Sintesi di complessi poliossometallati ibridi e multifunzionali

Polyoxometalates (POMs) are discrete polyanionic oxides that shows a wide variety of applications in material science, nanomedicine and catalysis. This thesis describes the synthesis and characterization of a small library of novel polyoxometalate-based hybrids, and includes the preliminary investigations about their possible applications. The preparation of the complexes was obtained through the bis-functionalization of the divacant Keggin polyanion [γ-SiW10O36]8-, which was selected as a model compound for its reproducible synthesis and stability. The decoration of this POM was obtained in one or two steps, and involved the reaction of trialkoxy organosilanes or organo phosphonic acids with the reactive oxygen atoms of the POM vacancy. The POM was thus decorated with the following pendants: -luminescent units (dansyl, pyrene, fluoresceine), to obtain POMs with sensing capabilities in the presence of ions (Cu2+) or apolar molecules (C60), as well as with promising applications in fluorescence imaging, for cell trafficking studies. -bioactive molecules (avidin, folic acid, tryptophan), to be used for the recognition of small molecules or biological targets, with potential application in oxidation catalysis and medicine. In all cases, the successful functionalization was confirmed by means of proton, carbon and heteronuclear NMR (P, Si, W), and ESI-MS and FTIR. The spectral features of the molecular hybrids, and the interactions between the POMs and target molecules/ions were highlighted by fluorescence and UV-Vis spectroscopy. Chiral derivative were also studied by circular dichroism.openTesi 3797Tesi non accessibile sino al 04/04/2014 per motivi correlati alla proprieta' intellettuale

Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members

Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnels, ligand binding residues and ligand binding energies. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels

Reevaluation of bromodomain ligands targeting BAZ2A

BAZ2A promotes migration and invasion in prostate cancer. Two chemical probes, the specific BAZ2-ICR, and the BAZ2/BRD9 cross-reactive GSK2801, interfere with the recognition of acetylated lysines in histones by the bromodomains of BAZ2A and of its BAZ2B paralog. The two chemical probes were tested in prostate cancer cell lines with opposite androgen susceptibility. BAZ2-ICR and GSK2801 showed different cellular efficacies in accordance with their unequal selectivity profiles. Concurrent inhibition of BAZ2 and BRD9 did not reproduce the effects observed with GSK2801, indicating possible off-targets for this chemical probe. On the other hand, the single BAZ2 inhibition by BAZ2-ICR did not phenocopy genetic ablation, demonstrating that bromodomain interference is not sufficient to strongly affect BAZ2A functionality and suggesting a PROTAC-based chemical ablation as an alternative optimization strategy and a possible therapeutic approach. In this context, we also present the crystallographic structures of BAZ2A in complex with the above chemical probes. Binding poses of TP-238 and GSK4027, chemical probes for the bromodomain subfamily I, and two ligands of the CBP/EP300 bromodomains identify additional headgroups for the development of BAZ2A ligands

Identification of a BAZ2A Bromodomain Hit Compound by Fragment Joining

The bromodomains of BAZ2A and BAZ2B (bromodomain adjacent to zinc finger domain proteins 2) are among the most hard to drug of the 61 human bromodomains. While little is known about the role of BAZ2B, there is strong evidence for the opportunity of targeting BAZ2A in various cancers. Here, a benzimidazole–triazole fragment that binds to the BAZ2A acetyl lysine pocket was identified by a molecular docking campaign and validated by competitive binding assays and X-ray crystallography. Another ligand was observed in close proximity by soaking experiments using the BAZ2A bromodomain preincubated with the benzimidazole–triazole fragment. The crystal structure of BAZ2A with the two ligands was employed to design a few benzimidazole–triazole derivatives with increased affinity. We also present the engineering of a BAZ2A bromodomain mutant for consistent, high-resolution crystallographic studies

Identification of a BAZ2A-Bromodomain Hit Compound by Fragment Growing

BAZ2A is an epigenetic regulator affecting transcription of ribosomal RNA. It is overexpressed in aggressive and recurrent prostate cancer, promoting cellular migration. Its bromodomain is characterized by a shallow and difficult-to-drug pocket. Here, we describe a structure-based fragment-growing campaign for the identification of ligands of the BAZ2A bromodomain. By combining docking, competition binding assays, and protein crystallography, we have extensively explored the interactions of the ligands with the rim of the binding pocket, and in particular ionic interactions with the side chain of Glu1820, which is unique to BAZ2A. We present 23 high-resolution crystal structures of the holo BAZ2A bromodomain and analyze common bromodomain/ligand motifs and favorable intraligand interactions. Binding of some of the compounds is enantiospecific, with affinity in the low micromolar range. The most potent ligand has an equilibrium dissociation constant of 7 μM and a good selectivity over the paralog BAZ2B bromodomain

Sintesi di complessi poliossometallati ibridi e multifunzionali

Polyoxometalates (POMs) are discrete polyanionic oxides that shows a wide variety of applications in material science, nanomedicine and catalysis. This thesis describes the synthesis and characterization of a small library of novel polyoxometalate-based hybrids, and includes the preliminary investigations about their possible applications. The preparation of the complexes was obtained through the bis-functionalization of the divacant Keggin polyanion [γ-SiW10O36]8-, which was selected as a model compound for its reproducible synthesis and stability. The decoration of this POM was obtained in one or two steps, and involved the reaction of trialkoxy organosilanes or organo phosphonic acids with the reactive oxygen atoms of the POM vacancy. The POM was thus decorated with the following pendants: -luminescent units (dansyl, pyrene, fluoresceine), to obtain POMs with sensing capabilities in the presence of ions (Cu2+) or apolar molecules (C60), as well as with promising applications in fluorescence imaging, for cell trafficking studies. -bioactive molecules (avidin, folic acid, tryptophan), to be used for the recognition of small molecules or biological targets, with potential application in oxidation catalysis and medicine. In all cases, the successful functionalization was confirmed by means of proton, carbon and heteronuclear NMR (P, Si, W), and ESI-MS and FTIR. The spectral features of the molecular hybrids, and the interactions between the POMs and target molecules/ions were highlighted by fluorescence and UV-Vis spectroscopy. Chiral derivative were also studied by circular dichroism

Моделювання методом молекулярної динаміки для аналізу структури та функцій ферментів Amadoriase

Background. Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes.Objective. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnel, ligand binding residues and ligand binding energies.Methods. We used a combination of homology modeling and molecular dynamics simulations to provide insights into the structural difference between the five enzymes of the FAOX family.Results. We first predicted the structure of the N1-1-FAOD and PnFPOX enzymes using homology modelling. Then, we used these models and the experimental crystal structures of Amadoriase I, Amadoriase II and FPOX-E to run extensive molecular dynamics simulations in order to compare the structures of these FAOX enzymes and assess their relevant interactions with two relevant ligands, f-val and f-lys.Conclusions. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels