Fipronil recognition by the FA1 site of human serum albumin

Fipronil is a broad‐spectrum pesticide widely used in agriculture, horticulture, and forestry. Because fipronil can cause a variety of toxic effects in animals and humans, its use is authorized as a pesticide in veterinary medicinal products for pets, but not for the treatment of livestock animals whose products are intended for consumption. Recently, however, the presence of fipronil residues has been detected in the eggs and meat of layer hens from farms located in different European countries. Given the relevance of fipronil toxicity for human health, it is important to gain information concerning its fate in the human body, including its binding mode to human serum albumin (HSA), the most abundant protein in plasma. Here, the inhibition of heme‐Fe(III) binding to the fatty acid site 1 (FA1) of HSA by fipronil is reported. Docking simulations support functional data, indicating that the FA1 site is the preferential cleft for fipronil recognition by HSA. The affinity of fipronil for HSA (K_f = 1.9 × 10^?6 M, at pH 7.3, and 20.0°C) may be relevant in vivo. Indeed, HSA could play a pivotal role in fipronil transport and scavenging, thus reducing the pesticide‐free plasmatic levels, with consequent reduced systemic toxicity. In turn, fipronil binding to the FA1 site of HSA could impair the recognition of endogenous and exogenous molecules.     

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.     

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.     

Warfarin modulates the nitrite reductase activity of ferrous human serum heme–albumin

Human serum heme–albumin (HSA–heme–Fe) displays reactivity and spectroscopic properties similar to those of heme proteins. Here, the nitrite reductase activity of ferrous HSA–heme–Fe [HSA–heme–Fe(II)] is reported. The value of the second-order rate constant for the reduction of $ {\text{NO}}_{2}^{ - } $ to NO and the concomitant formation of nitrosylated HSA–heme–Fe(II) (i.e., k _on) is 1.3 M^?1 s^?1 at pH 7.4 and 20 °C. Values of k _on increase by about one order of magnitude for each pH unit decrease between pH 6.5 to 8.2, indicating that the reaction requires one proton. Warfarin inhibits the HSA–heme–Fe(II) reductase activity, highlighting the allosteric linkage between the heme binding site [also named the fatty acid (FA) binding site 1; FA1] and the drug-binding cleft FA2. The dissociation equilibrium constant for warfarin binding to HSA–heme–Fe(II) is (3.1 ± 0.4) × 10^?4 M at pH 7.4 and 20 °C. These results: (1) represent the first evidence for the $ {\text{NO}}_{2}^{ - } $ reductase activity of HSA–heme–Fe(II), (2) highlight the role of drugs (e.g., warfarin) in modulating HSA(–heme–Fe) functions, and (3) strongly support the view that HSA acts not only as a heme carrier but also displays transient heme-based reactivity.     

Ibuprofen binding to secondary sites allosterically modulates the spectroscopic and catalytic properties of human serum heme-albumin

The ibuprofen primary binding site FA3-FA4 is located in domain III of human serum albumin (HSA), the secondary clefts FA2 and FA6 being sited in domains I and II. Here, the thermodynamics of ibuprofen binding to recombinant Asp1-Glu382 truncated HSA (tHSA)-heme-Fe(III) and nitrosylated tHSA-heme-Fe(II), encompassing domains I and II only, is reported. Moreover, the allosteric effect of ibuprofen on the kinetics of tHSA-heme-Fe(III)-mediated peroxynitrite isomerization and nitrosylated tHSA-heme-Fe(II) denitrosylation has been investigated. The present data indicate, for the first time, that the allosteric modulation of tHSA-heme and HSA-heme reactivity by ibuprofen depends mainly on drug binding to the FA2 and FA6 secondary sites rather than drug association with the FA3-FA4 primary cleft. Thus, tHSA is a valuable model with which to investigate the allosteric linkage between the heme cleft FA1 and the ligand-binding pockets FA2 and FA6, all located in domains I and II of (t)HSA.     

Binding of δ9-tetrahydrocannabinol and diazepam to human serum albumin

Cannabis is the most commonly used illicit drug worldwide. Cannabis users also appear to use other psychoactive drugs more frequently than noncannabis users. Here, Δ9-tetrahydrocannabinol (THC) and diazepam binding to human serum albumin (HSA) and HSA-heme is reported. THC binds to two different binding sites of HSA (K(d1) ≤ 10(-7) M and K(d2) = 10(-3)M) without affecting diazepam binding (K(d) = 1.2 × 10(-5) M). THC binding to the high-affinity site accounts for the low free fraction of the drug in plasma. Moreover, THC increases the affinity of heme for HSA. Accordingly, the affinity of THC for HSA-heme is higher than that for HSA. THC could bind to FA2 and FA7 sites, as substantiated by docking simulations; nevertheless, the observed allosteric effect(s) suggests that the primary binding site of THC is the FA2 cleft that positively modulates heme affinity. Possibly, the HSA conformational transition(s) induced by THC binding could account for drug delivery to the liver through receptor- mediated endocytosis.     

