La ciclofilina D come bersaglio farmacologico: suoi potenziali inibitori peptidici
Year: 2012
Type: dissertation
Abstract: The mitochondrial permeability transition pore (MPTP) plays a key role in the process of mitochondria-mediated cell death (apoptosis). Molecules able to inhibit the MPTP can therefore be potential therapeutic agents useful in treating conditions such as trauma, ischemia/reperfusion injury, Parkinson's and Alzheimer's disease and in general all those conditions in which oxidative stress is involved and leading to apoptosis. The cyclophilin D (CypD), a peptidyl-prolyl isomerase, is a mitochondrial matrix protein that is closely involved in the open-closed transition regulation of MPTP. The mechanism by which exerts its action is not yet known. There are, however, evidence highlighting its key role in the modulation of mitochondrial permeability. Like other cyclophilin family members, is targeted by cyclosporin A (CsA), a cyclic undecapeptide extracted from the fungus Tolypocladium inatum, which has revolutionized the practice of preventing transplant rejection. In fact, CsA is well known for his powerful immunosuppressant that is expressed through the formation of a ternary complex with a cyclophilin and calcineurin (Cn), a Ser/Thr phosphatase, Ca2+/calmodulin-dependent. Cn, through the dephosphorylation of nuclear factor of activated T cells, is heavily involved in signal pathways that activate the immune response. CsA interacts with a cyclophilin and the CsA-Cyp complex associates with Cn, inhibiting its enzymatic activity. Gene ablation studies show that CypD desensitizes MPTP. In addition, treatment with CsA improves, through interaction with CypD and then MPTP inhibition, the conditions of various diseases related to mitochondrial dysregulation in animal models. All these findings validate the hypothesis that inhibition of CypD is a good therapeutic strategy. The major problem in the use of CsA as an inhibitor of CypD is its immunosuppressive action, which limits its use as may be given only for short periods of time. Indeed, preliminary clinical studies performed on patients with Ullrich dystrophy and Bethlem myopathy have yielded encouraging results, but in a few weeks, the patients became immunosuppressed, and then exposed to the potential risk of contracting dangerous infections. Another problem is that CsA, in spite of its lipophilicity, does not reach adequate concentrations in the central nervous system, particularly in the brain after systemic administration, because it fails to adequately penetrate the blood-brain barrier. In light of these considerations it is clear that CypD is a drug target of interest and obtaining ligands with no activity against the Cn and can reach high concentrations in the central nervous system, is a condition for the development of promising of therapeutic agents that can control a variety of disorders or diseases. Antamanide (Anta) is a non-toxic ciclodecapeptide isolated from the fungus Amanita phalloides. Much of the research conducted on this molecule have been devoted to the study of his activities in relation to its ability to form complexes with alkali metals and alkaline earth metals, mainly Na+ and Ca2+, which were then structurally and functionally studied. As CsA and ciclolinopeptide A, a ciclononapeptide extracted from flax seed, the Anta has some immunosuppressive activity, which is however lower than that of CsA. The purpose of this work is to design, chemical synthesis and characterization, with NMR, atomic interactions with the protein, of peptide inhibitors of CypD and more specifically of the Anta and its derivatives. The objective is to identify new ligands that are not immunosuppressive. The Anta has been chosen as the starting point for the features common to CsA, for the fact of having biological activity and the ability to chelate Ca2+ ions that play an important role in the regulation of MPTP. For this reason we synthesized the Anta and some derivatives in which the Phe residue at position 6 and 9 are replaced by either Gly and Tyr residues. We also synthesized two octapeptidies lacking the Phe in position 5 and 6, and the Phe in position 9 has been replaced by Gly and Tyr. Finally we got for expression in E. coli the CypD marked with 15N, to perform two-dimensional heteronuclear NMR experiments. However, the great difficulties encountered in the refolding of the protein did not allow us to obtain valuable information that this technique could offer. Peptides were performed on the in vitro biological assays, which have shown as the Anta is an inhibitor of the mitochondrial transition pore and that this action is via inhibition of CypD. We have also shown that Anta protects from mitochondrial depolarization and cell death. Finally we can say that the Phe 6 and 9 are essential for the biological action on CypD, as all its derivatives have shown no activity.
Author: Nicola Antolini
Language: Italian
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W12702351