Synthesis and biological evaluation of a class of quinolone triazoles as potential antimicrobial agents and their interactions with calf thymus DNA

A novel series of quinolone triazoles were synthesized and characterized by IR, NMR, MS and HRMS spectra. All the newly prepared compounds were screened for their antimicrobial activities against seven bacteria and four fungi. Bioactive assay manifested that most of new compounds exhibited good or even stronger antibacterial and antifungal activities against the tested strains including multi-drug resistant MRSA in comparison with reference drugs Norfloxacin, Chloromycin and Fluconazole. The preliminary interactive investigations of compound 6b with calf thymus DNA by fluorescence and UV–vis spectroscopic methods revealed that compound 6b could effectively intercalate DNA to form compound 6b–DNA complex which might block DNA replication and thus exert its antimicrobial activities.     

Synthesis and bioactive evaluation of novel hybrids of metronidazole and berberine as new type of antimicrobial agents and their transportation behavior by human serum albumin

A series of novel hybrids of metronidazole and berberine as new type of antimicrobial agents were synthesized and characterized by ¹H NMR, ¹³C NMR, IR, MS and HRMS spectra. Bioactive assay manifested that most of the prepared compounds exhibited effective antibacterial and antifungal activities and some showed comparable or superior potency against Methicillin-resistant Staphylococcus aureus to reference drugs Norfloxacin, Chloromycin and Berberine. The transportation behavior of human serum albumin (HSA) to the highly active compound 5g was evaluated and revealed that the association of imidazole derivative 5g with HSA was spontaneous and the electrostatic interactions played important roles in the transportation of HSA to 5g. The calculated parameters indicated that compound 5g could be effectively stored and carried by HSA.     

Utilization of the non-covalent fluorescent dye, NanoOrange, as a potential clinical diagnostic tool: Nanomolar human serum albumin quantitation

The commercially available dye, NanoOrange, has been investigated as a potential tool for clinical diagnostics due to its low cost, ease of use, and ability to detect nanomolar concentrations of protein. Virtually non-fluorescent in dilute aqueous solutions, NanoOrange fluorescence is enhanced by at least an order of magnitude upon non-covalent interaction with proteins. These features, coupled with the requirement for high throughput assays in the clinical laboratory has prompted the development of two orthogonal NanoOrange approaches. Human serum albumin (HSA) was used as a model protein for the development of both 96-well microplate and capillary electrophoresis laser-induced fluorescence (CE–LIF) assay formats. Dye performance in five commonly used buffers of various concentrations and pH indicated considerable flexibility in assay buffer selection, with optimal performance at pH 9.0. A salt concentration study indicated that increasing NaCl concentration generally decreases fluorescence emission and can be minimized by pre-diluting biological samples to a final salt concentration of 20–80 mM. Titration of protein with NanoOrange resulted in optimal HSA–NanoOrange complex formation utilizing 1× and 2× NanoOrange in the 96-well microplate and CE–LIF approaches, respectively. A NanoOrange binding model based on rapid signal enhancement and zero order fluorescence emission kinetics is proposed. The utilization of NanoOrange in CE–LIF based human serum analysis results in a signal-to-background ratio improvement of up to two orders of magnitude.     

DNA-duplex linker for AFM-SELEX of DNA aptamer against human serum albumin

DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350 pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFM-SELEX.     

The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study

Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient F?rster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.     

Synthesis and activities of naphthalimide azoles as a new type of antibacterial and antifungal agents

Naphthalimide-derived azoles as a new type of antimicrobial agents were synthesized and evaluated for their efficiency in vitro against eight bacteria and two fungi by two fold serial dilution technique. Most title compounds exhibited good antimicrobial potency with low MIC values ranging from 1 to 16 μg/mL. Notably, some synthesized compounds displayed comparable or even better antibacterial and antifungal activities against some tested strains than the reference drugs Orbifloxacin, Chloromycin and Fluconazole, respectively.     

