The developing of molecules to target the opioid receptors and to regulate the insulin gene

During my PhD I worked with the group of Professor Severo Salvadori, I have investigated opiod system involved in the pain transmission, in particular MOP and NOP receptors. Presently various external opiates are administered like morphine for analgesic therapy, but they have a limit for clinical usefulness, because their tendency is to produce tolerance and dependence. The main project aim is to synthetize peptides bivalent ligands characterized by a NOP and MOP substrate with the deal to reduce the typical side effects of the current therapy. In order to obtain this peptide bivalent ligand was planned chemical strategies able to give them, in particular it was used Thio-Michael reaction and Thiol-ene reaction. Each reaction allows the introduction of the specific features into MOP pharmacophore and NOP pharmacophore involved in the reaction. Thio-Michael has allowed to obtain two peptides bivalent ligands: the first is characterized by a MOP pharmacophore as dermorphin functionalized with a Lysin with a maleimide function in lateral chain in C-terminal portion able to interact with a NOP pharmacophore as N/OFQ functionalized with a Cysteine in position 18 in order to afford the bivalent ligand. The second one is represented by a MOP pharmacophore as hexapeptide N-terminal of dinorphin with a Lysin with a maleimide function in lateral chain in position 8 able to react with a NOP pharmacophore as N/OFQ functionalized like previously seen in order to yield the bivalent ligand. The requirement to afford a good bivalent ligand is to maintain in vivo an equipotency between the substrate MOP and NOP. Both are been tested in vitro through the analysis of the intracellular calcium mobilization, compared to standard endogenous ligands, to observe the activity of the two pharmacophores inside the ligand with receptors transfected in CHO cells. Currently studies are underway in vitro tissues. The tests are performed from the group of Dr Girolamo Calò of Ferrara University. Thiol-ene reaction has not been successful to obtain peptide bivalent ligand, for these reasons it is decided to start an initial methodological study about Thiol-ene reaction. The aim was to understand the influence of the side chains of the aminoacids adjacent to the Cysteine, which aminoacid involved in the reaction. It was synthetized pentapeptades prototype with the introduction in position 3 and 4 different aminoacids with side chains variable to assess whether some of these are involved in side reactions sequence dependent. As alkene substrate it was used an olefin, such as the allyl-glucose. The peptides were analyzed by NMR in order to evaluate any changes of the chemical shift in the case of no success of reaction. Variable aminoacids are been investigated with characteristics hydrophilic, acidic and aromatic. All reactions tested have given the product of Thiol-ene and its byproduct sulfoxide. I spent 11 months of my PhD at Aberdeen University in the group of Professor Matteo Zanda and I worked in collaboration with Dr John Barrow in a study regarding the analysis of change of conformation of particular promoters through gel-based assay (EMSA). In particular the attention was focused on insulin-linked polymorphic region (ILPR) that is a regulatory sequence located upstream of the human insulin gene. It is characterized by multiple repeats of consensus sequence rich in guanine residues able to assume a particular conformation called G-quadruplex. DNA complex is stabilized by some cations and small molecules. This peculiar structure is very important in the complex system of the activation of insulin gene expression. Since insulin is directly related to the pathogenesis of diabetes, the potential regulatory role of the G-quadruplex in the insulin expression has received close attention. The main project aim is to alter the functionality of the ILPR, and subsequently alter INS gene expression using small molecules as ligands that will bind to and regulate the ILPR. The project was developed in different steps: -- Analysis of the appropriate concentration of oligonucleotides to proceed with the EMSA assay; -- Investigation of molecules known in literature and commercially available to have a good ability to stabilize G-quadruplex; -- Synthesis of a new compound triazine derivate to enhance the binding and stabilize the DNA complex. The small molecules chosen have shown a stabilization of G-quadruplexes in line with the data in literature. These data are also confirmed by CD analysis did from Glasgow University. The new triazine synthetized was only tested with EMSA assay and it has shown a slight ability to interact with the G-quadruplex. CD analysis of the new compound are under study.