Development and biological characterization of novel ligands for protein-protein interaction-based effects of prolyl oligopeptidase

Most neurodegenerative diseases (NDD) are characterized by accumulation of toxic protein species leading to the loss of movement and cognitive disorders. In the two most common NDD Parkinson´s disease (PD) and Alzheimer´s disease (AD), there are almost 60 million patients worldwide. In the U.S, there were one million PD patients and an estimated economic burden of 51.9 billion dollars in 2017. As there is no disease-modifying therapy available, NDD lead to loss of life quality and eventually to untimely death. Current drug therapies for PD can only relieve symptoms of the disease by increasing dopaminergic activity in the brain but they do not address the underlying mechanisms of the disease such as clearance of detrimental protein aggregates. In previous studies, prolyl oligopeptidase (PREP) has been shown to negatively regulate autophagy and enhance the aggregation of alpha-synuclein (αSyn). Thus, PREP inhibitors have been shown to reduce aggregation and increase clearance of αSyn aggregates via enhanced autophagy. These mechanisms are mediated by protein-protein -interactions (PPI) of PREP with other proteins. Several small molecular inhibitors of PREP have been developed to target memory disorders, cancer, and NDD. However, none of these PREP inhibitors are in therapeutical use. The aim of this study was to investigate the effect of these inhibitors in two neurodegeneration-related functions of PREP: autophagy and αSyn dimerization. A further aim was to synthesize novel PREP ligands targeted to modulate these PPI-mediated functions of PREP. In the first part of this study, a structurally diverse set of 12 potent PREP inhibitors were tested in in vitro assays of autophagy and αSyn dimerization. The study revealed that the least potent inhibitor with IC50-value of 1010 nM had the most prominent effect on reduction of αSyn dimers and enhancing autophagy, while some of the highly potent inhibitors with IC50-value ranging from 0.32 to 1.2 nM did not have an effect on autophagy or αSyn dimerization. In the second part of the study, novel 4-phenylbutanoyl-α-aminoacyl-2(S)-tetrazolyl-pyrrolidines were designed and synthesized to target αSyn dimerization. Synthesized compounds had IC50-values ranging from 12 nM to 200 000 nM, but they all reduced αSyn dimerization in a cellular assay. Furthermore, molecular docking studies showed that these novel compounds have another putative binding mode to PREP compared to typical PREP inhibitors. These findings combined with the findings from the first part of the study suggest that the PREP mediated reducing effect on αSyn dimerization and enhancing effect on autophagy is not dependent on the magnitude of PREP inhibition but more likely due to conformational stabilization in PREP caused by ligand binding. Another speculative explanation is that PREP has another binding site, which modulates the conformation of PREP and thereby the PPI mediated functions of PREP. In the third part of the study, a human αSyn transgenic mouse, carrying A30P and A53T point mutations in αSyn was characterized and evaluated as a model for early onset PD. The model displayed behavioral alternations at an early age with differences in dopaminergic neurotransmission in the nigrostriatal pathway and accumulation of oligomeric αSyn leading to decreased tyrosine hydroxylase in the striatum and substantia nigra. Furthermore, we evaluated the effect of seven days treatment by the novel PREP inhibitor, HUP-55, in this model. Results in this study showed that HUP-55 was able to decrease the level of oligomeric αSyn in the striatum and substantia nigra. In summary, this study revealed and highlighted the disconnected structure-activity relationships between inhibition of the proteolytic activity and the PPI mediated functions of PREP. Moreover, this finding led to the development of a novel PREP ligand that was successfully tested in a PD mouse model.  Useimmissa keskushermostoa rappeuttavissa sairauksissa havaitaan haitallisten proteiinien kertymistä aivoihin, mikä johtaa kognitiivisiin oireisiin sekä tahdonalaisten liikkeiden häiriöihin. Yleisimmät keskushermostoa rappeuttavat sairaudet ovat Alzheimerin tauti ja Parkinsonin tauti, joita sairastaa lähes 60 miljoona ihmistä maailmassa. Keskushermostoa rappeuttaviin sairauksiin ei ole parantavaa tai taudin etenemistä hidastavaa hoitoa, joten ne johtavat väistämättä elämänlaadun heikentymiseen ja lopulta ennenaikaiseen kuolemaan. Tällä hetkellä Parkinsonin taudin oireita pystytään lievittämään lisäämällä keskushermoston dopamiinin aktiivisuutta aivoissa, mutta tämä ei vaikuta taudin taustalla oleviin mekanismeihin kuten haitallisten proteiinikertymien poistumiseen aivoista. Aikaisemmissa tutkimuksissa on osoitettu, että prolyyli oligopeptidaasi (PREP) vähentää autofagiaa sekä lisää haitallisten alfasynukleiini-proteiinikertymien muodostumista. Toisaalta, PREP:n estämisen on osoitettu vähentävän alfasynukleiinin kertymistä keskushermostoon ja lisäävän sen puhdistumista autofagian avulla. Nämä prosessit johtuvat PREP:n ja muiden proteiinien välisistä suorista proteiini-proteiini vuorovaikutuksista. Lukuisia pienimolekyylisiä PREP-estäjiä onkin kehitetty muistihäiriöiden, syövän ja keskushermostoa rappeuttavien sairauksien hoitoon, mutta mikään näistä ei ole päätynyt markkinoille. Tämän tutkimuksen tarkoituksena oli tutkia PREP-estäjien vaikutusta kahteen keskushermoston rappeutumiseen liittyvään mekanismiin: autofagian säätelyyn ja alfasynukleiinin kertymiseen. Tämän tutkimuksen ensimmäisessä osassa selvitettiin 12 tehokkaan PREP-estäjän vaikutuksia autofagiaan ja alfasynukleiinin kertymiseen in vitro. Suurin vaikutus autofagiaan sekä alfasynukleiinikertymien vähentymiseen saatiin heikoimmalla PREP-estäjällä, samalla osa tehokkaista PREP-estäjistä eivät saaneet aikaan haluttuja vaikutuksia. Toisessa osassa tätä tutkimusta suunniteltiin ja syntetisoitiin uudenlaisia PREP-ligandeja, joilla pyrittiin vähentämään alfasynukleiinin kertymistä. Osa näistä yhdisteistä olivat heikkoja PREP-estäjiä, mutta kaikki yhdisteet vähensivät kuitenkin alfasynukleiinin kertymistä solukokeessa. Lisäksi molekyylien telakointi tietokoneavusteisesti osoitti, että nämä yhdisteet sitoutuvat eri tavalla PREP:iin kuin tyypilliset PREP-estäjät. Yhdistettynä ensimmäisen osakokeen havaintoihin, voidaan todeta, että PREP-välitteinen alfasynukleiinin kertymisen vähentyminen ja autofagian kiihtyminen ei ole riippuvainen PREP eston voimakkuudesta vaan todennäköisesti ligandin sitoutumisen aiheuttamasta stabilisaatiosta tiettyyn PREP konformaatioon. On myös mahdollista, että PREP:ssa on toinen sitoutumispaikka ligandeille, mikä säätelee PREP:in konformaatiota ja näin ollen proteiini-proteiini-vuorovaikutuksisten säätelemiä mekanismeja. Kolmannessa osakokeessa karaketerisoitiin siirtogeenisen hiirilinjan, joka kantoi A30P ja A53T pistemutaatioita alfasynukleiinigeenissä, ominaisuuksia mallintaa aikaisen vaiheen Parkinsonin tautia. Hiirillä havaittiin muutoksia käytöksessä sekä dopaminergisessa signaalinvälityksessä sekä alfasynukleiinin kertymisessä, joka johti hermosolujen vähentymiseen aivojuoviossa ja mustatumakkeessa. Lisäksi tutkimme uuden PREP-ligandi HUP-55:en vaikutuksia näihin hiiriin seitsemän päivän hoidon jälkeen. Havaittiin, että HUP-55 vähensi kertyneen alfasynukleiinin määrää aivojuoviossa sekä mustatumakkeessa. Yhteenvetona, tämä tutkimus paljasti epäjohdonmukaisuuden rakenneaktiivisuussuhteessa PREP:n aktiivisuuden estämisen sekä proteiini-proteiini-vuorovaikutus välitteisten PREP-vaikutusten välillä. Lisäksi tässä tutkimuksessa löytyi kokonaan uudenlainen PREP-ligandi, joka testattiin onnistuneesti Parkinsonin taudin hiirimallissa

Pientalon tekninen laatu. Tähtiluokitus. Opas pientalon rakennuttajille ja suunnittelijoille

Pientalon rakennuttaja, joka usein on hämmennyksissä lukuisten määräysten, ohjeiden, neuvojen ja sidosryhmien kanssa oman hankkeensa keskellä, tarvitsee asiantuntevaa opastusta  oman rakennushankkeensa ohjaukseen ja hallintaan. Tämän julkaisun avulla ympäristöministeriö haluaa tarjota pientalon rakennuttajille ja heidän suunnittelijoille ja muille yhteistyökumppaneille konkreettisen apuvälineen pientalohankkeen teknisen laadun hallintaan. Lukijaa ei rasiteta liiallisilla teknillisillä ja taloudellisilla neuvoilla. Oppaassa esitetään joukko yksinkertaisia ja helppoja valintakysymyksiä, joihin vastaamalla kyllä tai ei pientalorakennuttaja itse voi ohjata hankkeensa kosteudenkestävyyden, sisäilmaston laadun, energiankulutuksen ja ympäristövaikutukset haluamalleen tasolle. Kumppaneikseen hän tarvitsee osaavat ja vastuulliset suunnittelijat ja rakentajat. Jokaisen kestävästä, pitkäikäisestä ja laadukkaasta omakotitalosta haaveilevan tai sellaista suunnittelevan perheen kannatta tutustua tähän julkaisuun ja käyttää sitä hyväkseen. 