Comprehensive investigation of the non-covalent binding of MRI contrast agents with human serum albumin

Three techniques, electrospray mass spectrometry, ultrafiltration, and proton relaxometry, are compared in the context of the quantitative analysis of non-covalent binding between human serum albumin (HSA) and MRI contrast agents. The study of the affinity by proton relaxometry reveals the association constant and the number of interaction sites assuming that all sites are identical and independent. Ultrafiltration was adapted for the study of paramagnetic complexes. This technique confirmed the results obtained by relaxometry. Electrospray mass spectrometry, an original method able to study non-covalent binding because of its soft ionization process that allows for the survival of weak binding, provides qualitative and quantitative results. Electrospray mass spectrometry confirmed the affinity measured by proton relaxometry and ultrafiltration. This technique requires very small amounts of products and directly gives the stoichiometry of the association, information not easily obtained by classic techniques. Nevertheless, proton relaxometry remains a useful and mandatory technique for determining the enhancement of the relaxation subsequent to the binding although it demands large amounts of compounds. It is to be pointed out that even if the three techniques lead to a similar ranking of the affinity of the contrast agents for HSA, the absolute values of the association constants disagree as a result of the difference in the experimental conditions (presence of salt, native protein or desalted one, approximations in the fitting of the data, liquid or gas phases).     

The interaction of tylophorinidine,a potential leukemic drug,with bovine serum albumin

Association of tylophorinidine with bovine serum albumin was indioated by the quenching of the protein fluorescence above pH 7.0. The association constant, Ka, at pH 9.2 was calculated to be 5 × 10⁴ litres/mole. The Ka increased with increasing temperature, and the change in enthalpy, ΔH, was calculated to be 4.5 Kcals. The stoichionetry of binding obtained from Job's plot was 1:1. The data suggest involvement of tryptophan and hydrophobic forces in the albumintylophorinidine complex.     

Study on the interaction between human serum albumin and a novel bioactive acridine derivative using optical spectroscopy

The interaction of a novel bioactive agent N‐{[N‐(2‐dimethylamino) ethyl] acridine‐4‐carboxamide}‐α‐alanine [N‐(ACR‐4‐CA)‐α‐ALA] with human serum albumin (HSA) was investigated by fluorescence spectroscopy, UV–vis absorption and circular dichroism spectrophotometric techniques under simulative physiological conditions. The fluorescence quenching of HSA by addition of N‐(ACR‐4‐CA)‐α‐ALA is due to static quenching and hydrogen bonding. Moreover, hydrophobic interactions play a role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA as well. The number of binding sites, n, and the binding constant values, K_A, were noted to be 0.88 and 3.4 × 10⁴ L mol^?1 for N‐(ACR‐4‐CA)‐α‐ALA at 293 K. The binding distances and the energy transfer efficiency between N‐(ACR‐4‐CA)‐α‐ALA and protein were determined. The negative value of enthalpy change and positive value of entropy change in the present study indicated that both hydrogen bonding and hydrophobic forces played a major role in the binding of N‐(ACR‐4‐CA)‐α‐ALA to HSA. Copyright © 2010 John Wiley & Sons, Ltd.     

Gd(III) complexes as contrast agents for magnetic resonance imaging: a proton relaxation enhancement study of the interaction with human serum albumin

 The non-covalent interaction between human serum albumin (HSA) and DOTA-like Gd(III) complexes containing hydrophobic benzyloxymethyl (BOM) substituents has been thoroughly investigated by measuring the solvent proton relaxation rates of their aqueous solutions. The binding association constants (K _A) to HSA are directly related to the number of hydrophobic substituents present on the surface of the complexes. Furthermore, an estimation of ΔH° and ΔS° has been obtained by the temperature dependence of K _A. Assays performed with the competitor probes warfarin and ibuprofen established that the complexes interact with HSA through two nearly equivalent binding sites located in the subdomains IIA and IIIA of the protein. Strong relaxation enhancements, promoted by the formation of slowly tumbling paramagnetic adducts, have been measured at 20 MHz for complexes containing two and three hydrophobic substituents. The macromolecular adduct with the latter species has a relaxivity of 53.2±0.7 mM^–1 s^–1, which represents the highest value so far reported for a Gd(III) complex. The temperature dependence of the relaxivity for the paramagnetic adducts with HSA indicates long exchange lifetimes for the water molecules dipolarly interacting with the paramagnetic centre. This is likely to be related to the formation, upon hydrophobic interaction of the complexes with HSA, of a clathrate-like, second-coordination-sphere arrangement of water molecules. Besides affecting the dissociative pathway of the coordinated water molecule, this water arrangement may itself significantly contribute to enhancement of the bulk solvent relaxation rate. Received: 6 November 1995 / Accepted: 17 April 1996     

