Spectroscopic investigation on the binding of bioactive pyridazinone derivative to human serum albumin and molecular modeling

The interaction between a novel promising pyridazinone derivative (5-chloro-2-nitro-N-(4-(6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)benzamide (CNPB)) and human serum albumin (HSA) under physiological conditions has been investigated systematically by fluorescence spectroscopy, UV absorption spectroscopy, circular dichroism (CD) and molecular modeling. From the spectra obtained, it was observed that CNPB had a strong ability to quench the intrinsic fluorescence of HSA through a static quenching procedure. The site binding constants (K(b)) were 4.22 x 10(4) and 3.32 x 10(4)M(-1) at 290 and 300 K, respectively. The alterations of protein secondary structure in the presence of CNPB were qualitative and quantitative calculated by the results from CD and synchronous fluorescence. In addition, the thermodynamic standard enthalpy (DeltaH) and standard entropy (DeltaS) for the reaction were calculated to be -17.35 kJ mol(-1) and 9.57 J mol(-1)K(-1), respectively. These results showed that the binding of CNPB to HSA was mainly of hydrophobic interaction, but the hydrogen bonding and electrostatic interaction could not be excluded. Furthermore, the study of molecular modeling also indicated that CNPB could strongly bind to the site I (subdomain IIA) of HSA mainly by hydrophobic interaction and there were hydrogen bond interactions between CNPB and the residue His242.     

Spectroscopic and molecular modeling evidence of clozapine binding to human serum albumin at subdomain IIA

Various spectroscopy and molecular docking methods were used to examine the binding of Clozapine (CLZ) to human serum albumin (HSA) in this paper. By monitoring the intrinsic fluorescence of single Trp214 residue and performing Dansylamide (DNSA) displacement measurement, the specific binding of CLZ in the vicinity of Sudlow's Site I of HSA has been clarified. An apparent distance of 27.3 ? between the Trp214 and CLZ was obtained via fluorescence resonance energy transfer (FRET) method. In addition, the changes in the secondary structure of HSA after its complexation with CLZ ligand were studied with CD spectroscopy, which indicate that CLZ does not has remarkable effect on the structure of the protein. Moreover, thermal denaturation experiment shows that the HSA-CLZ complexes are conformationally more stable. Finally, the binding details between CLZ and HSA were further confirmed by molecular docking studies, which revealed that CLZ was bound at subdomain IIA through multiple interactions, such as hydrophobic effect, van der Waals forces and hydrogen bonding.     

Characterization of the structural changes of human serum albumin upon interaction with single-walled and multi-walled carbon nanotubes: spectroscopic and molecular modeling approaches

Research on Chemical Intermediates - Through the incorporation of spectorescopic and molecular methods of modeling, the researchers investigated the interaction between Carbon Nanotubes (CNTs) and...     

A molecular modeling study on binding of drugs to calmodulin

Summary Computer-graphical methods have been used to study the interaction between a series of drugs and calmodulin. Based on the X-ray crystallographic coordinates of the -C atoms of calmodulin, a molecular model of the helical sequences was built. The model has been used to derive two possible binding sites for phenothiazines and one binding site for penfluridol. The principal binding forces occur through contacts between acidic amino acids of calmodulin and the protonated side-chain nitrogen of the drugs as well as between a basic amino acid and the electronegative substituents of the aromatic rings. Calculated interaction energies show a good correlation with experimental inhibition data.     

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.     

Molecular modeling approaches for the prediction of the nonspecific binding of drugs to hepatic microsomes

Molecular modeling approaches for the prediction of the nonspecific binding of drugs to hepatic microsomes were examined using a published database of 56 compounds. Models generated were evaluated using an independent test set of 13 compounds. A pharmacophore approach identified structural features of drugs associated with nonspecific binding. A side-chain amino group and complementary hydrophobic domain were the principal features noted. The use of shape overlays, based on the pharmacophore, in conjunction with a chemical force field in the program ROCS, yielded discrimination between molecules classified as strong binders (experimental fraction unbound in microsomes<0.50) and those with a lower degree of binding (experimental fraction unbound in microsomes>0.50). In the initial data set of 56 molecules, 18 were classified as strong binders (on the basis of the above criteria), and all of those were recovered in the top 22 molecular hits from ROCS. Additionally, computationally generated values of log P were shown to provide a reasonable estimate of the fraction unbound in microsomes, providing the compounds were in their basic form at physiological pH.     

