Synthèse et caractérisation du Gd-C4-thyroxin-DTPA,un nouvel agent de contraste potentiel pour l’IRM. Étude de son interaction non covalente avec l’albumine sérique humaine

The synthesis and the physicochemical characterization of a new contrast agent for magnetic resonance imaging (MRI), Gd-C₄-thyroxin-DTPA, which has a high affinity for human serum albumin (HSA), are reported. The results show that this chelate is characterized by a relatively high relaxivity, which increases moreover with the concentration. This reflects an aggregation of the molecules in solution. It is also characterized by a better stability versus the transmetallation with the zinc ion than the parent compound, the Gd-DTPA (Magnevist^®, Bayer Schering Pharma). The study of its interaction with human serum albumin was performed by the proton relaxometry technique, which has revealed a relatively high affinity (Ka of the order of 10,000 M⁻¹, with two binding sites). Finally, competition experiments with ibuprofen and salicylate, of which the binding sites on HSA are known, were performed by the NMR diffusometry method. The results suggest that the chelate shares one of the binding sites of ibuprofen.     

Allosteric modulation of myristate and Mn(III)heme binding to human serum albumin. Optical and NMR spectroscopy characterization

Human serum albumin (HSA) is best known for its extraordinary ligand binding capacity. HSA has a high affinity for heme and is responsible for the transport of medium and long chain fatty acids. Here, we report myristate binding to the N and B conformational states of Mn(III)heme-HSA (i.e. at pH 7.0 and 10.0, respectively) as investigated by optical absorbance and NMR spectroscopy. At pH 7.0, Mn(III)heme binds to HSA with lower affinity than Fe(III)heme, and displays a water molecule coordinated to the metal. Myristate binding to a secondary site FAx, allosterically coupled to the heme site, not only increases optical absorbance of Mn(III)heme-bound HSA by a factor of approximately three, but also increases the Mn(III)heme affinity for the fatty acid binding site FA1 by 10-500-fold. Cooperative binding appears to occur at FAx and accessory myristate binding sites. The conformational changes of the Mn(III)heme-HSA tertiary structure allosterically induced by myristate are associated with a noticeable change in both optical absorbance and NMR spectroscopic properties of Mn(III)heme-HSA, allowing the Mn(III)-coordinated water molecule to exchange with the solvent bulk. At pH = 10.0 both myristate affinity for FAx and allosteric modulation of FA1 are reduced, whereas cooperation of accessory sites and FAx is almost unaffected. Moreover, Mn(III)heme binds to HSA with higher affinity than at pH 7.0 even in the absence of myristate, and the metal-coordinated water molecule is displaced. As a whole, these results suggest that FA binding promotes conformational changes reminiscent of N to B state HSA transition, and appear of general significance for a deeper understanding of the allosteric modulation of ligand binding properties of HSA.     

Synthesis of a new gadolinium complex with a high affinity for human serum albumin and its manifold physicochemical characterization by proton relaxation rate analysis, NMR diffusometry and electrospray mass spectrometry

A novel gadolinium complex, derived from Gd-DTPA (DTPA: diethylenetriaminepentaacetic acid) and sulfaphenazole, intended to be a potential MRI contrast agent and to interact with human serum albumin (HSA), was synthesized and characterized. Its relaxometric properties were evaluated in water, and its binding to HSA was investigated by three techniques: proton relaxation rate analysis, NMR diffusometry, and electrospray mass spectrometry. The complex has a higher relaxivity than the parent compound (r(1)=7.8s(-1)mM(-1) at 310K and 0.47T and 7.7s(-1)mM(-1) at 310K and 1.41T), a fast water exchange, and a very good stability versus zinc(II) transmetallation. All techniques agree with a high affinity of the complex for HSA, and competition experiments indicate that this contrast agent competes with ibuprofen for HSA.     