Chromatographic study of magnesium and calcium binding to immobilized human serum albumin

The use of immobilized human serum albumin (HSA) as a stationary phase in affinity chromatography has been shown to be useful in resolving optical antipodes or to investigate interactions between drugs and protein. However, to our knowledge, no inorganic ion binding has been studied on this immobilized protein type. To do this, the human serum albumin stationary phase was assimilated to a weak cation-exchanger by working with a mobile phase pH equal to 6.5. A study of the eluent ionic strength effect on ion retention was carried out by varying the buffer concentrations and the column temperatures. The thermodynamic parameters for magnesium and calcium transfer from the mobile to the stationary phase were determined from linear van’t Hoff plots. An enthalpy–entropy compensation study revealed that the type of interaction was independent of the mobile phase composition. A simple model based on the Gouy–Chapman theory was considered in order to describe the retention behavior of the test cations with the mobile phase ionic strength. From this theoretical approach, the relative charge densities of the human serum albumin surface implied in the binding process were estimated at different column temperatures.     

Dipeptidyl peptidase IV activity in commercial solutions of human serum albumin

Due to the heterogeneous nature of commercial human serum albumin (cHSA), other components, such as the protease dipeptidyl peptidase IV (DPP-IV), possibly contribute to the therapeutic effect of cHSA. Here, we provide evidence for the first time that DPP-IV activity contributes to the formation of aspartate–alanine diketopiperazine (DA-DKP), a known immunomodulatory molecule from the N terminus of human albumin. cHSA was assayed for DPP-IV activity using a specific DPP-IV substrate and inhibitor. DPP-IV activity was assayed at 37 and 60 °C because cHSA solutions are pasteurized at 60 °C. DPP-IV activity in cHSA was compared with other sources of albumin such as a recombinant albumin (rHSA). In addition, the production of DA-DKP was measured by negative electrospray ionization/liquid chromatography mass spectrometry (ESI⁻/LCMS). Significant levels of DPP-IV activity were present in cHSA. This activity was abolished using a specific DPP-IV inhibitor. Fully 70 to 80% DPP-IV activity remained at 60 °C compared with the 37 °C incubate. No DPP-IV activity was present in rHSA, suggesting that DPP-IV activity is present only in HSA produced using the Cohn fractionation process. The formation of DA-DKP at 60 °C was observed with the DPP-IV inhibitor significantly decreasing this formation. DPP-IV activity in cHSA results in the production of DA-DKP, which could account for some of the clinical effects of cHSA.     

Study on the interaction of two quinazoline derivatives as novel PI3K/mTOR dual inhibitors and anticancer agents to human serum albumin utilizing spectroscopy and docking

Interactions of human serum albumin (HSA) with two structurally similar quinazoline derivatives, S ₁ and S ₂ , which are potential anticancer drugs acting on PI3K/mTOR targets, were investigated in vitro utilizing multiple spectroscopy as well as molecular docking. The fluorescence quenching study demonstrated that HSA fluorescence could be statically quenched by S ₁ and S ₂ through the formation of an HSA–drug complex. Furthermore, the details of the binding site number, binding constant, as well as the thermodynamic parameters, were estimated at 298, 303, and 310 K. The results revealed that hydrogen bond interactions, as well as van der Waals forces, were the predominant factors responsible for binding HSA to S ₁ or S ₂ . Synchronous fluorescence and ultraviolet (UV) absorption spectra suggested that S ₁ and S ₂ had little effect on the polarity of the microenvironment and conformation of HSA. Energy transfer from HSA to S ₁ or S ₂ most probably occurred. The docking study revealed that S ₁ and S ₂ were able to bind to the hydrophobic cavity that was located in the HSA subdomain IIA and formed varying numbers of hydrogen bonds with amino acid residues nearby. Due to the subtle difference in the chemical structure, the binding of S ₁ and S ₂ to HSA was slightly different.     