Spectroscopic investigation and molecular modeling on porphyrin/PAMAM supramolecular adduct

Noncovalent adducts (TPPC@PAMAM) between meso-tetrakis(4-carboxyphenyl)porphyrin (TPPC) and polyamidoamine PAMAM dendrimer (generation 2.0) have been obtained by simply mixing the two components at different stoichiometric amount. The resulting species are readily soluble and stable in aqueous solution up to millimolar concentration. Electrostatic interactions between the anionic carboxylate groups of TPPC and the protonated amino groups of the PAMAM dendrimer play an important role in the stabilization of these adducts. UV/Vis absorption, steady state and time-resolved fluorescence emission and anisotropy measurements suggest the presence of equilibria involving different species as function of the [PAMAM]/[TPPC] ratio. At low ratios the observed spectroscopic behavior evidence the presence of H-aggregates, while at higher ratios well-defined species containing monomeric TPPC strongly interacting with the charged dendrimer are formed. Docking of the binary supramolecular adduct further supports the experimental results showing a favorable interaction with the porphyrin being completely included in the dendrimer. The interaction of the binary TPPC@PAMAM adduct (1/1 ratio) with calf-thymus DNA has been investigated through spectroscopic and photophysical techniques. All the experimental results point to the formation of a ternary complex between the binary adduct and the DNA backbone.     

Spectroscopic Investigation of a Synthetic Cyanine Amine Derivative upon Various Scaffolds

A detailed study of the supramolecular self-assembly of a cyanine amine derivative upon a variety of templates was conducted including investigation of charge properties, the rigidity of templates, and solubility. The amine derivative exhibited strong self-assembly on negatively-charged templates such as carboxymethyl amylose and carboxymethyl cellulose, and generated red-shifted absorption and enhanced fluorescence, which are characteristics of J-aggregation. Much less self-aggregation was observed for lower negatively-charged templates such as hyaluronic acid. The rigidity of templates had a great impact on self-aggregation in terms of no appreciable J- or H-aggregation observed on rigid Laponite clay. Cyanine amine existed in the molecular state with self-aggregates. For the carboxymethyl cellulose/cyanine amine system, the order of mixing each component impacted the aggregation of cyanine amine: carboxymethyl cellulose to cyanine amine mainly led to J-aggregation. In contrast, H-aggregation was dominant in cyanine amine to carboxymethyl cellulose.     

Studies on the binding of vinpocetine to human serum albumin by molecular spectroscopy and modeling

The interaction between vinpocetine(VPC) and human serum albumin(HSA) in physiological buffer(pH 7.40) was investigated by fluorescence,FT-IR,UV-vis absorption and molecular modeling.VPC effectively quenched the intrinsic fluorescence of HSA via static quenching.The binding site number n and apparent binding constant K_a,corresponding thermodynamic parametersΔG,ΔH andΔS at different temperatures were calculated.The synchronous fluorescence and FT-IR spectra were used to investigate the structural change of HSA molecules with addition of VPC.Molecular modeling indicated that VPC could bind to the site I of HSA and hydrophobic interaction was the major acting force,which was in agreement with the binding mode study.     