Carbon-13 NMR Spectra of Sodium d-Gluco- and d-Galactopyranuronates in the Presence of Lanthanide Ions

Carbon-13 NMR spectra of sodium d-gluco- and d-galactopyranuronates were measured in the presence of lanthanum, europium, praseodymium, or neodymium ions in deuterium oxide. The lanthanide-induced shifts of all the carbon signals were divided into three components based on complex-formation, contact, and pseudocontact effects. The last effects on C-1 in the α-anomers were exceedingly greater than those in the corresponding β-anomers. Carbon-13 spin-lattice relaxation times and their reduction induced by gadolinium ion were also measured and the binding sites of the ion were estimated, which showed marked differences between the two anomers and suggested the formation of bidentate complexes, involving linkages to both the ring and the carboxyl oxygen only between the α-anomers and the lanthanide ions.     

Interaction of ochratoxin A with human serum albumin. Binding sites localized by competitive interactions with the native protein and its recombinant fragments

Competitive interactions of ochratoxin A (OTA) and several other acidic compounds were utilized to gain insight into the localization of binding sites and the nature of binding interactions between anionic species and human serum albumin (HSA). Depolarization of OTA fluorescence in the presence of a competing anion was used to quantify ligand-protein interactions. The results obtained were rationalized in terms of OTA displacement from its major binding site. Based on their ability to displace OTA, two distinct groups of the anionic ligands were revealed. The first group contained structurally diverse compounds that shared a common binding site in subdomain IIA (Sudlow Site I). The second group consisted of three non-steroidal anti-inflammatory drugs, which showed much lower affinity to Site I than the OTA dianion. The major site for these drugs was located in domain III. Fluorescence spectroscopy measurements of OTA, warfarin (WAR) and naproxen (NAP) complexes with recombinant proteins corresponding to the domains of HSA (D1-D3) revealed binding to all domains but with different affinities. The binding constants for OTA and WAR decreased in the series D2z.Gt;D3>D1. In contrast, NAP showed the most favorable interaction with D3 and comparable affinities to the two remaining domains. The OTA binding constant for D2, 7.9 x 10(5) M(-1), was smaller than the largest constant for HSA by a factor of approximately 7. The binding constant for OTA with D3, 1.1 x 10(5) M(-1), was very close to that of the secondary binding site for HSA.     

COLCEMID INHIBITION OF CELL GROWTH AND THE CHARACTERIZATION OF A COLCEMID-BINDING ACTIVITY IN SACCHAROMYCES CEREVISIAE

Under restricted culture conditions, the growth and division of Saccharomyces cerevisiae was inhibited by the antimitotic drug Colcemid; in contrast, the related drug colchicine had no effect. The difference in the sensitivity of yeast to these two agents was not dependent on their ability to permeate the cell but rather reflected an inherent difference in the affinity of the two drugs for a cellular-binding site. The binding moiety was characterized by gel filtration as a macromolecule of approximately 110,000 mol wt with an affinity constant for Colcemid of 0.5 x 10⁴ liters per mole; in addition, this macromolecule was retained by diethylaminoethyl (DEAE) ion exchangers. On the basis of these properties, the Colcemid-binding substance in S. cerevisiae cells was provisionally identified as microtubule subunits.     

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.     

Distribution of injected MRI contrast agents in mouse livers studied by confocal and SIMS microscopy

OBJECTIVE: To localize magnetic resonance imaging (MRI) contrast agents injected intravenously into mouse livers. STUDY DESIGN: Parallel studies were performed on fluorescent europium and nonfluorescent, paramagnetic gadolinium and on a product combining nanoparticles of Fe and Texas Red to obtain combined information on the distribution of these molecules inside the liver. The distribution of different superparamagnetic iron oxides was also studied because the size of these new compounds is not always convenientfor microcirculation studies. RESULTS: Europium and Texas Red can be detected by confocal microscopy. Europium, iron and gadolinium can be detected by secondary ion mass spectrometry (SIMS) microscopy. Studies confirmed the complementarity of both microscopies. They also confirmed the possibility of using europium as a model of gadolinium to analyze thefate of MRI contrast agents. CONCLUSION: The methodology can be used on mice injected intravenously and analyzed by confocal and SIMS microscopy to localize MRI contrast agents inside cellular and tissue specimens of mice.     

