THE NEUROPEPTIDE KYOTORPHIN AND ITS DERIVATIVES: UNVEILING ROUTES AND TARGETS

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Kyotorphin and its derivatives : unveiling routes and targets

Publication . Perazzo, Juliana Rodrigues; Castanho, Miguel Augusto Rico Botas, 1967-

The endogenous neuropeptide kyotorphin (L-Tyr-L-Arg, KTP) has remarkable analgesic activity, up to 4.2 fold more potent than endogenous opioids when administered directly into the brain, but is inefficient after systemic administration. Our group has designed and studied several KTP analogues, among them KTP-amide (LTyr L-Arg-NH2, KTP-NH2), which is analgesic, anti-inflammatory and neuroprotective after systemic administration. Despite this interesting set of biological properties, what makes of KTP-NH2 an interesting candidate for pharmacological development, the lack of knowledge on its mechanism of action is limiting. We need to improve our knowledge on the molecular basis of action of the peptide so then we can execute an industrial development plan for the drug. Understanding the relationships between the structure of the drug and its function, efficacy and pharmacokinetics as well as the identification and characterization of molecular targets will contribute to progress in that area. The aim of this thesis is contribute to understand the mechanism of action of KTP-NH2 and to identify molecular targets of this peptide. To achieve such aim, we have designed and studied KTP-NH2 derivatives to elucidate new aspects of the structure-activity relationship; and we looked for specific targets that could explain KTP-NH2 biological activities. First, we introduced selective changes in the chemical structure of KTP-NH2 by introducing D-amino acids residues and/or N-terminal methylation, in order to improve lipophilicity and resistance to enzymatic degradation. We found that D-Tyr-L-Arg-NH2 (KTP-NH2-DL) has a pronounced anti-inflammatory effect, but insignificant analgesic action probably due to its low permeation through lipid membranes. KTP-NH2 has a similar behavior, except for its potent analgesic action in several pain models. The results support the hypothesis that KTP-NH2 uses a specific transporter to cross the blood brain barrier (BBB), which is not efficient to transport KTP-NH2 analogues. Methylated KTP-NH2 isomers, LL and LD, are very permeable through lipid membranes and probably diffuse passively across the BBB. They are also more resistant to enzymatic degradation. Therefore, they have a prolonged analgesic effect in comparison with KTP NH2, but the pro-inflammatory activity jeopardizes their pharmacological potential. In a second phase, to elucidate the mechanism of action of KTP-NH2 at the molecular level, we used an approach based on intravital microscopy (IVM) and pharmacological inhibitors. We have demonstrated that KTP-NH2 is able to decrease, within 10 minutes, the number of rolling leukocytes induced either by lipopolysaccharide (LPS) or CXCL-1, suggesting a nongenomic mechanism of action. In the inflammation model induced by LPS, KTP-NH2 failed to decrease the number of rolling leukocytes in mice pre-treated either with metyrapone, an inhibitor of glucocorticoid (GC) synthesis, or with an inhibitor of interleukin-1 receptor (IL-1R). In addition, KTP-NH2 was not as effective reducing the number of rolling leukocytes of LPS-stimulated mice pre-treated with an inhibitor of heme-oxygenase-1 (HO-1) or with an inhibitor of interleukin-10 (IL-10), but pre-treatment of the LPS-stimulated animals with an inhibitor of MyD88 did not affect the action of KTP-NH2. In the inflammation model induced with CXCL-1, pre-treatment with metyrapone did not affect the anti-inflammatory action of KTP-NH2. Thus, we concluded that KTP-NH2 has dual action: a GC-mediated action, which is dominant in full-fledged inflammation models, and a GC-independent mechanism, which is predominant in models in which leukocyte rolling is stimulated but inflammation is not totally developed. We also tested the hypothesis that KTP-NH2 could act as an inhibitor of enkephalinases. Unlike the endogenous dipeptide KTP, KTP-NH2 did not inhibit angiotensin-converting enzyme (ACE) neither dipeptidyl-peptidase III (DPP3). However, we do not rule out the possibility that KTP-NH2 can inhibit other untested enkephalinases. The work in this thesis suggests that KTP-NH2 is a multifunctional peptide that has both central and peripheral actions. KTP-NH2 presents a very fast (10 minutes) anti inflammatory mechanism that blocks the expression/activation of adhesion molecules on the endothelium and we propose that GCs might be the molecular link to explain the analgesics, anti-inflammatory and neuroprotective effects of the peptide.

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Fundação para a Ciência e a Tecnologia

Número da atribuição

PD/BD/52225/2013