Intracellular protein binding patterns of the anticancer ruthenium drugs KP1019 and KP1339

The ruthenium compound KP1019 has demonstrated promising anticancer activity in a pilot clinical trial. This study aims to evaluate the intracellular uptake/binding patterns of KP1019 and its sodium salt KP1339, which is currently in a phase I–IIa study. Although KP1339 tended to be moderately less cytotoxic than KP1019, IC₅₀ values in several cancer cell models revealed significant correlation of the cytotoxicity profiles, suggesting similar targets for the two drugs. Accordingly, both drugs activated apoptosis, indicated by caspase activation via comparable pathways. Drug uptake determined by inductively coupled plasma mass spectrometry (ICP-MS) was completed after 1 h, corresponding to full cytotoxicity as early as after 3 h of drug exposure. Surprisingly, the total cellular drug uptake did not correlate with cytotoxicity. However, distinct differences in intracellular distribution patterns suggested that the major targets for the two ruthenium drugs are cytosolic rather than nuclear. Consequently, drug–protein binding in cytosolic fractions of drug-treated cells was analyzed by native size-exclusion chromatography (SEC) coupled online with ICP-MS. Ruthenium–protein binding of KP1019- and KP1339-treated cells distinctly differed from the platinum binding pattern observed after cisplatin treatment. An adapted SEC-SEC-ICP-MS system identified large protein complexes/aggregates above 700 kDa as initial major binding partners in the cytosol, followed by ruthenium redistribution to the soluble protein weight fraction below 40 kDa. Taken together, our data indicate that KP1019 and KP1339 rapidly enter tumor cells, followed by binding to larger protein complexes/organelles. The different protein binding patterns as compared with those for cisplatin suggest specific protein targets and consequently a unique mode of action for the ruthenium drugs investigated.     

Serum-protein interactions with anticancer Ru(III) complexes KP1019 and KP418 characterized by EPR

The compounds imidazolium [trans-[RuCl₄(1H-imidazole)₂] (KP418) and indazolium [trans-RuCl₄(1H-indazole)₂] (KP1019) both show significant anticancer activity, with the latter recently having completed phase I clinical trials. An important component of this success has been associated with targeted delivery of the complexes to cancer cells by serum proteins. In this study, electron paramagnetic resonance (EPR) measurements, combined with incubation under physiological conditions, and separation of protein-bound fractions, have been used to characterize the interactions of these complexes with human serum albumin (hsA), human serum transferrin (hsTf) apoprotein, and whole human serum. The strong EPR signals observed in these experiments demonstrate that both complexes are primarily retained in the 3+ oxidation state in the presence of serum components. Rapid, noncovalent binding of KP1019 was observed in the presence of both hsA and serum, indicating that the predominant interactions occur within the hydrophobic binding sites of hsA. This sequestering process correlates with the low levels of side effects observed in clinical trials of the complex. At longer incubation times, the noncovalently bound complexes are converted slowly to a protein-coordinated form. Noncovalent interactions are not observed in the presence apo-hsTf, where only slow binding of KP1019 via ligand exchange with the protein occurs. By contrast, hydrophobic interactions of KP418 with hsA only occur with the aquated products of the complex, a process that also dominates in serum. In the presence of apo-hsTf, KP418 interacts directly with the protein through exchange of ligands, as observed with KP1019.     

The sulfur-cyanolysis sites of serum albumin: metabolite competition studies

In the presence of a source of sulfane sulfur, a cyanolysis reaction catalyzed by serum albumin may contribute to cyanide detoxication. The active site for this catalysis by serum albumin has been investigated in competition studies with ligands that have known albumin binding sites. Despite complications caused by the occurrence of multiple primary and secondary sites for many ligands, the results show that the primary sites for bilirubin, steroids, indoles, aspirin, and palmitate are distinct from that for sulfur. Laurate is a tight-binding partial inhibitor of the cyanolysis reaction, competitive with cyanide rather than with sulfur. In view of the formal mechanism previously established for the catalyzed reaction, this result indicates that the sulfur-cyanolysis site is probably near the site occupied by laurate.     