Modulation of heme and myristate binding to human serum albumin by anti-HIV drugs. An optical and NMR spectroscopic study

Human serum albumin (HSA) has an extraordinary ligand-binding capacity, and transports Fe(III)heme and medium- and long-chain fatty acids. In human immunodeficiency virus-infected patients the administered drugs bind to HSA and act as allosteric effectors. Here, the binding of Fe(III)heme to HSA in the presence of three representative anti-HIV drugs and myristate is investigated. Values of the dissociation equilibrium constant K(d) for Fe(III)heme binding to HSA were determined at different myristate concentrations, in the absence and presence of anti-HIV drugs. Nuclear magnetic relaxation dispersion profiles of HSA-Fe(III)heme were measured, at different myristate concentrations, in the absence and presence of anti-HIV drugs. Structural bases for anti-HIV drug binding to HSA are provided by automatic docking simulation. Abacavir and nevirapine bind to HSA with K(d) values of 1 x 10(-6) and 2 x 10(-6) M, respectively. Therefore, at concentrations used in therapy (in the 1-5 x 10(-6) M range) abacavir and nevirapine bind to HSA and increase the affinity of heme for HSA. In the presence of abacavir or nevirapine, the affinity is not lowered by myristate. FA7 should therefore be intended as a secondary binding site for abacavir and nevirapine. Binding of atazanavir is limited by the large size of the drug, although preferential binding may be envisaged to a site positively coupled with FA1 and FA2, and negatively coupled to FA7. As a whole, these results provide a foundation for the comprehension of the complex network of links modulating HSA-binding properties.     

1H-NMR studies of synthetic peptide analogues of calcium-binding site III of rabbit skeletal troponin C: effect on the lanthanum affinity of the interchange of aspartic acid and asparagine residues at the metal ion coordinating positions

The present work determines the contribution of liganding aspartic acid (Asp) residues, at the +X, +Y, and +Z metal ion coordinating positions, to the lanthanum(3+) (La3+) ion binding affinity of synthetic analogues of calcium-binding site III of rabbit skeletal troponin C. Eight 13-residue synthetic analogues were prepared by solid-phase synthesis; the primary sequences of these analogues represent all possible combinations having aspartic acid and asparagine at the +X, +Y, and +Z positions. High-field proton nuclear magnetic resonance (NMR) spectroscopy was used to monitor the binding of the La3+ ion to each of the analogues. Comparison of the chemical shift changes showed large variations in the magnitude of the shift; these were reflected in the La3+ ion association constants determined for each analogue. The association constants ranged from 9.1 x 10(2) M-1 to 2.5 x 10(5) M-1. It was observed that those analogues with the larger number of acidic residues to coordinate the La3+ ion yielded the higher association constants. The La3+ ion binding results demonstrate that the Asp residues at the positions of study contribute equally and in an additive manner to the association constant and that the presence of neighboring Asp residues at either the +X and +Y, the +Y and +Z, or the +X and +Y and +Z metal ion coordinating positions introduced dentate-dentate repulsion, which, acts as to detract from the La3+ ion association constant of the analogues.     

Synthesis,characterization, and binding assessment with human serum albumin of three bipyridine lanthanide(III) complexes

In this work, the terbium(III), dysprosium(III), and ytterbium(III) complexes containing 2, 2′-bipyridine (bpy) ligand have been synthesized and characterized using CHN elemental analysis, FT-IR, UV–Vis and ¹H-NMR techniques and their binding behavior with human serum albumin (HSA) was studied by UV–Vis, fluorescence and molecular docking examinations. The experimental data indicated that all three lanthanide complexes have high binding affinity to HSA with effective quenching of HSA fluorescence via static mechanism. The binding parameters, the type of interaction, the value of resonance energy transfer, and the binding distance between complexes and HSA were estimated from the analysis of fluorescence measurements and Förster theory. The thermodynamic parameters suggested that van der Waals interactions and hydrogen bonds play an important role in the binding mechanism. While, the energy transfer from HSA molecules to all these complexes occurs with high probability, the order of binding constants (BpyTb > BpyDy > BpyYb) represents the importance of radius of Ln³⁺ ion in the complex-HSA interaction. The results of molecular docking calculation and competitive experiments assessed site 3 of HSA, located in subdomain IB, as the most probable binding site for these ligands and also indicated the microenvironment residues around the bound mentioned complexes. The computational results kept in good agreement with experimental data.     

