Investigating the inclusion properties of aromatic amino acids complexing beta-cyclodextrins in model peptides.

Cyclodextrins are commonly used as complexing agents in biological, pharmaceutical, and industrial applications since they have an effect on protein thermal and proteolytic stability, refolding yields, solubility, and taste masking. ß-cyclodextrins (ß-CD), because of their cavity size are a perfectly suited complexing agent for many common guest moieties. In the case of peptide-cyclodextrin and protein-cyclodextrin host-guest complexes the aromatic amino acids are reported to be the principal responsible of the interaction. For these reasons, we have investigated the inclusion properties of nine designed tripeptides, obtained permuting the position of two L-alanines (Ala, A) with that of one L-tryptophan (Trp, W), L-phenylalanine (Phe, F), or L-tyrosine (Tyr, Y), respectively. Interestingly, the position of the aromatic side-chain in the sequence appears to modulate the ß-CD:peptide binding constants, determined via UV-Vis and NMR spectroscopy, which in turn assumes values higher than those reported for the single amino acid. The tripeptides containing a tyrosine showed the highest binding constants, with the central position in the Ac-AYA-NH2 peptide becoming the most favorite for the interaction. A combined NMR and Molecular Docking approach permitted to build detailed complex models, highlighting the stabilizing interactions of the neighboring amino acids backbone atoms with the upper rim of the ß-CD.

Towards understanding the molecular recognition process in prokaryotic zinc-finger domain.

Eukaryotic Cys2His2 zinc finger domain is one of the most common and important structural motifs involved in protein-DNA interaction. The recognition motif is characterized by the tetrahedral coordination of a zinc ion by conserved cysteine and histidine residues. We have characterized the prokaryotic Cys2His2 zinc finger motif, included in the DNA binding region (Ros87) of Ros protein from Agrobacterium tumefaciens, demonstrating that, although possessing a similar zinc coordination sphere, this domain presents significant differences from its eukaryotic counterpart. Furthermore, basic residues flanking the zinc binding region on either side have been demonstrated, by Electrophoretic Mobility Shift Assay (EMSA) experiments, to be essential for Ros DNA binding. In spite of this wealth of knowledge, the structural details of the mechanism through which the prokaryotic zinc fingers recognize their target genes are still unclear. Here, to gain insights into the molecular DNA recognition process of prokaryotic zinc finger domains we applied a strategy in which we performed molecular docking studies using a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) simulations data. The results demonstrate that the MD ensemble provides a reasonable picture of Ros87 backbone dynamics in solution. The Ros87-DNA model indicates that the interaction involves the first two residue of the first α-helix, and several residues located in the basic regions flanking the zinc finger domain. Interestingly, the prokaryotic zinc finger domain, mainly with the C-terminal tail that is wrapped around the DNA, binds a more extended recognition site than the eukaryotic counterpart. Our analysis demonstrates that the introduction of the protein flexibility in docking studies can improve, in terms of accuracy, the quality of the obtained models and could be particularly useful for protein showing high conformational heterogeneity as well as for computational drug design applications.

ß-Cyclodextrin inclusion complex to improve physicochemical properties of pipemidic acid: characterization and bioactivity evaluation.

The aptitude of cyclodextrins (CDs) to form host-guest complexes has prompted an increase in the development of new drug formulations. In this study, the inclusion complexes of pipemidic acid (HPPA), a therapeutic agent for urinary tract infections, with native ß-CD were prepared in solid state by kneading method and confirmed by FT-IR and 1H NMR. The inclusion complex formation was also characterized in aqueous solution at different pH via UV-Vis titration and phase solubility studies obtaining the stability constant. The 1:1 stoichiometry was established by a Job plot and the inclusion mechanism was clarified using docking experiments. Finally, the antibacterial activity of HPPA and its inclusion complex was tested on P. aeruginosa, E. coli and S. aureus to determine the respective EC50s and EC90s. The results showed that the antibacterial activity of HPPA:ß-CD against E. coli and S. aureus is higher than that of HPPA. Furthermore, HPPA and HPPA:ß-CD, tested on human hepatoblastoma HepG2 and MCF-7 cell lines by MTT assay, exhibited, for the first time, antitumor activities, and the complex revealed a higher activity than that of HPPA. The use of ß-CD allows an increase in the aqueous solubility of the drug, its bioavailability and then its bioactivity.

Physicochemical characterization and cytotoxic activity evaluation of hydroxymethylferrocene:ß-cyclodextrin inclusion complex.

An inclusion complex of hydroxymethylferrocene (FeMeOH) with ß-cyclodextrin (ß-CD) was prepared in the solid state by different techniques such as physical mixture, coprecipitation, kneading and freeze-drying. The formation of the inclusion complex was confirmed by X-ray Powder Diffractometry and Fourier Transform-Infrared spectroscopy. In aqueous solution, the 1:1 stoichiometry was established by a Job plot. The inclusion complex formation was also investigated by NMR and the stability constant (Kb) of the complex was determined to be 478 M⁻¹, which is in agreement with that obtained with UV-Vis tritation (Kb = 541.3 M⁻¹). The phase solubility study showed a diagram classified as Bs type and that the solubility of FeMeOH was slightly increased in the presence of ß-CD. Furthermore, utilizing phase solubility diagram data, the Kb was estimated to be equal to 528.0 M⁻¹. The cytotoxic activity of FeMeOH and its complexation product with ß-CD was determined using the MTT-assay on MDA-MB-231 cell line, showing that the inclusion complex has a higher capability of inhibiting cell growth compared to that of pure FeMeOH.