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.     

Titanocene anticancer complexes and their binding mode of action to human serum albumin: a computational study

Due to the pivotal role played by human serum albumin (HSA) in the transport and cytotoxicity of titanocene complexes, a docking study has been performed on a selected set of titanocene complexes to aid in the current understanding of the potential mode of action of these titanocenes upon binding HSA. Analysis of the docking results has revealed potential binding at the known drug binding sites in HSA and has provided some explanation for the specificity and subsequent cytotoxicity of these titanocenes. Additionally, a new alternative binding site for these titanocenes has been postulated.     

Determining binding sites of drugs on human serum albumin using FIA-QCM

A simple and effective method was developed for determining binding sites of drugs on human serum albumin (HSA) by independent binding or competitive displacement of bilirubin using flow injection analysis-quartz crystal microbalance (FIA-QCM) system. Both independent and competitive bindings were entirely monitored in real time. Bilirubin as a site I-binding ligand was pre-bound to HSA sensor so as to occupy the drug-binding site I. When the model site II-binding drugs (ibuprofen, ketoprofen and flurbiprofen) were injected into the bilirubin pre-bound HSA system, the frequency continuously decreased by 6Hz, 4Hz and 5Hz, respectively, which was the same as that of their individual binding to HSA sensor. It indicated that the drug binding to site II was independent and did not interfere with bilirubin binding. However, when the model site I-binding drugs (iodipamide and magnesium salicylate) were introduced into the system, the frequency remained unchanged in the initial several minutes and then rapidly decreased by 4Hz for iodipamide and increased by 4Hz for magnesium salicylate. This phenomenon revealed site I-binding drugs competitively bound to HSA against bilirubin and displaced the pre-bound bilirubin. The results demonstrate FIA-QCM can be a valid approach for monitoring the dynamic interaction between drugs and HSA in real time further identifying drug-binding sites without the need of labels.     

Influence of ascorbic acid on the activity of the investigational anticancer drug KP1019

Ascorbic acid has been previously discussed to have antitumor potential through its interaction with transition metal ions such as iron and copper. Furthermore, ascorbic acid may act as a reducing agent for Ru(III) compounds such as indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019), an investigational anticancer drug which is supposed to be activated by reduction, prior to binding to cellular target proteins. Therefore, we investigated the influence of ascorbic acid on the activity of this antitumor metal complex in cell culture studies. We show that co-incubation of equicytotoxic, constant amounts of KP1019 with high concentrations of ascorbic acid (50–700 μM) increases cytotoxicity of the ruthenium anticancer drug in the human colon carcinoma cell line SW480, human cervical carcinoma KB-3-1 cells, and the multidrug-resistant subline KBC-1, whereas addition of low concentrations (2.7–50 μM) has a strong chemoprotective effect in the human colon carcinoma cell line SW480, but not in multidrug-resistant KBC-1 cells. Although cellular uptake of KP1019 is not altered, ascorbic acid induce stronger interaction of the ruthenium compound with DNA both in SW480 cells and under cell-free conditions with plasmid DNA. Even if DNA interactions probably play a subordinate role in vivo given the extensive protein binding of the compound, our data exemplify that ascorbic acid enhances the reactivity of KP1019 with biomolecules. Moreover, we demonstrate that the levels of KP1019-generated reactive oxygen species are markedly decreased by co-incubation with ascorbic acid. Conclusively, our results indicate that application of high doses of ascorbic acid might increase the anticancer effects of KP1019.     