Using Molecular Dynamics to Predict Factors Affecting Binding Strength and Magnetic Relaxivity of MRI Contrast Agents

We demonstrate the use of molecular dynamics and molecular mechanics methods to calculate properties and behavior of metal-chelate complexes that can be used as MRI contrast agents. Static and dynamic properties of several known agents were calculated and compared with experiment. We calculated the static properties such as the q-values (number of inner shell waters) and binding distances of chelate atoms to the metal ion for a set of chelates with known X-ray structure. The dynamic flexibility of the chelate arms was also calculated. These computations were extended to a series of exploratory chelate structures in order to estimate their potential as MRI contrast agents. We have also calculated for the first time the NMR relaxivity of an MRI contrast agent using a long (5 nsec) molecular dynamics simulation. Our predictions are promising enough that the method should prove useful for evaluating novel candidate compounds before they are synthesized. One novel static property, the projected area of chelate atoms onto a virtual surface centered on the metal ion (gnomonic projection), was found to give an effective measure of how well the chelate atoms use the free space around the metal ion.     

Studies by biointeraction chromatography of binding by phenytoin metabolites to human serum albumin

Biointeraction studies based on high performance affinity chromatography were used to investigate the binding of human serum albumin (HSA) to two major phenytoin metabolites: 5-(3-hydroxyphenyl)-5-phenylhydantoin (m-HPPH) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH). This was initially examined by conducting self-competition zonal elution experiments in which m-HPPH or p-HPPH were placed in both the mobile phase and injected sample. It was found that each metabolite had a single major binding site on HSA. Competitive zonal elution experiments using l-tryptophan, warfarin, digitoxin, and cis-clomiphene as site-selective probes indicated that m-HPPH and p-HPPH were interacting with the indole-benzodiazepine site of HSA. The estimated association equilibrium constants for m-HPPH and p-HPPH at this site were 3.2 (+/-1.2)x10(3) and 5.7 (+/-0.7)x10(3)M(-1), respectively, at pH 7.4 and 37 degrees C. Use of these metabolites as competing agents for injections of phenytoin demonstrated that m-HPPH and p-HPPH had direct competition with this drug at the indole-benzodiazepine site. However, the use of phenytoin as a competing agent indicated that this drug had additional negative allosteric interactions on the binding of these metabolites to HSA. These results agreed with previous studies on the binding of phenytoin to HSA and its effects on the interactions of HSA with site-selective probes for the indole-benzodiazepine site.     

