Finding new drugs for CNS diseases by targeting proteases: the case of matrix metalloproteinase-9 and prolyl oligopeptidase

Abstract

[eng] Central nervous system (CNS) diseases are a broad category of conditions limiting health and the ability to function that affects nearly one in six people of the world’s population. Proteases are involved in a large number of diseases, which difficult therapeutic treatment since it requires the design of highly selective molecules that inhibits or activate these enzymes in addition to have the capacity to cross the Blood-brain barrier (BBB). Over the last decades, several proteases have been identified as potential targets to treat some CNS related disorders. This is the case of the prolyl oligopeptidase (POP) and the matrix metalloproteinase type 9 (MMP-9), which have been related to the cognitive impairment associate with schizophrenia (CIAS) and epilepsy development. POP, is a cytosolic serine endopeptidase that hydrolyses post-proline bonds of small peptides, it is expressed ubiquitous in the human body, but it has an increased expression and activity in the CNS. POP inhibition is known to have cognitive enhancing and neuroprotective effects in scopolamine-treated rats and an increase activity of POP has been described in plasma from schizophrenia patients, but its mechanism of action is not well understood. POP has been suggested to be involved in protein-protein interactions in addition to its enzymatic role. In this regard, several studies report suggest that POP interacts with other proteins expressed in neurons and accelerates alpha-synuclein protein aggregation. There are other studies that reported that Akt pathway could have some role in Schizophrenia patients. Following bibliography evidences and previous experimental results obtained in the group, I performed a multiplex assay of this pathway and other related experiments to analyse the role of POP in the Akt pathway. In these experiments it was confirmed that POP is involved in this pathway and that POP selective inhibition affects PP2A activity. On the other hand, by means of immunoprecipitations and western blots, I analyzed the potential direct interaction of POP with some proteins such as alpha-tubulin, GAP43, alpha-synuclein or PP2A and its possible involvement in the autophagic processes. All the experiments were performed in cells cultures or in mice in the presence or absence of the POP inhibitors IPR-19 and IPR-166, developed previously in the company.

Matrix metalloproteinases (MMPs) are a family of zinc dependent endopeptidases that play an important role in tissue remodelling in physiological and pathological conditions through cleaving extracellular matrix proteins. The MMP subfamily of gelatinases is composed by the MMP-2 and MMP-9, which have reported to have a pivotal role in synaptic circuit remodelling, neuroinflammation and BBB maintenance. MMP-9 has been documented to drive the epileptogenesis process in several preclinical models of epilepsy, showing an up-regulation of the protease after a brain insult. High levels of MMP-9 have been also reported in human brain tissue of patients with epilepsy. It is well known that MMP-9 is involved in synaptic plasticity, learning and memory and is recognized as capable to degrade extracellular matrix (ECM) proteins. Despite the robust evidence of the role of MMP-9 in the development of epilepsy and their potential use as a therapeutic target, currently there are no selective MMP-9 inhibitors because the high structural homology between MMPs, which has challenged its use as therapeutic target. The company has developed a family of potent and selective gelatines inhibitors with the capacity to cross blood-brain barrier (BBB). However, these inhibitors have low proteolytically stability in plasma and are not active after oral administration. In this thesis, I have been involved in a team who designed and synthesised new gelatinase inhibitors with a higher rat plasma stability maintaining selectivity, potency and permeability across the BBB to obtain a lead candidate. After the synthesis and several in vitro assays, the two most promising candidates were selected to perform a pharmacokinetics (PK) and solubility test. Unfortunately, the pharmacokinetics were not satisfactory but first preliminary results of solubility and possible formulations were obtained.

[cat] Les malalties del sistema nerviós central (SNC) són una àmplia categoria de condicions que limiten la salut i la capacitat de funcionar que afecta gairebé una de cada sis persones. Per algunes malalties del SNC, la diana terapèutica és una proteasa, les quals són difícils d’atacar ja que el fàrmac ha de ser altament específic perquè inhibeixi o activi aquests enzims i han de creuar la barrera hematoencefàlica. En els últims anys, s’han descobert algunes proteases prometedores com a dianes per tractar malalties del SNC com la prolil oligopeptidasa (POP) o la metal·loproteinases de matriu-9 (MMP-9). La POP, és una serina endopeptidasa citosòlica que hidrolitza els enllaços post-prolina de pèptids curts amb una expressió i activitat augmentades al SNC. Se sap que la inhibició del POP té efectes neuroprotectors i de millora cognitiva en rates tractades amb escopolamina i s'ha descrit un augment de l'activitat del POP en el plasma de pacients amb esquizofrènia. No obstant això, el rol fisiològic i patològic de la POP encara no es coneix del tot. També s’ha descrit que interactua directament amb altres proteïnes involucrades en el SNC, així com també la seva possible participació en la via de senyalització de la Akt. En aquesta tesis, s’han dut a terme una sèrie d’experiments utilitzant els inhibidors de la POP IPR-19 i IPR-166. En concret, s’ha confirmat l’ implicació directa d’aquesta proteïna en la via de la Akt i que la seva inhibició afecta a l’activitat de la proteïna PP2A, encara que de manera indirecta. També s’ha pogut demostrar la interacció directa de la POP amb la proteïna alfa-tubulina i clatrina, i que hi ha indicis de que pot participar en processos d’autofàgia. En la literatura s’ha descrit un increment en els nivells d’expressió i activitat de gelatinases (MMP-2 i MMP-9) en diferents models animals d’epilèpsia, així com també en humans amb aquesta condició, sent la MMP-9 les més sobre expressada. Per això, un inhibidor selectiu de la MMP-9, podria ser beneficial pel tractament d’aquesta malaltia. En aquesta tesis, s’han dissenyat i sintetitzat una sèrie d’inhibidors de la MMP-9 compatibles amb l’administració per via oral i estables en plasma de rata, a més de ser selectius per la diana. Tota aquesta avaluació s’ha dut a terme mitjançant assajos in vitro i els tres candidats més prometedors van ser seleccionats per fer una farmacocinètica en rosegadors.