum albumin

The specific binding of two model drugs for photodynamic therapy, namely chlorin p6 and purpurin 18 in the vicinity of Sudlow's Site I of HSA has been investigated by monitoring the intrinsic fluorescence of single tryptophanyl residue and by competitive binding with warfarin. The distance from the tryptophanyl residue has been ascertained by FRET from Trp to the chlorins and has been found to indicate a binding to Sudlow's Site I. The principal driving force for the interaction is found to be the hydrophobic effect. The main mechanism of protein fluorescence quenching was static. Time-resolved fluorescence results of competitive binding with warfarin are found to confirm that they bind to the warfarin binding site.     

Identification and Characterization of a Single High‐Affinity Fatty Acid Binding Site in Human Serum Albumin

A single high‐affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐C₁₂ fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site‐specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA‐binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow‐sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X‐ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug‐protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.     

Spectrophotofluorimetric Study of the Interaction between Trazodone Hydrochloride and Bovine Serum Albumin

Abstract

The binding of trazodone hydrochloride (TZH), an antidepressant drug, to bovine serum albumin (BSA) has been investigated by fluorescence spectroscopic analysis. The fluorescence emission of BSA (λ_em=350 nm) was quenched by TZH while that of this ligand was enhanced (λ_em=443 nm). The spectral behavior was consistent with the static quenching mechanism, and the apparent binding constant, K _a (1.05×10⁴ l mol^?1) as well as binding site number, n (～1), were estimated. Thermodynamic parameters obtained from the measured data at different temperatures showed that the binding of TZH to BSA involved predominantly hydrophobic interactions as well as smaller contributions from electrostatic forces. Phenylbutazone and ibuprofen were utilized as competitive markers for sites I and II, respectively, in the interaction of TZH with BSA. This competitive displacement procedure indicated that the likely binding was site I, i.e., subdomain IIA, and this was supported by the observation that up to 50% of this site marker, phenylbutazone, could be exchanged with TZH whilst only a few percent of ibuprofen were so affected.     

Studies of Interaction between Ergosta‐4,6,8(14),22‐tetraen‐3‐one (Ergone) and Human Serum Albumin by Molecular Spectroscopy and Modeling

Our previous experimental results have shown that ergosta‐4,6,8(14),22‐tetraen‐3‐one (ergone) is one of the main bioactive components of Polyporus umbellatus. The efficacy of ergone binding to human serum albumin (HSA) is critical for pharmacokinetic behavior of ergone. The interactions between ergone and HSA under simulative physiological conditions were investigated by the methods of fluorescence spectroscopy, absorption and circular dichroism spectroscopy. Fluorescence data revealed that the fluorescence quenching of HSA by ergone was the result of the formation of the ergone‐HSA complex. According to the modified Stern‐Volmer equation, the binding constants (K_a) between ergone and HSA were determined. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated to be 0.989 kJ mol^‐1 and 11.214 J mol^‐1 K^‐1, indicating that the hydrogen bonds and hydrophobic interactions played a dominant role in the binding of ergone to HSA. The conformational investigation showed that the presence of ergone decreased the α‐helical content of HSA and induced the slight unfolding of the polypeptides of protein. Furthermore, displacement experiments using warfarin and ibuprofen indicated that ergone could bind to site I of HSA, which was also in agreement with the results of the molecular modeling.     

Competitive binding of small molecules with biopolymers: a fluorescence spectroscopy and chemometrics study of the interaction of aspirin and ibuprofen with BSA

The interaction of aspirin and ibuprofen with bovine serum albumin (BSA) was studied by spectrofluorimetry under simulated physiological conditions. Both aspirin and ibuprofen quenched the intrinsic fluorescence of BSA and the binding ratios obtained were 2 : 1 for aspirin-BSA and 3 : 1 for ibuprofen-BSA interactions, respectively. The thermodynamic parameters (ΔH, ΔS and ΔG) obtained from the fluorescence spectroscopy data showed that the binding of aspirin to BSA involved van der Waals interactions and hydrogen bonds. Competitive experiments using warfarin and diazepam as site markers indicated that aspirin was mainly located in the hydrophobic pocket of site II of the protein as well as to a small extent in site I. Furthermore, the competitive interaction of the aspirin and ibuprofen with BSA, which was studied with the use of the three-way excitation-emission fluorescence spectra and a parallel factor analysis (PARAFAC) chemometrics method, showed that the competitive effect of ibuprofen was stronger than that of aspirin, i.e. the former molecule replaced the aspirin from the aspirin-BSA complex.     