1H-NMR study on the interactions of human serum albumin with free fatty acid

Binding of free fatty acid (FFA) to human serum albumin (HSA) was studied by 1H-NMR spectroscopy. Addition of FFA to defatted HSA at a mole ratio (FFA/HSA) up to 4 caused a small change in the NMR spectrum of HSA. The integrated intensity of sharp signals of the histidine C2 proton region of HSA decreased as the mole ratio was increased from 0 to 4 for both medium chain (lauric acid) and long chain (palmitic acid, stearic acid, and oleic acid) FFA's. By contrast, when the mole ratio was increased above 4, several histidine C2 proton signals coalesced and sharpened. Therefore, the HSA molecule appears to have a different conformation on binding with more than 4 FFA molecules, which allows increased local motions of HSA. By analyzing the NMR difference spectra of HSA with various amounts of FFA, the conformational change of HSA was investigated in more detail. The difference spectrum between [HSA + 2FFA] and [HSA + FFA] was almost the same as the difference spectrum between [HSA + FFA] and [HSA], which suggests that one primary site binds a pair of FFA molecules. These results are consistent with those of a spectroscopic study with polyene fatty acids (Berde, C.B., et al. (1979) J. Biol. Chem. 254, 391-400). The existence of a bimolecular complex of FFA molecules in aqueous solution may facilitate this type of binding. Similarly, it was found that the third and fourth FFA molecules were bound to a secondary site on HSA, because the difference spectrum between [HSA + 4FFA] and [HSA + 3FFA] was nearly equal to the difference spectrum between [HSA + 3FFA] and [HSA + 2FFA]. Further addition of FFA resulted in a drastic spectral change of HSA. The NMR difference spectrum between HSA solutions with perdeuterated FFA and those with undeuterated FFA gave the 1H-NMR spectra of FFA molecules bound to HSA. Titration of FFA revealed that, in the binding to the primary site of HSA, the carboxyl group of FFA is tightly bound to the protein, whereas the methyl group is not so firmly bound. In contrast, in the binding to low affinity sites, the methyl group is bound to HSA as tightly as other portions of the molecule.     

Towards MRI contrast agents of improved efficacy. NMR relaxometric investigations of the binding interaction to HSA of a novel heptadentate macrocyclic triphosphonate Gd(III)-complex

 A novel heptacoordinating ligand consisting of a thirteen-membered tetraazamacrocycle containing the pyridine ring and bearing three methylenephosphonate groups (PCTP-[13]) has been synthesized. Its Gd(III) complex displays a remarkably high longitudinal water proton relaxivity (7.7 mM^–1 s^–1 at 25  °C, 20 MHz and pH 7.5) which has been accounted for in terms of contributions arising from (1) one water molecule bound to the metal ion, (2) hydrogen-bonded water molecules in the second coordination sphere, or (3) water molecules diffusing near the paramagnetic chelate. Variable-temperature ¹⁷O-NMR transverse relaxation data indicate that the residence lifetime of the metal-bound water molecule is very short (8.0 ns at 25  °C) with respect to the Gd(III) complexes currently considered as contrast agents for magnetic resonance imaging. Furthermore, GdPCTP-[13] interacts with human serum albumin (HSA), likely through electrostatic forces. By comparing water proton relaxivity data for the GdPCTP-[13]-HSA adduct, measured as a function of temperature and magnetic field strength, with those for the analogous adduct with GdDOTP (a twelve-membered tetraaza macrocyclic tetramethylenephosphonate complex lacking a metal-bound water molecule), it has been possible to propose a general picture accounting for the main determinants of the relaxation enhancement observed when a paramagnetic Gd(III) complex is bound to HSA. Basically, the relaxation enhancement in these systems arises from (1) water molecules in the hydration shell of the macromolecule and protein exchangeable protons which lie close to the interaction site of the paramagnetic complex and (2) the metal bound water molecule(s). As far as the latter contribution is concerned, the interaction with the protein causes an elongation of the residence lifetime of the metal-bound water molecule, which limits, to some extent, the potential relaxivity enhancement expected upon the binding of the paramagnetic complex to HSA. Received: 27 January 1997 / Accepted: 12 May 1997     

Flunitrazepam, a 7-nitro-1,4-benzodiazepine that is unable to bind to the indole-benzodiazepine site of human serum albumin

Benzodiazepine (BDZ) is generally thought to bind to site II of human serum albumin (HSA), also known as the indole-BDZ site, which is located at subdomain III A of the molecule. However, differences in the binding characteristics of BDZ drugs with HSA have been reported. The photolabeling profiles of HSA with [(3)H]flunitrazepam (FNZP) in the presence and absence of diazepam (DZP) were shown to be identical, suggesting that each drug primarily binds to different regions. The results of fluorescent probe displacement experiments showed that FNZP failed to decrease the fluorescence of dansylsarcosine to an extent similar to that of DZP. In the photoinhibition experiment, site I and site II ligands failed to inhibit the photoincorporation of [(3)H]FNZP to HSA. In order to evaluate the photolabeling specificity of FNZP, an attempt was made to photolabel alpha(1)-acid glycoprotein (AGP) which also binds BDZ with similar affinity as HSA. The effect of myristate (MYR) and DZP on the FNZP photolabeling of these two major drug binding plasma proteins was examined. Photoincorporation was inhibited when HSA was photolabeled with [(3)H]FNZP in the presence of MYR but not in the presence of DZP. Conversely, DZP inhibited the photolabeling of [(3)H]FNZP to AGP. These results suggest that FNZP interacts with HSA at regions which are not located in the preformed binding pocket of subdomain III A.     