Influence of aspirin and iron(III) tetrasulfonated phthalocyanine on bilirubin binding by human serum albumin

The interaction of bilirubin with aspirin-modified human serum albumin (HSA) and the influence of iron tetrasulfonated phthalocyanine on bilirubin binding by the native protein has been studied by difference spectroscopy and circular dichroism measurements. Spectroscopic studies of the systems containing bilirubin and aspirin-modified HSA compared to the analogous systems with the native protein have shown that selective acetylation of albumin at lysine 199 inhibits bilirubin binding by this protein. In both cases, interaction between bilirubin and albumin leads to complex formation at a molar ratio of ligand to protein of 2:1. The studies of the reaction of bilirubin with fragments of albumin produced by reaction with CNBr have demonstrated that one of the strong bilirubin binding sites is located in the M fragment and is close to the high-affinity binding site of aspirin. The other one was found in fragment C. Acetylation of albumin brings about marked conformational change in the protein, which probably accounts for the decrease in its ability to react with anti-HSA antibody. Bilirubin does not change the secondary structure of albumin but, like aspirin, lowers its antigenicity. It has been suggested that the decrease in antigenic properties in this case results from cooperation of the closely neighboring antigenic and bilirubin-binding sites. The studies of the influence of iron(III) tetrasulfonated phthalocyanine on bilirubin binding by HSA suggest that there is no competition between strong sites for iron(III) tetrasulfonated phthalocyanine and bilirubin, but these compounds compete for some of the weaker sites.     

Spectroscopic and thermodynamic studies on the binding of gadolinium(III) to human serum transferrin

A wide variety of thermodynamic, kinetic, and spectroscopic studies have demonstrated differences between the two metal-binding sites of transferrin. In the present investigation, we have further assessed these differences with respect to the binding of gadolinium, evaluated by UV difference spectrophotometry, electron paramagnetic resonance (EPR) titration, EPR difference spectroscopy in conjunction with urea gel electrophoresis, and equilibrium dialysis. Combinations of these studies establish that only one site of the protein binds Gd(III) sufficiently firmly to be characterized. In order to reveal which of the two sites accepts Gd(III), we made use of monoferric transferrins preferentially loaded with Fe(III) at either site in EPR spectroscopic studies. Because of the overlap of signals, difference spectroscopy was required to distinguish resonances arising from Fe(III) and Gd(III) specifically complexed to the protein. When iron is bound to the C-terminal site, leaving the N-terminal site free for binding of gadolinium, the difference spectrum shows no evidence of specific binding. However, when iron is bound to the N-terminal site, the difference spectrum shows a resonance line at g' = 4.1 indicative of specific binding, thus implicating the C-terminal site in the binding of Gd(III). The effective stability constant for the binding of Gd(III) to this site of transferrin at pH 7.4 and ambient pCO2 is 6.8 X 10(6) M-1. At physiological pCO2, the formation of nonbinding carbonato complexes of Gd(III) precludes a substantial role for transferrin in the transport of the lanthanide in vivo.     

Reactions of gold(III) complexes with serum albumin.

The reactions of a few representative gold(III) complexes -[Au(ethylenediamine)2]Cl3, [Au(diethylentriamine)Cl]Cl2, [Au(1,4,8,11-tetraazacyclotetradecane)](ClO4)2Cl, [Au(2,2',2'-terpyridine)Cl]Cl2, [Au(2,2'-bipyridine)(OH)2][PF6] and the organometallic compound [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)][PF6]- with BSA were investigated by the joint use of various spectroscopic methods and separation techniques. Weak metal-protein interactions were revealed for the [Au(ethylenediamine)2]3+ and [Au(1,4,8,11-tetraazacyclotetradecane)]3+ species, whereas progressive reduction of the gold(III) centre was observed in the cases of [Au(2,2'-bipyridine)(OH)2]+ and [Au(2,2',2'-terpyridine)Cl]2+. In contrast, tight metal-protein adducts are formed when BSA is reacted with either [Au(diethylentriamine)Cl]2+ and [Au(6-(1,1-dimethylbenzyl)-2,2'-bipyridine-H)(OH)]+. Notably, binding of the latter complex to serum albumin results in the appearance of characteristic CD bands in the visible spectrum. It is suggested that adduct formation for both of these gold(III) complexes occurs through coordination at the level of surface histidines. Stability of these gold(III) complexes/serum albumin adducts was tested under physiologically relevant conditions and found to be appreciable. Metal binding to the protein is tight; complete detachment of the metal from the protein has been achieved only after the addition of excess potassium cyanide. The implications of the present results for the pharmacological activity of these novel cytotoxic agents are discussed.     

Impacts of hydrophobicity and ionicity of phendione-based cobalt(II)/(III) complexes on binding with bovine serum albumin

Abstract

For efficient designing of metallodrugs, it is imperative to analyse the binding affinity of those drugs with drug-carrying serum albumins to comprehend their structure–activity correlation for biomedical applications. Here, cobalt(II) and cobalt(III) complexes comprising three phendione ligands, [Co(phendione)₃]Cl₂ (1) and [Co(phendione)₃]Cl₃ (2), where, phendione = 1,10-phenanthroline-5,6-dione, has been chosen to contrast the impact of their hydrophobicity and ionicity on binding with bovine serum albumin (BSA) through spectrophotometric titrations. The attained hydrophobicity values using octanol/water partition coefficient method manifested that complex 1 is more hydrophobic than complex 2, which could be attributed to lesser charge on its coordination sphere. The interaction of complexes 1 and 2 with BSA using steady state fluorescence studies revealed that these complexes quench the intrinsic fluorescence of BSA through static mechanism, and the extent of quenching and binding parameters are higher for complex 2. Further thermodynamics of BSA-binding studies revealed that complexes 1 and 2 interact with BSA through hydrophobic and hydrogen bonding/van der Waals interactions, respectively. Further, UV–visible absorption, circular dichroism and synchronous fluorescence studies confirmed the occurrence of conformational and microenvironmental changes in BSA upon binding with complexes 1 and 2. Molecular docking studies have also shown that complex 2 has a higher binding affinity towards BSA as compared to complex 1. This sort of modification of ionicity and hydrophobicity of metal complexes for getting desirable binding mode/strength with drug transporting serum albumins will be a promising pathway for designing active and new kind of metallodrugs for various biomedical applications.

Communicated by Ramaswamy H. Sarma     

Studies on the synthesis,characterization, human serum albumin binding and biological activity of single chain surfactant–cobalt(III) complexes

The interaction of surfactant–cobalt(III) complexes [Co(bpy)(dien)TA](ClO₄)₃ · 3H₂O (1) and [Co(dien)(phen)TA](ClO₄)₃ · 4H₂O (2), where bpy = 2,2′‐bipyridine, dien = diethylenetriamine, phen = 1,10‐phenanthroline and TA = tetradecylamine with human serum albumin (HSA) under physiological conditions was analyzed using steady state, synchronous, 3D fluorescence, UV/visabsorption and circular dichroism spectroscopic techniques. The results show that these complexes cause the fluorescence quenching of HSA through a static mechanism. The binding constant (K_b) and number of binding‐sites (n) were obtained at different temperatures. The corresponding thermodynamic parameters (?G°, ?H° and ?S°) and E_a were also obtained. According to Förster's non‐radiation energy transfer theory, the binding distance (r) between the complexes and HSA were calculated. The results of synchronous and 3D fluorescence spectroscopy indicate that the binding process has changed considerably the polarity around the fluorophores, along with changes in the conformation of the protein. The antimicrobial and anticancer activities of the complexes were tested and the results show that the complexes have good activities against pathogenic microorganisms and cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of spin-labelled fatty acids and hematoporphyrin binding sites interactions in serum albumin