Studies on the binding of nevadensin to human serum albumin by molecular spectroscopy and modeling

The binding of nevadensin to human serum albumin (HSA) in aqueous solution was investigated for the first time by molecular spectroscopy and modeling at pH 7.4. Spectrophotometric observations are rationalized in terms of a static quenching process and binding constant (K_a, K_b) and the number of binding sites (n ≈ 1) were evaluated by fluorescence quenching methods. Thermodynamic data showed that nevadensin was included in the hydrophobic cavity of HSA mainly via hydrophobic interactions. The value of 3.09 nm for the distance r between the donor (HSA) and acceptor (nevadensin) was derived from the fluorescence resonance energy transfer. Spectrophotometric techniques were also applied to investigate the structural information of HSA molecules on the binding of nevadensin and the results showed that the binding of nevadensin to HSA did not change significantly molecular conformation of HSA in our experimental conditions. Furthermore, the study of molecular modeling also indicated that nevadensin could strongly bind to the site I (subdomain IIA) of HSA mainly by a hydrophobic interaction and there are hydrogen bond interactions between nevadensin and the residues Arg-218, Arg-222, Lys-195, and Asp-451. As compared to the other flavonoids, the flavonoids containing methoxy groups which are in aromatic rings can bind to HSA with higher affinity.     

Spectroscopic evidence for the formation of a four-coordinate Co2+ cobalamin species upon binding to the human ATP:cobalamin adenosyltransferase

The human adenosyltransferase hATR converts exogenous cobalamin into coenzyme B12 by transferring the adenosyl group from cosubstrate ATP to a transiently formed Co1+cobalamin (Co1+Cbl) species. A particularly puzzling aspect of hATR function is that the midpoint potential for Co2+Cbl --> Co1+Cbl reduction is below that of readily available biological reductants. Our magnetic circular dichroism and electron paramagnetic resonance spectroscopic studies reported here reveal that, in the absence of ATP, the interaction between Co2+Cbl and hATR promotes partial conversion of the cofactor to its "base-off" form in which a water molecule occupies the lower axial position. This interaction becomes much stronger in the presence of ATP, leading to the formation of an unprecedented Co2+Cbl species with spectroscopic signatures consistent with an essentially four-coordinate, square-planar Co2+ center. This unusual Co2+Cbl coordination is expected to raise the Co2+/1+ reduction potential well into the physiological range.     

A molecular mechanics approach to modeling protein-ligand interactions: relative binding affinities in congeneric series

We introduce the "Prime-ligand" method for ranking ligands in congeneric series. The method employs a single scoring function, the OPLS-AA/GBSA molecular mechanics/implicit solvent model, for all stages of sampling and scoring. We evaluate the method using 12 test sets of congeneric series for which experimental binding data is available in the literature, as well as the structure of one member of the series bound to the protein. Ligands are "docked" by superimposing a common stem fragment among the compounds in the series using a crystal complex from the Protein Data Bank and sampling the conformational space of the variable region. Our results show good correlation between our predicted rankings and the experimental data for cases in which binding affinities differ by at least 1 order of magnitude. For 11 out of 12 cases, >90% of such ligand pairs could be correctly ranked, while for the remaining case, Factor Xa, 76% of such pairs were correctly ranked. A small number of compounds could not be docked using the current protocol because of the large size of functional groups that could not be accommodated by a rigid receptor. CPU requirements for the method, involving CPU minutes per ligand, are modest compared with more rigorous methods that use similar force fields, such as free energy perturbation. We also benchmark the scoring function using series of ligands bound to the same protein within the CSAR data set. We demonstrate that energy minimization of ligands in the crystal structures is critical to obtain any correlation with experimentally determined binding affinities.     

5,10-Methylene-5,6,7,8-tetrahydrofolate conformational transitions upon binding to thymidylate synthase: molecular mechanics and continuum solvent studies

Summary We applied the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) approach to evaluate relative stability of the extended (flat) and C-shaped (bent) solution conformational forms of the 5,10-methylene-5,6,7,8-tetrahydrofolate (mTHF) molecule in aqueous solution. Calculations indicated that both forms have similar free energies in aqueous solution but detailed energy components are different. The bent solution form has lower intramolecular electrostatic and van der Waals interaction energies. The flat form has more favorable solvation free energy and lower contribution from the bond, angle and torsion angle molecular mechanical internal energies. We exploit these results and combine them with known crystallographic data to provide a model for the progressive binding of the mTHF molecule, a natural cofactor of thymidylate synthase (TS), to the complex forming in the TS-catalyzed reaction. We propose that at the time of initial weak binding in the open enzyme the cofactor molecule remains in a close balance between the flat and bent solution conformations, with neither form clearly favored. Later, thymidylate synthase undergoes conformational change leading to the closure of the active site and the mTHF molecule is withdrawn from the solvent. That effect shifts the thermodynamic equilibrium of the mTHF molecule toward the bent solution form. At the same time, burying the cofactor molecule in the closed active site produces numerous contacts between mTHF and protein that render change in the shape of the mTHF molecule. As a result, the bent solution conformer is converted to more strained L-shaped bent enzyme conformer of the mTHF molecule. The strain in the bent enzyme conformation allows for the tight binding of the cofactor molecule to the productive ternary complex that forms in the closed active site, and facilitates the protonation of the imidazolidine N10 atom, which promotes further reaction. *To whom correspondence should be addressed. Fax: +4822-822-5342; E-mail: a.jarmula@nencki.gov.pl     