DNA/HSA interaction and nuclease activity of an iron(III) amphiphilic sulfonated corrole

The DNA binding of amphiphilic iron(III) 2,17‐bis(sulfonato)‐5,10,15‐tris(pentafluorophenyl)corrole complex (Fe–SC) was studied using spectroscopic methods and viscosity measurements. Its nuclease‐like activity was examined by using pBR322 DNA as a target. The interaction of Fe–SC with human serum albumin (HSA) in vitro was also examined using multispectroscopic techniques. Experimental results revealed that Fe–SC binds to ct‐DNA via an outside binding mode with a binding constant of 1.25 × 10⁴ M^–1. This iron corrole also displays good activity during oxidative DNA cleavage by hydrogen peroxide or tert‐butyl hydroperoxide oxidants, and high‐valent (oxo)iron(V,VI) corrole intermediates may play an important role in DNA cleavage. Fe–SC exhibits much stronger binding affinity to site II than site I of HSA, indicating a selective binding tendency to HSA site II. The HSA conformational change induced by Fe–SC was confirmed by UV/Vis and CD spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Cantharidin poisoning associated with specific binding site in liver

Cantharidin, the potent vesicant and toxicant of blister beetles, was prepared as a radioligand to probe its mechanism of action. [3H]Cantharidin interacts in a saturable and specific manner with a binding site in mouse liver cytosol with apparent Kd and Bmax values of 30 nM and 1.8 pmol/mg protein, respectively. Comparisons of cantharidic acid, the related herbicide endothal, and 20 other oxabicycloheptane-dicarboxylic acids show that their potency as inhibitors of [3H]cantharidin binding is closely correlated with their intraperitoneal toxicity to mice. This binding site is also inhibited in vivo by toxic doses of cantharidin. The [3H]cantharidin binding site in mouse liver cytosol therefore represents, or serves as a model for, the site of toxic action of cantharidin and structurally-related compounds.     

Drug binding to Sudlow's site I impairs allosterically human serum heme-albumin-catalyzed peroxynitrite detoxification

Heme endows human serum albumin (HSA) with globin-like reactivity and spectroscopic properties. Here, the effect of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, tolbutamide, and warfarin on peroxynitrite isomerization to NO(3) (-) by ferric HSA-heme (HSA-heme-Fe(III)) is reported. Drugs binding to Sudlow's site I impair dose-dependently peroxynitrite isomerization by HSA-heme-Fe(III). The allosteric modulation of HSA-heme-Fe(III)-mediated peroxynitrite isomerization by drugs has been ascribed to the pivotal role of Tyr150, a residue that either provides a polar environment in Sudlow's site I or protrudes into the heme cleft (i.e., the fatty acid site 1, FA1), depending on ligand occupancy of either sites.     

Probing the mechanism of interaction of metoprolol succinate with human serum albumin by spectroscopic and molecular docking analysis

In the present work, the mechanism of the interaction between a β1 receptor blocker, metoprolol succinate (MS) and human serum albumin (HSA) under physiological conditions was investigated by spectroscopic techniques, namely fluorescence, Fourier transform infra‐red spectroscopy (FT‐IR), fluorescence lifetime decay and circular dichroism (CD) as well as molecular docking and cyclic voltammetric methods. The fluorescence and lifetime decay results indicated that MS quenched the intrinsic intensity of HSA through a static quenching mechanism. The Stern–Volmer quenching constants and binding constants for the MS–HSA system at 293, 298 and 303 K were obtained from the Stern–Volmer plot. Thermodynamic parameters for the interaction of MS with HSA were evaluated; negative values of entropy change (ΔG°) indicated the spontaneity of the MS and HSA interaction. Thermodynamic parameters such as negative ΔH° and positive ΔS° values revealed that hydrogen bonding and hydrophobic forces played a major role in MS–HSA interaction and stabilized the complex. The binding site for MS in HSA was identified by competitive site probe experiments and molecular docking studies. These results indicated that MS was bound to HSA at Sudlow's site I. The efficiency of energy transfer and the distance between the donor (HSA) and acceptor (MS) was calculated based on the theory of Fosters' resonance energy transfer (FRET). Three‐dimensional fluorescence spectra and CD results revealed that the binding of MS to HSA resulted in an obvious change in the conformation of HSA. Cyclic voltammograms of the MS–HSA system also confirmed the interaction between MS and HSA. Furthermore, the effects of metal ions on the binding of MS to HSA were also studied.     