The disaccharide anthracycline MEN 10755 binds human serum albumin to a non-classical drug binding site

The interaction of the novel disaccharide anthracycline MEN 10755 with human serum albumin (HSA) was investigated by visible absorption and fluorescence spectroscopies and by ultrafiltration. Notably, MEN 10755 binds serum albumin far stronger than doxorubicin. Albumin binding results into a drastic quenching of the intrinsic fluorescence of MEN 10755; a binding constant of 1.1 x 10(5) was determined from fluorescence data. To localize the HSA binding site of MEN 10755 competition experiments were carried out with ligands that are selective for the different drug binding sites of the protein. No relevant competition effects were seen in the case of warfarin, diazepam and hemin, known ligands of sites I, II and III, respectively. Modest effects were observed following addition of palmitic acid that targets the several fatty acid binding sites of the protein. In contrast, extensive displacement of the bound anthracycline was achieved upon addition of ethacrinic acid. On the basis of these results, it is proposed that MEN 10755 binds serum albumin tightly to a non-canonical surface binding site for which it competes specifically with ethacrinic acid.     

Human serum albumin binding assay based on displacement of a non selective fluorescent inhibitor

In this paper, we describe a fluorescent antibacterial analog, 6, with utility as a competition probe to determine affinities of other antibacterial analogs for human serum albumin (HSA). Analog 6 bound to HSA with an affinity of 400+/-100 nM and the fluorescence was environmentally sensitive. With 370 nm excitation, environmental sensitivity was indicated by a quenching of the 530 nm emission when the probe bound to HSA. Displacement of dansylsarcosine from HSA by 6 indicated it competed with compounds that bound at site II (ibuprofen binding site) on HSA. Analog 6 also shifted the NMR peaks of an HSA bound oleic acid molecule that itself was affected by compounds that bound at site II. In addition to binding at site II, 6 interacted at site I (warfarin binding site) as indicated by displacement of dansylamide and the shifting of NMR peaks of an HSA bound oleic acid molecule affected by warfarin site binding. Additional evidence for multiple site interaction was discovered when a percentage of 6 could be displaced by either ibuprofen or phenylbutazone. A competition assay was established using 6 to determine relative affinities of other antibacterial inhibitors for HSA.     

The HSA affinity of warfarin and flurbiprofen determined by fluorescence anisotropy measurements of camptothecin

The determination of affinity of warfarin and flurbiprofen to human serum albumin (HSA) by fluorescence anisotropy measurements of carboxylate form of camptothecin (CPT-C) is the subject of this paper. A simple method based on measurements of fluorescence anisotropy of CPT-C allows to determine the affinity constant of CPT-C to HSA by computation of the fraction of bound CPT-C molecules with HSA It was observed, that adding of competing drug to plasma significant reduces the rate of increase of CPT-C fluorescence anisotropy with increase of albumin concentration and, the affinity constant of CPT-C to HSA decreases. The hypothesis of interactions between competing drug and CPT-C is presented. The results of these studies suggest that CPT-C displaces other drug from protein binding site and the degree of this displacement depends on concentration of drug and drug-HSA binding affinity. The presented in this paper biosystems research allows to estimate the affinity constant of warfarin and flurbiprofen. It was also confirmed that despite that most of drugs bind predominantly to Site I or Site II of HSA (only one of these sites is high-affinity site), at elevated concentrations, part of drug molecules can be bound to low-affinity site of HSA.