Internal rotation in enaminonitriles: Investigation by dynamic NMR spectroscopy and molecular modeling

Rotational isomerism in enaminonitriles was studied using dynamic NMR spectroscopy and molecular modeling. It was found that the barrier to rotation about C_vinyl-NH bond was higher for enaminonitriles derived from aliphatic amines than that of enaminonitriles derived from aromatic amines. It was also found that the rotational isomerism about the C_ar-C_vinyl bond also exists in enaminonitriles.     

Spectroscopic approaches to the study of the interaction of aluminum with humic substances

Aluminum ion (Al³⁺) in the ‘free’ (aquo) state is becoming increasingly prevalent in environmental waters, especially fresh waters, as a consequence of acid rain and other environmental processes. As Al³⁺ ion is known to affect markedly a wide range of biological systems, and since the presence of Al³⁺ in humans has been linked to a number of human diseases, it is important to understand the speciation of Al³⁺ ion in natural waters. Since some of the most important complexation agents for Al³⁺ in both fresh and sea waters are members of the complex humic substances group, it is important to understand the manner in which Al³⁺ interacts with this class of molecules, especially since binding of Al³⁺ to these molecules can effectively increase the bioavailability of this toxic metal ion to biological systems. The objective of this review is to present the current state of our understanding of aqueous aluminum complexation with the most acidic members (and therefore the most likely candidates for serving as Al³⁺ complexing agents) of the humic substances group, the fulvic acids. Much of the current knowledge has been revealed by comprehensive fluorescence titration analyses. Some additional information has come from other experimental approaches, including infrared spectroscopy, nuclear magnetic resonance spectroscopy, and a variety of electrochemical approaches. In this review, we also report on the results of our recent fluorescence and IR spectroscopy survey of the interaction of metals from of all three Nieboer and Richardson categories of environmental metals (Class A, Class B and Intermediate Class) with the fulvic acid sub-group of the humic substances. This has proven helpful in understanding some of the unique spectral behaviors of the Al³⁺-fulvic acid complex vis-a-vis fulvic acid complexes with many other metal ions. The results of our fluorescence and IR experiments with the model compounds, such as salicylic and phthalic acids, have allowed confirmation of the important roles played by both salicylic acid-like sites and phthalic acid-like sites in the unique complexation of Al³⁺ to humic substances, and help to explain some of the observed spectroscopic changes associated with Al³⁺ ion complexation to humic material. From the current work, it seems clear that major sources of the deviation in spectral properties between Al³⁺ and many other metal ions (across all three Nieboer and Richardson categories) are the unusually high value of its charge density and relatively low propensity for involvement in covalent bonding interactions (i.e. a very high ionic index combined with a relatively low covalent index in the Nieboer and Richardson classification of environmental metals), as well as affinity for certain functional groups.     