Study on the molecular recognition action of lamivudine by human serum albumin

In this work, the interaction of an anti‐HIV drug lamivudine and human serum albumin (HSA) was studied by multispectroscopic and molecular modeling methods. The fluorescence emission spectra showed that the fluorescence of HSA was quenched by lamivudine through static mechanism with HSA‐lamivudine complex produced at ground state. According to the binding equilibriums observed at 4 different temperatures, the number of binding site, binding constant, enthalpy change, entropy change, and Gibbs free energy change of the interaction were calculated. The results indicated that there was only 1 main binding site under present concentration condition, and then, the location of this binding site was ascertained by molecular probe experiments using warfarin and ibuprofen as site markers. The interaction was a spontaneous and exothermic process. Hydrogen bonds and van der Waals force were the major power that fixed lamivudine on Sudlow's site I in subdomain IIA of HSA molecule. The distance between donor and acceptor was determined by Förster's nonradiative fluorescence resonance energy transfer theory. Circular dichroism spectra exhibited the alteration of HSA's secondary structures. Molecular modeling investigation revealed the structure of HSA‐lamivudine complex, including the conformation of lamivudine in binding site, the amino residues close to lamivudine, and the interaction forces between receptor and ligand. The study may be beneficial to therapists in understanding the distribution of lamivudine in vivo and explaining its drug‐resistant mechanism in clinical diagnosis.     

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.     

A fluorescent fatty acid probe, DAUDA, selectively displaces two myristates bound in human serum albumin

11-(Dansylamino) undecanoic acid (DAUDA) is a dansyl-type fluorophore and has widely used as a probe to determine the binding site for human serum albumin (HSA). Here, we reported that structure of HSA-Myristate-DAUDA ternary complex and identified clearly the presence of two DAUDA molecules at fatty acid (FA) binding site 6 and 7 of HSA, thus showing these two sites are weak FA binding sites. This result also show that DAUDA is an appropriate probe for FA site 6 and 7 on HSA as previous studied, but not a good probe of FA binding site 1 that is likely bilirubin binding site on HSA.     

Reversible binding of ethacrynic acid to human serum albumin: Difference circular dichroism study

The reversible binding of ethacrynic acid was characterized by a difference circular dichroism method. A 2/1 stoichiometry was determined for the [drug]/[HSA] (human serum albumin) complex. The reversible binding of ethacrynic acid to HSA determines direct competition with ligands that selectivity bind to site II and to the fatty acid site. Furthermore, indirect competition was shown for ligands for site I (anticooperative) and to site III (cooperative). Chirality 11:33–38, 1999. © 1999 Wiley‐Liss, Inc.     

Binding and relaxometric properties of heme complexes with cyanogen bromide fragments of human serum albumin

The spectroscopic and reactivity properties of hemin complexes formed with cyanogen bromide fragments B (residues 1-123), C (124-298), A (299-585), and D (1-298) of human serum albumin (HSA) have been investigated. The complex hemin-D exhibits binding, spectral, circular dichroism, and reactivity characteristics very similar to those of hemin-HSA, indicating that fragment D contains the entire HSA domain involved in heme binding. The characteristics of the other hemin complexes are different, and a detailed investigation of the properties of hemin-C has been carried out because this fragment contains the HSA binding region of several important drugs. Hemin-C contains a low-spin Fe(III) center, with two imidazole ligands, but the complex undergoes a reversible structural transition at basic pH leading to a high-spin, five-coordinated Fe(III) species. This change determines a marked increase in the relaxation rate of water protons. Limited proteolysis experiments and mass spectral analysis carried out on fragment C and hemin-C show that the region encompassing residues Glu-208 to Trp-214 is protected from activity of proteases in the complex and, therefore, is involved in the interaction with hemin. A structural model of fragment C enables us to propose that His-242 and His-288 are the axial ligands for the Fe(III) center.     

Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus

Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain.     

