No abstract

Tetracycline (TC) derivatives comprise a group of bacteriostatic antibiotics extensively used in human and veterinary medicine and in acquaculture, and as animal growth promoters due to their safe toxicological profile.1) More recently, they have been proposed as anti-amyloidogenic drugs since they can interfere with protein conformational changes and self-assemblies associated with protein misfolding and deposition diseases. 2-4)Despite their popular use, TC derivatives are highly unstable and some degradation products formed during the storage or normal use of the drugs are pharmacologically toxic or inactive compounds.5) Anhydrotetracycline (AHTC) is one of the major degradation products of TC that has been linked to several side effects of the drug, e.g., cutaneous phototoxicity 6) and Fanconi-type syndrome. 7) AHTC has also proven to be more toxic than TC against environmentally relevant bacteria. 8)AHTC can be produced by dehydration in acid media, 5,8) by photolysis in mild reducing conditions 6,9,10) or during high-temperature treatments.11) Removal of a water molecule from the C5a-C6 positions of TC by treatment with warm mineral acid gives the anhydroderivative (Fig. 1).12) Dehydration leads to significant changes in aromatization and planarity of the tetracyclic moiety, 13,14) showing variations in potency, 15) mechanism of action 1) and lipophilicity 16,17) as compared to the parent compound. Changes in chemical configuration can also modulate the interaction of drugs with serum proteins. 18)Serum albumins are the main carriers of physiological metabolites and pharmacological drugs in blood. Protein binding has a significant impact on the pharmacokinetics and biological activity of drugs and modulates the toxicity of bound substances, attenuates adverse reactions and prolongs the in vivo half-life of therapeutic agents. 18-21)The facts outlined above prompted us to investigate the interaction of AHTC with bovine serum albumin (BSA) using three biophysical techniques. Binding affinity and number of interacting ligands were assessed by fluorimetric titration. . Drug binding was enthalpically and entropically driven and seemed to involve hydrophobic interactions. AHTC fluorescence enhancement and hypsochromic shifts observed upon binding suggested a low-polarity location excluded from water for the bound drug. Our data are useful for evaluating the biodisponibility of the pharmacophore and the dynamic distribution of the toxic derivative.

In the present work we studied the catalytic activity of E. coli β-Gal confined in a nanoporous silicate matrix (Eβ-Gal) at different times after the beginning of the sol-gel polymerization process. Enzyme kinetic experiments with two substrates (ONPG and PNPG) that differed in the rate-limiting steps of the reaction mechanism for their β-Gal-catalyzed hydrolysis, measurements of transverse relaxation times (T2) of water protons through 1H-NMR, and scanning electron microscopy analysis of the gel nanostructure, were performed. In conjunction, results provided evidence that water availability is crucial for the modulation observed in the catalytic activity of β-Gal as long as water participate in the rate limiting step of the reaction (only with ONPG). In this case, a biphasic rate vs. substrate concentration was obtained exhibiting one phase with catalytic rate constant (kcA), similar to that observed in solution, and another phase with a higher and aging-dependent catalytic rate constant (kcB). More structured water populations (lower T2) correlates with higher catalytic rate constants (kcB). The T2-kcB negative correlation observed along the aging of gels within the 15-days period assayed reinforces the coupling between water structure and the hydrolysis catalysis inside gels.