Spectroscopic study and antioxidant activity of the inclusion complexes of cyclodextrins and amlodipine besylate drug

The aim of the study was to synthesize and characterization the inclusion complexes of amlodipine besylate (AML) drug with β-cyclodextrin (β-CD) and γ-cyclodextrin (γ-CD) which has antioxidating activity property. The guest/host interaction of AML with β-CD and γ-CD in order to complexation drug in β-CD and γ-CD were investigated. The interaction inclusion complexes was characterized by fourier transform infrared and ultraviolet–visible spectroscopies. The formation constant was calculated by using a modified Benesi–Hildebrand equation at 25 °C. The stoichiometry of inclusion complexes was found to be 1:1 for β-CD and γ-CD with AML drug. The antioxidant activity of AML drug and its inclusion complexes were determined by the scavenging of stable radical 2,2′-diphenyl-1-picrylhydrazyl (DPPH^·). Kinetic studies of DPPH^· with AML and CDs complexes were done. The experimental results confirmed the forming of AML complexes with CDs also these indicated that the AML/β-CD and AML/γ-CD inclusion complexes was the most reactive than its free form into antioxidant activity.     

Hydroxycamptothecin deactivation rates and binding to model membranes and HSA determined by fluorescence spectra analysis

Camptothecins (CPTs) are fluorescent compounds exhibiting anticancer activity. They can exist in two forms, a lactone and a carboxylate. In neutral and base solution, lactone forms hydrolyse and convert into carboxylates. Only the lactone forms of CPTs are biologically active. Because of strong affinity of the carboxylate form of the parent drug camptothecin to human serum albumin (HSA), this protein promotes the deactivation of this compound. On the other hand, the lactone forms of camptothecins do not hydrolyse and are stabilized when bound to membranes. The following three hydroxycamptothecins, 10 hydroxycamptothecin (10-OH-CPT), 7-ethyl-10-hydroxy-camptothecin (SN-38) and 7-tert-butyldimethylsil-10-hydroxycamptothecin (DB-67) were studied. Factor analysis of a set of fluorescence excitation spectra recorded during lactone hydrolysis facilitated the high-throughput determination of the deactivation rates of camptothecin and each hydroxycamptothecin in phosphate buffered saline. The fluorescence spectra of hydroxycamptothecins diluted in HSA solution or suspended in DMPC liposomes were recorded, and the association constants of these drugs to membranes and plasma proteins were calculated. Among the analysed agents, DB-67 exhibited the most desirable properties including low affinity of the carboxylate form for albumin and high affinity of its lactone form for model membranes.     

Through Bridge Spin Coupling in Homo- and Heterobimetallic Porphyrin Dimers upon Stepwise Oxidations: A Spectroscopic and Theoretical Investigation

We have described copper(II)-iron(III) and copper(II)-manganese(III) heterobimetallic porphyrin dimers and compared them with the corresponding homobimetallic analogs. UV-visible spectra are very distinct in the heterometallic species while electrochemical studies demonstrate that these species, as compared to the homobimetallic analog, are much easier to oxidize. Combined Mössbauer, EPR, NMR, magnetic and UV-visible spectroscopic studies show that upon 2e-oxidation of the heterobimetallic complexes only ring-centered oxidation occurs. The energy differences between HOMO and LUMO are linearly dependent with the low-energy NIR band obtained for the 2e-oxidized complexes. Also, strong electronic communication between two porphyrin rings through the bridge facilitates coupling between various unpaired spins present while the coupling model depends on the nature of metal ions used. While unpaired spins of Fe(III) and the porphyrin π-cation radical are strongly antiferromagnetically coupled, such coupling is rather weak between Mn(III) and a porphyrin π-cation radical. Moreover, the coupling between two π-cation radicals are much stronger in the 2e-oxidized complexes of dimanganese(III) and copper(II)-manganese(III) porphyrin dimers as compared to their diiron(III) and copper(II)-iron(III) analogs. Furthermore, coupling between the unpaired spins of a π-cation radical and copper(II) is much stronger in the 2e-oxidized complex of copper(II)-iron(III) porphyrin dimer as compared to its copper(II)-manganese(III) analog. The Mulliken spin density distributions in 2e-oxidized homo- and heterobimetallic complexes show symmetric and asymmetric spread between the two macrocycles, respectively. In both the 2e-oxidized heterobimetallic complexes, the Cu(II) porphyrin center acts as a charge donor while Fe(III)/Mn(III) porphyrin center act as a charge acceptor. The experimental observations are also strongly supported by DFT calculations.