Cytotoxic palladium(II) complexes of 8-aminoquinoline derivatives and the interaction with human serum albumin

Palladium(II) complexes are potential antitumor metallodrugs for their chemical resemblance to platinum(II) complexes. Two palladium(II) complexes (1 and 2) in the formula of [PdLⁿCl] [L¹ = N-(tert-butoxycarbonyl)-l-methionine-N′-8-quinolylamide, L² = L-alanine-N′-8-quinolylamide] have been synthesized accordingly. The structures of the complexes were fully characterized by X-ray crystallography. The palladium(II) center in 1 is coordinated by two N atoms and an S atom from L¹ with one chloride anion as the leaving group; while that in 2 is coordinated by three N atoms from L² with one chloride anion as the leaving group. The interaction between complex 1 and human serum albumin (HSA) has been investigated using fluorescence and circular dichroism spectroscopies. The complex seems to react with HSA chiefly through hydrophobic and electrostatic interactions, and it does not alter the α-helical nature of HSA. The cytotoxicity of these complexes has been tested against the human cervical cancer (HeLa), human mammary cancer (MCF-7), and human lung cancer (A-549) cell lines. Complex 1 displays a cytotoxic activity comparable to that of cisplatin, but complex 2 is less active than cisplatin.     

Solute complexation degree with human serum albumin: biochromatographic approach

A mathematical model was developed for the study of the D,L-dansylamino acid retention mechanism in reversed-phase liquid chromatography using a C18 column as a stationary phase and human serum albumin (HSA) as an eluent modifier. The solute retention factor is dependent on the HSA concentration in the eluent as well as the binding constant of the guest-HSA complex. A determination of the degree of complexation n(c) (the percent of the complexed guest) could be carried out. Different Van 't Hoff plot shapes of the degree of complexation were observed with different eluent pH, confirming a change in the solute complexation mechanism for physiological pH (between 7-7.5). Enthalpy-entropy compensation was also analysed in relation to this mathematical model to confirm the solute complexation behavior with HSA. These results finally confirmed that at physiological pH and temperature (approximately 35 degrees C) values the HSA was in a favorable structural conformation for its binding with a great majority of drugs.     

Direct observation of covalent adducts with Cys34 of human serum albumin using mass spectrometry

The interactions of the unpaired thiol residue (Cys34) of human serum albumin (HSA) with low-molecular-weight thiols and an Au(I)-based antiarthritic drug have been examined using electrospray ionization mass spectrometry. Early measurements of the amount of HSA containing Cys34 as the free thiol suggested that up to 30% of circulating HSA bound cysteine as a mixed disulfide. It has also been suggested that reaction of HSA with cysteine, occurs only on handling and storage of plasma. In our experiments, there were three components of HSA in freshly collected plasma from normal volunteers, HSA, HSA+cysteine, and HSA+glucose in the ratio approximately 50:25:25. We addressed this controversy by using iodoacetamide to block the free thiol of HSA in fresh plasma, preventing its reaction with plasma cysteine. When iodoacetamide was injected into a vacutaner tube as blood was collected, the HSA was modified by iodoacetamide, with 20-30% present as the mixed disulfide with cysteine (HSA+cys). These data provide strong evidence that 20-30% of HSA in normal plasma contains one bound cysteine. Reaction of HSA with [Au(S(2)O(3))(2)](3-) resulted in formation of the adducts HSA+Au(S(2)O(3)) and HSA+Au. Reaction of HSA with iodoacetamide prior to treatment with [Au(S(2)O(3))(2)](3-) blocked the formation of gold adducts.     