Spectroscopic study on the inherent binding information of cationic perfluorinated surfactant with bovine serum albumin

UV-vis, FT-IR, fluorescence and synchronous fluorescence spectra are applied to discuss the inherent binding information of model protein bovine serum albumin (BSA) with perfluorinated surfactant trimethyl-1-propanaminium iodide (FC-134). According to the results analyzed from Stern-Volmer equation, FC-134 can quench the fluorescence intensity of BSA via a dynamic quenching mechanism with complex formation. The thermodynamic parameters are calculated, revealing that hydrophobic force is the main interaction driven force. The binding constants and number of binding sites are also obtained. With the aid of site markers—warfarin and ibuprofen, we first report that FC-134 primarily binds to tryptophan residue Trp-214 of BSA within site I (sub-domain IIA).     

Interaction Between Toddalolatone and Human Serum Albumin

A combination of fluorescence, UV–Vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) spectroscopic and molecular modeling approaches was employed to investigate the interaction between toddalolactone (TDT) and human serum albumin (HSA) at physiological buffer conditions (pH 7.4). Fluorescence titration suggests that the mechanism of the fluorescence quenching of HSA is static, resulting from the formation of a TDT–HSA complex. Binding parameters calculated from the modified Stern–Volmer equation show that TDT binds to HSA with high affinity. Negative enthalpy change and positive entropy change values suggest that the binding process is primarily driven by hydrophobic interactions and hydrogen bonds. The binding of TDT to HSA results in an increase in the surface hydrophobicity of HSA. The binding distance between the Trp-214 residue (donor) and TDT (acceptor) was determined to be 4.18 nm based on the Förster theory of non-radioactive energy transfer. Displacement studies of site markers reveal that the binding site of TDT to HSA is located in the subdomain IIA (Sudlow’s site I). Furthermore, the molecular docking results corroborate and illustrate the specific binding mode and binding site. Analysis of UV–Vis absorption, CD and FT-IR spectra demonstrated that TDT induced a small alteration of the protein’s conformation.     

A spectroscopic study on the interaction between the anticancer drug erlotinib and human serum albumin

The interaction between erlotinib and human serum albumin (HSA) in simulated physiological conditions was investigated by spectroscopic methods. The results revealed that erlotinib caused the fluorescence quenching of HSA through a static quenching procedure. The binding constants at 293, 298, 303 and 308 K were obtained as 2.53 × 10⁵, 8.13 × 10⁴, 3.59 × 10⁴ and 1.93 × 10⁴ M^?1, respectively. There may be one binding site of erlotinib on HSA at 298 K. The thermodynamic parameters indicated that the interaction between erlotinib and HSA was driven mainly by hydrogen bonding or van der Waals forces. Synchronous fluorescence spectra, UV–Vis spectra, circular dichroism and Fourier Transform infrared spectroscopy results showed erlotinib binding slightly changed the conformation of HSA with secondary structural content changes. Förster resonance energy transfer study revealed high possibility of energy transfer with erlotinib-Trp-214 distance of 3.48 nm. The results of the present study may provide valuable information for studying the distribution, toxicological and pharmacological mechanisms of erlotinib in vivo.     

嘧啶衍生物与人血清白蛋白的相互作用

在模拟生理条件下,运用荧光光谱、激光闪光光解(LFP)和分子对接等技术研究了8种具有抗肿瘤活性的嘧啶衍生物(PDs,其中PDs A 5-FU为成药,PDs B-H为实验室自制)与人血清白蛋白(HSA)的相互作用.利用Stern-Volmer方程和激光闪光光解技术分析了PDs对HSA的荧光猝灭机制,PDs A和B为静态猝灭,PDs G和H为动态猝灭.用双倒数曲线法得出5种PDs与HSA的结合常数Ka和结合位点数n,在测定条件下5种PDs与载体结合位点数均为1,且均以弱结合力结合,通过热力学参数ΔH,ΔS和ΔG推测出PDs B,C和E与HSA之间的作用力为静电作用力和疏水作用力,PDs A和D与HSA之间的作用力是氢键和范德华力,分子对接结果与其一致.根据F9rster非辐射能量转移理论(FRET)分析了HSA和PDs之间的结合距离(r),其结果均小于4 nm,符合能量转移理论.进一步利用同步荧光、三维荧光和圆二色光谱考察了PDs与HSA结合过程中HSA空间构象的变化,结果显示,仅PDs A和C对HSA的芳香族氨基酸周围的疏水性略有增强作用.体外实验结果表明,HSA可以作为优良的载体来运输和储存PDs A~E,这为嘧啶衍生物的后续研究提供了可参考的实验数据.     

