A potentiometric and spectrofluorimetric approach to unravel inhibitory effects of semi- and thiosemicarbazones on mushroom tyrosinase activity.

The inhibitory effects on mushrooms tyrosinase activity of some semi- and thiosemicarbazones were investigated. While the semicarbazones are inactive, the thiosemicarbazones are, in general, more active than the reference (kojic acid, IC50 = 70 µM), with maximum activity obtained with benzaldehyde thiosemicarbazone (IC50 = 7 µM). These inhibitors probably act by coordination of the copper(II) metal ions in the active site of tyrosinase: effectively, potentiometric studies conducted in water solutions confirm that the most active thiosemicarbazone is a good ligand for copper(II) ions. The tyrosinase CD spectra do not show any significant difference by addition of an inhibitor or an inactive compound. On the contrary, interesting results were obtained by spectrofluorimetric titrations of mushrooms tyrosinase aqueous solutions with some of the investigated compounds, giving helpful information about possible mechanism of action. The thiosemicarbazones here reported are not cytotoxic on human fibroblasts and do not activate cells in a pro-inflammatory way.

Antiviral activity and metal ion-binding properties of some 2-hydroxy-3-methoxyphenyl acylhydrazones.

Here we report on the results obtained from an antiviral screening, including herpes simplex virus, vaccinia virus, vesicular stomatitis virus, Coxsackie B4 virus or respiratory syncytial virus, parainfluenza-3 virus, reovirus-1 and Punta Toro virus, of three 2-hydroxy-3-methoxyphenyl acylhydrazone compounds in three cell lines (i.e. human embryonic lung fibroblast cells, human cervix carcinoma cells, and African Green monkey kidney cells). Interesting antiviral EC50 values are obtained against herpes simplex virus-1 and vaccinia virus. The biological activity of acylhydrazones is often attributed to their metal coordinating abilities, so potentiometric and microcalorimetric studies are here discussed to unravel the behavior of the three 2-hydroxy-3-methoxyphenyl compounds in solution. It is worth of note that the acylhydrazone with the higher affinity for Cu(II) ions shows the best antiviral activity against herpes simplex and vaccinia virus (EC50 ~ 1.5 µM, minimal cytotoxic concentration = 60 µM, selectivity index = 40).

Hydroxyphenyl thiosemicarbazones as inhibitors of mushroom tyrosinase and antibrowning agents.

Tyrosinase is a metalloenzyme involved in o-hydroxylation of monophenols and oxidation of o-diphenols to o-quinones, with formation of brown or black pigments (melanines). Tyrosinase inhibitors are of great interest in medicine and cosmetics (skin whitening compounds), but also in food and beverage industry (antibrowning agents). Here we report on the activity as mushroom tyrosinase inhibitors of a series of hydroxyphenyl thiosemicarbazones (1-5): one of them revealed an inhibitory activity stronger than kojic acid, used as reference. Enzymatic inhibition activity was confirmed by colorimetric measurements on small wheels of Fuji apples treated with the hydroxyphenyl thiosemicarbazones. The mechanism of action of compounds 1-5 was investigated by molecular modelling and by studying in solution their speciation with Cu(II) ions, the ions in the active site of the enzyme. Finally, compounds 1-5 were tested on human fibroblasts: they are not cytotoxic and they do not activate cells in a pro-inflammatory way.

A versatile salicyl hydrazonic ligand and its metal complexes as antiviral agents.

Acylhydrazones are very versatile ligands and their coordination properties can be easily tuned, giving rise to metal complexes with different nuclearities. In the last few years, we have been looking for new pharmacophores able to coordinate simultaneously two metal ions, because many enzymes have two metal ions in the active site and their coordination can be a successful strategy to inhibit the activity of the metalloenzyme. As a part of this ongoing research, we synthesized the acylhydrazone H2L and its complexes with Mg(II), Mn(II), Co(II), Ni(II), Cu(II) and Zn(II). Their characterization, both in solution--also by means of potentiometric studies--and in the solid state, evidenced the ability of the o-vanillin hydrazone scaffold to give rise to different types of metal complexes, depending on the metal and the reaction conditions. Furthermore, we evaluated both the free ligand and its metal complexes in in vitro studies against a panel of diverse DNA- and RNA-viruses. In particular, the Mg(II), Mn(II), Ni(II) and Zn(II) complexes had EC50 values in the low micromolar range, with a pronounced activity against vaccinia virus.

A software for the estimation of binding parameters of biochemical equilibria based on statistical probability model.

An algorithm is proposed for the estimation of binding parameters for the interaction of biologically important macromolecules with smaller ones from electrometric titration data. The mathematical model is based on the representation of equilibria in terms of probability concepts of statistical molecular thermodynamics. The refinement of equilibrium concentrations of the components and estimation of binding parameters (log site constant and cooperativity factor) is performed using singular value decomposition, a chemometric technique which overcomes the general obstacles due to near singularity. The present software is validated with a number of biochemical systems of varying number of sites and cooperativity factors. The effect of random errors of realistic magnitude in experimental data is studied using the simulated primary data for some typical systems. The safe area within which approximate binding parameters ensure convergence has been reported for the non-self starting optimization algorithms.

Hydrophobic hydration processes thermal and chemical denaturation of proteins.

The hydrophobic hydration processes have been analysed under the light of a mixture model of water that is assumed to be composed by clusters (W(5))(I), clusters (W(4))(II) and free water molecules W(III). The hydrophobic hydration processes can be subdivided into two Classes A and B. In the processes of Class A, the transformation A(-ξ(w)W(I)→ξ(w)W(II)+ξ(w)W(III)+cavity) takes place, with expulsion from the bulk of ξ(w) water molecules W(III), whereas in the processes of Class B the opposite transformation B(-ξ(w)W(III)-ξ(w)W(II)→ξ(w)W(I)-cavity) takes place, with condensation into the bulk of ξ(w) water molecules W(III). The thermal equivalent dilution (TED) principle is exploited to determine the number ξ(w). The denaturation (unfolding) process belongs to Class A whereas folding (or renaturation) belongs to Class B. The enthalpy ΔH(den) and entropy ΔS(den) functions can be disaggregated in thermal and motive components, ΔH(den)=ΔH(therm)+ΔH(mot), and ΔS(den)=ΔS(therm)+ΔS(mot), respectively. The terms ΔH(therm) and ΔS(therm) are related to phase change of water molecules W(III), and give no contribution to free energy (ΔG(therm)=0). The motive functions refer to the process of cavity formation (Class A) or cavity reduction (Class B), respectively and are the only contributors to free energy ΔG(mot). The folded native protein is thermodynamically favoured (ΔG(fold)≡ΔG(mot)<0) because of the outstanding contribution of the positive entropy term for cavity reduction, ΔS(red)≫0. The native protein can be brought to a stable denatured state (ΔG(den)≡ΔG(mot)<0) by coupled reactions. Processes of protonation coupled to denaturation have been identified. In thermal denaturation by calorimetry, however, is the heat gradually supplied to the system that yields a change of phase of water W(III), with creation of cavity and negative entropy production, ΔS(for)≪0. The negative entropy change reduces and at last neutralises the positive entropy of folding. In molecular terms, this means the gradual disruption by cavity formation of the entropy-driven hydrophobic bonds that had been keeping the chains folded in the native protein. The action of the chemical denaturants is similar to that of heat, by modulating the equilibrium between W(I), W(II), and W(III) toward cavity formation and negative entropy production. The salting-in effect produced by denaturants has been recognised as a hydrophobic hydration process belonging to Class A with cavity formation, whereas the salting-out effect produced by stabilisers belongs to Class B with cavity reduction. Some algorithms of denaturation thermodynamics are presented in the Appendices.

Hydrophobic hydration processes. General thermodynamic model by thermal equivalent dilution determinations.

The "hydrophobic hydration processes" can be satisfactorily interpreted on the basis of a common molecular model for water, consisting of two types of clusters, namely W(I) and W(II) accompanied by free molecules W(III). The principle of thermal equivalent dilution (TED) is the potent tool (Ergodic Hypothesis) employed to monitor the water equilibrium and to determine the number xi(w) of water molecules W(III) involved in each process. The hydrophobic hydration processes can be subdivided into two Classes: Class A includes those processes for which the transformation A(-xi(w)W(I)-->xi(w)W(II)+xi(w)W(III)+cavity) takes place with the formation of a cavity, by expulsion of xi(w) water molecules W(III) whereas Class B includes those processes for which the opposite transformation B(-xi(w)W(II)-xi(w)W(III)-->xi(w)W(I)-cavity) takes place with reduction of the cavity, by condensation of xi(w) water molecules W(III). The number xi(w) depends on the size of the reactants and measures the extent of the change in volume of the cavity. Disaggregating the thermodynamic functions DeltaH(app) and DeltaS(app) as the functions of T (or lnT) and xi(w) has enabled the separation of the thermodynamic functions into work and thermal components. The work functions DeltaG(Work), DeltaH(Work) and DeltaS(Work) only refer specifically to the hydrophobic effects of cavity formation or cavity reduction, respectively. The constant self-consistent unitary (xi(w)=1) work functions obtained from both large and small molecules indicate that the same unitary reaction is taking place, independent from the reactant size. The thermal functions DeltaH(Th) and DeltaS(Th) refer exclusively to the passage of state of water W(III). Essential mathematical algorithms are presented in the appendices.

Thermodynamics of micelle formation in water, hydrophobic processes and surfactant self-assemblies.

The critical micelle concentration (c.m.c.) for four cationic surfactants, alkyl-trimethyl-ammonium bromides, was determined as a function of temperature by conductivity measurements. The values of the standard free energy of micellisation DeltaG degrees(mic) at different temperatures were calculated by using a pseudo-phase transition model. Then, from the diagram (-DeltaG degrees(mic)/T)=f(1/T), the thermodynamic functions DeltaH(app) and DeltaS(app) were calculated. From the plots DeltaH(app)=f(T) and DeltaS(app) = f(ln T) the slopes DeltaC(p) = n(w(H))C(p,w) and DeltaC(p)=n(w(S))C(p,w) were calculated, with the numbers n(w(H)) and n(w(S)) negative and equal and therefore defined simply as n(w). The number n(w)<0, indicating condensed water molecules, depends on the reduction of cavity that takes place as a consequence of the coalescence of the cavities previously surrounding the separate aliphatic or aromatic moieties. The analysis, based on a molecular model consisting of three forms of water, namely W(I), W(II), and W(III), respectively, was extended to several other types of surfactants for which c.m.c. data had been published by other authors. The results of this analysis form a coherent scheme consistent with the proposed molecular model. The enthalpy for all the types of surfactant is described by DeltaH(app)= -3.6 + 23.1xi(w)-xi(w)C(p,w)T and the entropy by DeltaS(app)= +10.2+428xi(w)-xi(w)C(p,w) ln T where xi(w)= |n(w)| represents the number of molecules W(III) involved in the reaction. The term Deltah(w)= +23.1 kJ mol(-1) xi(w)(-1) indicates an unfavourable endothermic contribution to enthalpy for reduction of the cavity whereas the term Deltas(w)= +428 J K(-1) mol(-1) xi(w)(-1) represents a positive entropy contribution for reduction of the cavity, what is the driving force of hydrophobic association. The processes of non polar gas dissolution in water and of micelle formation were found to be strictly related: they are, however, exactly the opposite of one another. In micelle formation no intermolecular electronic short bond is formed. We propose, therefore, to substitute the term "hydrophobic bond" with that of "hydrophobic association".