Investigation of the interaction of aurantio‐obtusin with human serum albumin by spectroscopic and molecular docking methods

The interaction between human serum albumin (HSA) and aurantio‐obtusin was investigated by spectroscopic techniques combined with molecular docking. The Stern–Volmer quenching constants (K_SV) decreased from 8.56 × 10⁵ M^?1 to 5.13 × 10⁵ M^?1 with a rise in temperatures from 289 to 310 K, indicating that aurantio‐obtusin produced a static quenching of the intrinsic fluorescence of HSA. Time‐resolved fluorescence studies proved again that the static quenching mechanism was involved in the interaction. The sign and magnitude of the enthalpy change as well as the entropy change suggested involvement of hydrogen bonding and hydrophobic interaction in aurantio‐obtusin–HSA complex formation. Aurantio‐obtusin binding to HSA produced significant alterations in secondary structures of HSA, as revealed from the time‐resolved fluorescence, Fourier transform infrared (FT‐IR) spectroscopy, three‐dimensional (3D) fluorescence and circular dichroism (CD) spectral results. Molecular docking study and site marker competitive experiment confirmed aurantio‐obtusin bound to HSA at site I (subdomain IIA).     

Cantharidin world on islands: Species diversity of canthariphilous arthropods in the Izu–Ogasawara Arc

Cantharidin is a defense chemical produced by only two beetle families, Meloidae and Oedemeridae. This compound also functions as an attractant for certain groups of arthropods (canthariphilous arthropods). Cantharidin‐producing beetles and canthariphilous arthropods build a cantharidin‐mediated community, called cantharidin world. In this study, to clarify the canthariphilous arthropod community formed on small islands, the arthropods were collected by cantharidin‐baited traps and compared among the mainland, peninsula and 11 small islands of the Izu–Ogasawara Arc, Japan. Control traps without cantharidin were also used to collect non‐canthariphilous arthropods. Cantharidin‐producing beetles were distributed across the study area, but the number of species was reduced on islands. We identified 10 species of five families as canthariphilous arthropods in this study area: two species of Anthicidae and Pyrochroidae (Coleoptera), four species of Ceratopogonidae (Diptera), three species of Formicidae (Hymenoptera) and one species of Podoctidae (Opiliones). The number of canthariphilous species was not correlated with island size (area) but was negatively correlated with the distance of island from the mainland. These tendencies were also seen in non‐canthariphilous fauna. The canthariphilous arthropods increased with the number of cantharidin‐producing beetle species, but the non‐canthariphilous fauna did not. Thus, the cantharidin world on islands seems to be affected not only by the geographic characteristics of islands, particularly the degree of isolation from the mainland, but also by the diversity of cantharidin‐producing members within it.     

Insight into the interaction of antitubercular and anticancer compound clofazimine with human serum albumin: spectroscopy and molecular modelling

The binding of clofazimine to human serum albumin (HSA) was investigated by applying optical spectroscopy and molecular docking methods. Fluorescence quenching data revealed that clofazimine binds to protein with binding constant in the order of 10⁴ M^?1, and with the increase in temperature, Stern–Volmer quenching constants gradually decreased indicating quenching mode to be static. The UV–visible spectra showed increase in absorbance upon interaction of HSA with clofazimine which further reveals formation of the drug–albumin complex. Thermodynamic parameters obtained from fluorescence data indicate that the process is exothermic and spontaneous. Forster distance (R_o) obtained from fluorescence resonance energy transfer is found to be 2.05 nm. Clofazimine impelled rise in α-helical structure in HSA as observed from far-UV CD spectra while there are minor alterations in tertiary structure of the protein. Clofazimine interacts strongly with HSA inducing secondary structure in the protein and slight alterations in protein topology as suggested by dynamic light scattering results. Moreover, docking results indicate that clofazimine binds to hydrophobic pocket near to the drug site II in HSA.     

Heme impairs allosterically drug binding to human serum albumin Sudlow's site I

Human serum albumin (HSA), the most prominent protein in plasma, is best known for its exceptional ligand (e.g., heme and drugs) binding capacity. Here, the binding of chlorpropamide, digitoxin, furosemide, indomethacin, phenylbutazone, sulfisoxazole, and tolbutamide to HSA and ferric heme-HSA is reported. Moreover, ferric heme binding to HSA in the absence and presence of drugs has been investigated. Values of the association equilibrium constant for drug binding to Sudlow’s site I of ferric heme-HSA (ranging between 1.7 × 10³ and 1.6 × 10⁵ M⁻¹) are lower by one order of magnitude than those for drug binding to ferric heme-free HSA (ranging between 1.9 × 10⁴ and 1.8 × 10⁶ M⁻¹). According to linked functions, the value of the association equilibrium constant for heme binding to HSA decreases from 7.8 × 10⁷ M⁻¹, in the absence of drugs to 7.0 × 10⁶ M⁻¹, in the presence of drugs. These findings represent a clear-cut evidence for the allosteric inhibition of drug binding to HSA Sudlow’s site I by the heme. According to linked functions, drugs impair allosterically heme binding to HSA. These results appear to be relevant in the drug therapy and management.