Inclusion of a Catechol-Derived Hydrazinyl-Thiazole (CHT) in ß-Cyclodextrin Nanocavity and Its Effect on Antioxidant Activity: A Calorimetric, Spectroscopic and Molecular Docking Approach.

The aim of the present research was to obtain a supramolecular complex between a strong antioxidant compound previously reported by our group, in order to extend its antioxidant activity. The formation of the inclusion complex of a catechol hydrazinyl-thiazole derivative (CHT) and ß-cyclodextrin in aqueous solution has been investigated using isothermal titration calorimetry (ITC), spectroscopic and theoretical methods. The stoichiometry of this inclusion complex was established to be equimolar (1:1) and its equilibrium constant was determined. An estimation of the thermodynamic parameters of the inclusion complex showed that it is an enthalpy and entropy-driven process. Our observations also show that hydrophobic interactions are the key interactions that prevail in the complex. 1H NMR spectroscopic method was employed to study the inclusion process in an aqueous solution. Job plots derived from the 1H NMR spectral data demonstrated 1:1 stoichiometry of the inclusion complex in a liquid state. A 2D NMR spectrum suggests the orientation of the aromatic ring of CHT inside the ß-CD cavity. The antiradical activity of the complex was evaluated and compared with free CHT, indicating a delayed activity compared with free CHT. To obtain additional qualitative and visual insight into the particularity of CHT and ß-CD interaction, molecular docking calculations have been performed.

Antioxidant Activity Evaluation and Assessment of the Binding Affinity to HSA of a New Catechol Hydrazinyl-Thiazole Derivative.

Polyphenols have attained pronounced attention due to their ability to provide numerous health benefits and prevent several chronic diseases. In this study, we designed, synthesized and analyzed a water-soluble molecule presenting a good antioxidant activity, namely catechol hydrazinyl-thiazole (CHT). This molecule contains 3',4'-dihydroxyphenyl and 2-hydrazinyl-4-methyl-thiazole moieties linked through a hydrazone group with very good antioxidant activity in the in vitro evaluations performed. A preliminary validation of the CHT developing hypothesis was performed evaluating in silico the bond dissociation enthalpy (BDE) of the phenol O-H bonds, compared to our previous findings in the compounds previously reported by our group. In this paper, we report the binding mechanism of CHT to human serum albumin (HSA) using biophysical methods in combination with computational studies. ITC experiments reveal that the dominant forces in the binding mechanism are involved in the hydrogen bond or van der Waals interactions and that the binding was an enthalpy-driven process. NMR relaxation measurements were applied to study the CHT-protein interaction by changing the drug concentration in the solution. A molecular docking study added an additional insight to the experimental ITC and NMR analysis regarding the binding conformation of CHT to HSA.

Study of the binding affinity between imatinib and α-1 glycoprotein using nuclear spin relaxation and isothermal titration calorimetry.

Imatinib is a selective tyrosine kinase inhibitor, successfully used for the treatment of chronic myelogenous leukaemia and gastrointestinal stromal tumors. Binding of drugs to proteins influence their pharmacokinetic and pharmacodynamics action. In the blood, the drug is distributed in the body in the free form or bound to plasma protein. Albumin and α-1 glycoprotein (AGP) are plasma proteins with the highest affinity for drug substances. Drugs which are weak acids mainly bind to plasma albumin, while drugs that are bases have affinity for α-1 glycoprotein. The main goal of this study is to quantitatively evaluate the interaction between imatinib mesylate (IMT) and α-1 glycoprotein to characterize the nature and forces underlying the formation of a molecular complex. Relaxation experiments provide quantitative information about the relationship between the binding affinity and structure of IMT. Thus, association constant was determined as Ka = 873.36 M-1. The ITC data revealed that the binding was an entropy driven process and the association constant Ka = 3.22 × 103 M-1, with a 1:1 stoichiometry. The results obtained by NMR and ITC were complemented with a molecular docking study.

Affinity and Effect of Anticancer Drugs on the Redox Reactivity of Hemoglobin.

Hemoglobin's redox reactivity is affected by anticancer drugs of the antitubulin class. Direct binding of these drugs to hemoglobin, with biomedically relevant affinities, is demonstrated. While this interaction is mostly allosteric, in the case of docetaxel, a direct redox reaction is also observed-correlating well with structural differences between the four compounds. A role for Tyr145 in this reactivity is proposed, in line with previous observations of the importance of this amino acid in the reactivity of Hb toward agents of oxidative stress. A susceptibility of vinorelbin (and to a lower extent of paclitaxel) toward peroxide and peroxidase is shown.