Electron paramagnetic resonance (EPR) was used to investigate the spin-labelled fatty acid (SLFA) binding equilibrium to human (HSA) and bovine (BSA) serum albumin. The number of 5-doxyl stearate (5-DS) and 16-doxyl stearate (16-DS) binding sites on HSA and BSA were found to be equal, while the association constants, KA values (especially those of the primary binding site) were different. The applied EPR spectra analysis permitting a quantitative distinguishing between slow macromolecular rotation (pi c) and fast anisotropic motion (steric restriction, S) of bound SLFA, allowed SLFA oxazolidinyl ring mobility to be estimated. The 5-DS nitroxide radical is completely immobilized within the HSA protein matrix (S approximately 1.0, pi c approximately 56 +/- 1 ns). The 5-DS when bound to BSA exhibited the presence of more extensive fluctuations (lower S and pi c values) and its immersion depth with respect to BSA surface was calculated to be 4 +/- 2 A. The 16-DS oxazolidinyl radical bound to HSA was found to undergo moderated fluctuations (both S and pi c are smaller with respect to 5-DS) and it is buried deeper within the protein core (rimm = 10 +/- 2 A with respect to BSA surface). The tetrapyrrole ligands hematoporphyrin (Hp) and hematoporphyrin derivative (HpD) were found to induce well detectable changes in the SLFA binding patterns to serum albumin. The action mode was determined to be different for 16-DS (primary) and 5-DS (secondary) serum albumin binding sites: (i) 5-DS is extruded from several binding sites accompanied by an increase in KA in the remaining ones; (ii) simultaneous binding of 16-DS and Hp consists of cooperative and non-cooperative phases (both the number of the independent sites and the parameter of cooperativity, alpha, being dependent on Hp/HSA ratio); (iii) in principal the mobilities of 5-DS and 16-DS bound to HSA are changed, depending on the porphyrin/HSA ratio; and (iv) the effective immersion depth of the paramagnetic centres with respect to the protein surface is increased when Hp is present as a second ligand (rimm = 7 +/- 2 and 16 +/- 2 A for 5-DS and 16-DS, respectively).     

Experimental and computational studies on the binding of diazinon to human serum albumin

In the present research, the binding properties of diazinon (DZN), as an organophosphorus herbicide, to human serum albumin (HSA) were investigated using combination of spectroscopic, electrochemistry, and molecular modeling techniques. Changes in the UV–Vis and FT-IR spectra were observed upon ligand binding along with a significant degree of tryptophan fluorescence quenching on complex formation. The obtained results from spectroscopic and electrochemistry experiments along with the computational studies suggest that DZN binds to residues located in subdomains IIA of HSA with binding constant about 1410.9 M^?1 at 300 K. From the thermodynamic parameters calculated according to the van’t Hoff equation, the enthalpy change ΔH° and entropy change ΔS° were found to be ?16.695 and 0.116 KJ/mol K, respectively. The primary binding pattern is determined by hydrophobic interaction and hydrogen binding occurring in so-called site I of HSA. DZN could slightly alter the secondary structure of HSA. All of experimental results are supported by computational techniques such as docking and molecular dynamics simulation using a HSA crystal model.     

Competition of antibacterial drugs for binding sites of human serum albumin

Simultaneous binding of two drugs to human serum albumin (HSA) was studied by flow microcalorimetry. The following drug pairs were used: sulfadimethoxine and cefazolin. Sulfadimethoxine and dicloxacillin, sulfadimethoxine and chlortetracycline. A procedure for estimating the calorimetric titration curves in competing binding of the drugs to the HSA homogeneous active site is described. Comparison of the theoretical and experimental titration curves enabled detection of the ligand competition for the biopolymer binding site. It was shown that sulfadimethoxine displaced cefazolin in the HSA active site, the nature of the HSA association with dicloxacillin and sulfadimethoxine was independent and binding of doxycycline or chlortetracycline to HSA had no influence on sulfadimethoxine interaction with protein.     

Relations between high-affinity binding sites of markers for binding regions on human serum albumin.