A solid-phase adsorption method for PEGylation of human serum albumin and staphylokinase: preparation,purification and biochemical characterization

A solid-phase adsorption method was developed to circumvent the disadvantage of the conventional liquid-phase PEGylation, i.e. the heterogeneity of the PEGylated products. The model proteins, human serum albumin (HSA) and staphylokinase (SAK), were adsorbed on the ion exchange chromatography media, followed by PEGylation with succinimidyl carbonate (SC)-mPEG5K and salt elution. Since PEGylation with SC-PEG5K alters the positive charge of the proteins, Q-Sepharose Big Beads and DEAE Sepharose Fast Flow were used for adsorption of HSA and SAK, respectively. Size exclusion chromatography and SDS-PAGE studies demonstrated that solid-phase PEGylation of proteins generate monoPEGylated proteins with the yield of 35–47%. Circular dichroism and intrinsic fluorescence studies showed that solid-phase PEGylation led to little conformational change of the proteins. Solid-phase PEGylation resulted in 35% loss in the biological activity of SAK, which is lower than the liquid-phase PEGylation (70%).     

Interaction of cromolyn sodium with human serum albumin: a fluorescence quenching study

The interaction between cromolyn sodium (CS) and human serum albumin (HSA) was investigated using tryptophan fluorescence quenching. In the discussion of the mechanism, it was proved that the fluorescence quenching of HSA by CS is a result of the formation of a CS–HSA complex. Quenching constants were determined using the Sterns–Volmer equation to provide a measure of the binding affinity between CS and HSA. The thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures were calculated. The distance r between donor (Trp²¹⁴) and acceptor (CS) was obtained according to fluorescence resonance energy transfer (FRET). Furthermore, synchronous fluorescence spectroscopy data and UV–vis absorbance spectra have suggested that the association between CS and HSA changed the molecular conformation of HSA and the electrostatic interactions play a major role in CS–HSA association.     

Alteration of redox state of human serum albumin in patients under anesthesia and invasive surgery

Human serum albumin is a mixture of mercapt- (HMA, reduced form) and nonmercaptalbumin (HNA, oxidized form). We studied the mercapt↔nonmercapt conversion of human serum albumin, which reflects the redox state of the extracellular fluids, in cardiac and other common surgical patients using high-performance liquid chromatography. Mean values of [(HMA)/(HMA + HNA)] ± standard deviation [f_HMA ± σ], for patients who received common surgery (group 1) and cardiac surgery (group 2) at the start of anesthesia were0.636±0.50(n=83) and 0.615±0.062(n=14), respectively. f_HMA values were markedly lower than those for healthy male adults of 0.750±0.028(n=28). f_HMA values increased at 24 h after the start of anesthesia and decreased on the 4th postoperative day in most of the patients. These postoperative changes were prominent in surgical cardiac patients. Although f_HMA values after the 7th postoperative day recovered to those at the start of anesthesia in almost all of common surgical patients, those in cardiac surgical patients, never recovered even on the 21st postoperative day.     

Combined multispectroscopic and molecular docking investigation on the interaction between strictosamide and human serum albumin

The interaction between strictosamide (STM) and human serum albumin (HSA) was investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, three‐dimensional fluorescence spectroscopy, ultraviolet‐visible absorption spectroscopy, circular dichroism spectroscopy and molecular modeling under physiological pH 7.4. STM effectively quenched the intrinsic fluorescence of HSA via static quenching. The binding site number n and apparent binding constant K_a were determined at different temperatures by fluorescence quenching. The thermodynamic parameters, enthalpy change (ΔH) and entropy change (ΔS) for the reaction were calculated as ?3.01 kJ/mol and 77.75 J/mol per K, respectively, which suggested that the hydrophobic force played major roles in stabilizing the HSA–STM complex. The distance r between donor and acceptor was obtained to be 4.10 nm according to Förster's theory. After the addition of STM, the synchronous fluorescence and three‐dimensional fluorescence spectral results showed that the hydrophobicity of amino acid residues increased and the circular dichroism spectral results showed that the α‐helix content of HSA decreased (from 61.48% to 57.73%). These revealed that the microenvironment and conformation of HSA were changed in the binding reaction. Furthermore, the study of molecular modeling indicated that STM could bind to site I of HSA and the hydrophobic interaction was the major acting force, which was in agreement with the binding mode study. Copyright © 2013 John Wiley & Sons, Ltd.