The high affinity of small-molecule antioxidants for hemoglobin.

Hemoglobin has previously been shown to display ascorbate peroxidase and urate peroxidase activity, with measurable Michaelis-Menten parameters that reveal a particularly low Km for ascorbate as well as for urate - lower than the respective in vivo concentrations of these antioxidants in blood. Also, direct detection of a hemoglobin-ascorbate interaction was possible by monitoring the 1H-NMR spectrum of ascorbate in the presence of hemoglobin. The relative difference in structures between ascorbate and urate may raise the question as to exactly what the defining structural features would be, for a substrate that binds to hemoglobin with high affinity. Reported here are Michaelis-Menten parameters for hemoglobin acting as peroxidase against a number of other substrates of varying structures - gallate, caffeate, rutin, 3-hydroxyflavone, 3,6-dihydroxyflavone, quercetin, epicatechin, luteolin - all with high affinities (some higher than those of physiologically-relevant redox partners of Hb - ascorbate and urate). Moreover, this high affinity appears general to animal hemoglobins. 1H-NMR and 13C-NMR spectra reveal a general pattern wherein small hydrophilic antioxidants appear to all have their signals affected, presumably due to binding to hemoglobin. Fluorescence and calorimetry measurements confirm these conclusions. Docking calculations confirm the existence of binding sites on hemoglobin and on myoglobin for ascorbate as well as for other antioxidants. Support is found for involvement of Tyr42 in binding of three out of the four substrates investigated in the case of hemoglobin (including ascorbate and urate, as blood-contained relevant substrates), but also for Tyr145 (with urate and caffeate) and Tyr35 (with gallate).

Calorimetric and spectroscopic studies of the interaction between zidovudine and human serum albumin.

A quantitative analysis of the interaction between zidovudine (AZT) and human serum albumin (HSA) was achieved using Isothermal titration calorimetry (ITC) in combination with fluorescence and 1H NMR spectroscopy. ITC directly measure the heat during a biomolecular binding event and gave us thermodynamic parameters and the characteristic association constant. By fluorescence quenching, the binding parameters of AZT-HSA interaction was determined and location to binding site I of HSA was confirmed. Via T1 NMR selective relaxation time measurements the drug-protein binding extent was evaluated as dissociation constants Kd and the involvement of azido moiety of zidovudine in molecular complex formation was put in evidence. All three methods indicated a very weak binding interaction. The association constant determined by ITC (3.58×102M-1) is supported by fluorescence quenching data (2.74×102M-1). The thermodynamic signature indicates that at least hydrophobic and electrostatic type interactions played a main role in the binding process.

Size dependence of molecular self-assembling in stacked aggregates. 2. Heat exchange effects.

Many aromatic molecules self-associate in aqueous solutions into dimers, trimers, and higher order one-dimensional aggregates. They self-assemble due to the π-π stacking interactions in supramolecular structures with a length distribution determined by the size-dependent association equilibrium constants. Due to their low solubility, in many experiments the aromatic molecules are dissolved in their cationic form. The presence of the localized positive charge promotes a new type of binding led by the cation-π interaction. Accordingly, in the case of aromatic molecules dissolved in water in their cationic form, at least two types of aggregates may appear: the rodlike aggregates stabilized by π-π stacking, and dimers coupled by the cation-π interaction. The heat exchange which accompanies the association (dissociation) processes offers the most direct way to determine the characteristic thermodynamic parameters. The heat exchange that arises at each dissociation event has been measured by isothermal titration calorimetry (ITC) on a test molecule whose self-association properties are already known: ciprofloxacin hydrochloride. The fitting protocol relies on the recently developed DK self-association model which describes in a rigorous manner the size distribution of molecular aggregates in solution.

The artifactual nature of stavudine binding to human serum albumin. A fluorescence quenching and isothermal titration calorimetry study.

The interaction between stavudine, a nucleoside reverse transcriptase inhibitor and human serum albumin (HSA), was investigated by fluorescence quenching technique and isothermal titration calorimetry (ITC). A good linearity of albumin fluorescence quenching in the presence of stavudine was determined. Analyzing these data we obtained for the dissociation constant the value K(d)=(18.18 ± 0.46) × 10(-5)M. However, due to contradictory results obtained in ITC experiments, we checked the fluorescence quenching data for the inner-filter effect, the main confounding factor in the observed quenching. Based on the UV-vis absorption data we have corrected the observed fluorescence intensities and concluded, in accordance with ITC results, that stavudine binding to HSA is negligible and the observed quenching effect is entirely caused by a failure to correct for the inner-filter effect.