Binding of warfarin, digitoxin, diazepam, salicylate and Phenol Red, individually or in different pair combinations, to defatted human serum albumin at ligand/protein molar ratios less than 1:1 was studied at pH 7.0. The binding was determined by ultrafiltration. Some of the experiments were repeated with the use of equilibrium dialysis in order to strengthen the results. Irrespective of the method used, all ligands bind to one high-affinity binding site with an association constant in the range 10(4)-10(6) M-1. High-affinity binding of the following pair of ligands took place independently: warfarin-Phenol Red, warfarin-diazepam, warfarin-digitoxin and digitoxin-diazepam. Simultaneous binding of warfarin and salicylate led to a mutual decrease in binding of one another, as did simultaneous binding of digitoxin and Phenol Red. Both effects could be accounted for by a coupling constant. The coupling constant is the factor by which the primary association constants are affected; in these examples of anti-co-operativity the factor has a value between 0 and 1. In the first example it was calculated to be 0.8 and in the latter 0.5. Finally, digitoxin and salicylate were found to compete for a common high-affinity binding site. The present findings support the proposal of four separate primary binding sites for warfarin, digitoxin (and salicylate), diazepam and Phenol Red. An attempt to correlate this partial binding model for serum albumin with other models in the literature is made.     

DNA binding studies of ruthenium(II) complexes containing asymmetric tridentate ligands

Absorption spectroscopy, fluorescence spectroscopy and viscosity measurements have been used to characterize the DNA binding of [Ru(tpy)(dppt)](2+) (tpy=2,2':6',2"-terpyridine, dppt=3-(1,10-phenanthrolin-2-yl)-5,6-diphenyl-as-triazine), [Ru(tpy)(pta)](2+) (pta=3-(1,10-phenanthrolin-2-yl)-as-triazino[5,6-f]acenaphthylene) and [Ru(tpy)(ptp)](2+) (ptp=3-(1,10-phenanthrolin-2-yl)-as-triazino[5,6-f]-phenanthrene). The results indicate that [Ru(tpy)(pta)](2+) and [Ru(tpy)(ptp)](2+) bind with CT-DNA in an intercalative mode, while [Ru(tpy)(dppt)](2+) binds with DNA by partial intercalation. The ligand planarity of the complex has a significant effect on DNA binding affinity increases in the order [Ru(tpy)(dppt)](2+)<[Ru(tpy)(pta)](2+)<[Ru(tpy)(ptp)](2+).     

Preclinical characterization of anticancer gallium(III) complexes: solubility, stability, lipophilicity and binding to serum proteins

The discovery and development of gallium(III) complexes capable of inhibiting tumor growth is an emerging area of anticancer drug research. A range of novel gallium coordination compounds with established cytotoxic efficacy have been characterized in terms of desirable chemical and biochemical properties and compared with tris(8-quinolinolato)gallium(III) (KP46), a lead anticancer gallium-based candidate that successfully finished phase I clinical trials (under the name FFC11), showing activity against renal cell cancer. In view of probable oral administration, drug-like parameters, such as solubility in water, saline and 0.5% dimethyl sulfoxide, stability against hydrolysis, measured as the rate constant of hydrolytic degradation in water or physiological buffer using a capillary zone electrophoresis (CZE) assay, and the octanol-water partition coefficient (logP) providing a rational estimate of a drug's lipophilicity, have been evaluated and compared. The differences in bioavailability characteristics between different complexes were discussed within the formalism of structure-activity relationships. The reactivity toward major serum transport proteins, albumin and transferrin, was also assayed in order to elucidate the drug's distribution pathway after intestinal absorption. According to the values of apparent binding rate constants determined by CZE, both KP46 and bis(2-acetylpyridine-4,4-dimethyl-3-thiosemicarbazonato-N,N,S)gallium(III) tetrachlorogallate(III) (KP1089) bind to transferrin faster than to albumin. This implies that transferrin would rather mediate the accumulation of gallium antineoplastic agents in solid tumors. A tendency of being faster converted into the protein-bound form found for KP1089 (due possibly to non-covalent binding) seems complementary to its greater in vitro antiproliferative activity.