Investigating the interaction of juglone (5-hydroxy-1, 4-naphthoquinone) with serum albumins using spectroscopic and in silico methods

The interaction between juglone at the concentration range of 10–110 µM and bovine serum albumin (BSA) or human serum albumin (HSA) at the constant concentration of 11 µM was investigated by fluorescence and UV absorption spectroscopy under physiological-like condition. Performing the experiments at different temperatures showed that the fluorescence intensity of BSA/HSA was decreased in the presence of juglone by a static quenching mechanism due to the formation of the juglone–protein complex. The binding constant for the interaction was in the order of 10³ M^?1, and the number of binding sites for juglone on serum albumins was determined to be equal to one. The thermodynamic parameters including enthalpy (ΔH), entropy (ΔS) and Gibb’s free energy (ΔG) changes were obtained by using the van’t Hoff equation. These results indicated that van der Waals force and hydrogen bonding were the main intermolecular forces stabilizing the complex in a spontaneous association reaction. Moreover, the interaction of BSA/HSA with juglone was verified by UV absorption spectra and molecular docking. The results of synchronous fluorescence, UV–visible and CD spectra demonstrated that the binding of juglone with BSA/HSA induces minimum conformational changes in the structure of albumins. The increased binding affinity of juglone to albumin observed in the presence of site markers (digoxin and ibuprofen) excludes IIA and IIIA sites as the binding site of juglone. This is partially in agreement with the results of molecular docking studies which suggests sub-domain IA of albumin as the binding site.     

A novel lophine-based fluorescence probe and its binding to human serum albumin

The binding of a lophine-based fluorescence probe, 4-[4-(4-dimethylaminophenyl)-5-phenyl-1H-imidazol-2-yl]benzoic acid methyl ester (DAPIM) with human serum albumin (HSA) was investigated by fluorescence spectroscopy under physiological conditions. While DAPIM shows extreme low fluorescence in aqueous solution, DAPIM binding with HSA emits strong fluorescence at 510 nm. The binding constant and binding number determined by Scatchard plot was 3.65 × 10⁶ M⁻¹ and 1.07, respectively. Competitive binding between DAPIM and other ligands such as warfarin, valproic acid, diazepam and oleic acid, were also studied fluorometrically. The results indicated that the primary binding site of DAPIM to HSA is site II at subdomain IIIA. DAPIM can be a useful fluorescence probe for the characterization of drug-binding sites. In addition to the interaction study, because the fluorescence intensity of DAPIM increased in proportion to HSA concentration, its potential in HSA assay for serum sample was also evaluated.     

HSA-Lys-161 covalent bound fluorescent dye for in vivo blood drug dynamic imaging and tumor mapping

The specific combination of human serum albumin and fluorescent dye will endow superior performance to a coupled fluorescent platform for in vivo fluorescence labeling. In this study, we found that lysine-161 in human serum albumin is a covalent binding site and could spontaneously bind a ketone skeleton quinoxaline–coumarin fluorescent dye with a specific turn-on fluorescence signal for the first time. Supported by the abundant drug binding domains in human serum albumin, drugs such as ibuprofen, warfarin and clopidogrel could interact with the fluorescent dye labeled human serum albumin to feature a substantial enhancement in fluorescence intensity (6.6-fold for ibuprofen, 4.5-fold for warfarin and 5-fold for clopidogrel). The drug concentration dependent fluorescence intensity amplification realized real-time, in situ blood drug concentration monitoring in mice, utilizing ibuprofen as a model drug. The non-invasive method avoided continuous blood sample collection, which fundamentally causes suffering and consumption of experimental animals in the study of pharmacokinetics. At the same time, the coupled fluorescent probe can be efficiently enriched in tumors in mice which could map a tumor with a high-contrast red fluorescence signal and could hold great potential in clinical tumor marking and surgical resection.

HSA lysine-161 covalent bound quinoxaline–coumarin based fluorescent dye realized in situ blood drug concentration monitoring and tumor visualization.     

Allosteric and competitive displacement of drugs from human serum albumin by octanoic acid, as revealed by high-performance liquid affinity chromatography, on a human serum albumin-based stationary phase.

A chiral stationary phase for high-performance liquid chromatography, based upon immobilized human serum albumin (HSA), was used to investigate the effect of octanoic acid on the simultaneous binding of a series of drugs to albumin. Octanoic acid was found to bind with high affinity to a primary binding site, which in turn induced an allosteric change in the region of drug binding Site II, resulting in the displacement of compounds binding there. Approximately 80% of the binding of suprofen and ketoprofen to HSA was accounted for by binding at Site II. Octanoic acid was found to also bind to a secondary site on HSA, with much lower affinity. This secondary site appeared to be the warfarin-azapropazone binding area (drug binding Site I), as both warfarin and phenylbutazone were displaced in a competitive manner by high levels of octanoic acid. The enantioselective binding to HSA exhibited by warfarin, suprofen and ketoprofen was found to be due to differential binding of the enantiomers at Site I; the primary binding site for suprofen and ketoprofen was not enantioselective.