Behavioural and dopaminergic changes in double mutated human A30P*A53T alpha-synuclein transgenic mouse model of Parkinson´s disease.

Alpha-synuclein (aSyn) is the main component of Lewy bodies, the histopathological marker in Parkinson's disease (PD), and point mutations and multiplications of the aSyn coding SNCA gene correlate with early onset PD. Therefore, various transgenic mouse models overexpressing native or point-mutated aSyn have been developed. Although these models show highly increased aSyn expression they rarely capture dopaminergic cell loss and show a behavioural phenotype only at old age, whereas SNCA mutations are risk factors for PD with earlier onset. The aim of our study was to re-characterize a transgenic mouse strain carrying both A30P and A53T mutated human aSyn. Our study revealed decreased locomotor activity for homozygous transgenic mice starting from 3 months of age which was different from previous studies with this mouse strain that had behavioural deficits starting only after 7-9 months. Additionally, we found a decreased amphetamine response in locomotor activity and decreased extracellular dopaminergic markers in the striatum and substantia nigra with significantly elevated levels of aSyn oligomers. In conclusion, homozygous transgenic A30P*A53T aSyn mice capture several phenotypes of PD with early onset and could be a useful tool for aSyn studies.Peer reviewe

Prolyl oligopeptidase inhibition activates autophagy via protein phosphatase 2A

Prolyl oligopeptidase (PREP) is a serine protease that has been studied particularly in the context of neurode-generative diseases for decades but its physiological function has remained unclear. We have previously found that PREP negatively regulates beclinl-mediated macroautophagy (autophagy), and that PREP inhibition by a small-molecule inhibitor induces clearance of protein aggregates in Parkinson's disease models. Since autophagy induction has been suggested as a potential therapy for several diseases, we wanted to further characterize how PREP regulates autophagy. We measured the levels of various kinases and proteins regulating beclin1-autophagy in HEK-293 and SH-SY5Y cell cultures after PREP inhibition, PREP deletion, and PREP overexpression and restoration, and verified the results in vivo by using PREP knock-out and wild-type mouse tissue where PREP was restored or overexpressed, respectively. We found that PREP regulates autophagy by interacting with protein phosphatase 2A (PP2A) and its endogenous inhibitor, protein phosphatase methylesterase 1 (PME1), and activator (protein phosphatase 2 phosphatase activator, PTPA), thus adjusting its activity and the levels of PP2A in the intracellular pool. PREP inhibition and deletion increased PP2A activity, leading to activation of deathassociated protein kinase 1 (DAPK1), beclin1 phosphorylation and induced autophagy while PREP overexpression reduced this. Lowered activity of PP2A is connected to several neurodegenerative disorders and cancers, and PP2A activators would have enormous potential as drug therapy but development of such compounds has been a challenge. The concept of PREP inhibition has been proved safe, and therefore, our study supports the further development of PREP inhibitors as PP2A activators.Peer reviewe

Prolyl oligopeptidase inhibition reduces alpha-synuclein aggregation in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25α to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25α. Similar to earlier studies, p25α increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25α transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25α did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 µM KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells.Peer reviewe

Removal of proteinase K resistant alpha Syn species does not correlate with cell survival in a virus vector-based Parkinson's disease mouse model

Parkinson's disease (PD) is characterized by degeneration of nigrostriatal dopaminergic neurons and accumu-lation of alpha-synuclein (alpha Syn) as Lewy bodies. Currently, there is no disease-modifying therapy available for PD. We have shown that a small molecular inhibitor for prolyl oligopeptidase (PREP), KYP-2047, relieves alpha Syn-induced toxicity in various PD models by inducing autophagy and preventing alpha Syn aggregation. In this study, we wanted to study the effects of PREP inhibition on different alpha Syn species by using cell culture and in vivo models.We used Neuro2A cells with transient alpha Syn overexpression and oxidative stress or proteasomal inhibition -induced alpha Syn aggregation to assess the effect of KYP-2047 on soluble alpha Syn oligomers and on cell viability. Here, the levels of soluble alpha Syn were measured by using ELISA, and the impact of KYP-2047 was compared to anle138b, nilotinib and deferiprone. To evaluate the effect of KYP-2047 on alpha Syn fibrillization in vivo, we used unilateral nigral AAV1/2-A53T-alpha Syn mouse model, where the KYP-2047 treatment was initiated two-or four -weeks post injection.KYP-2047 and anle138b protected cells from alpha Syn toxicity but interestingly, KYP-2047 did not reduce soluble alpha Syn oligomers. In AAV-A53T-alpha Syn mouse model, KYP-2047 reduced significantly proteinase K-resistant alpha Syn oligomers and oxidative damage related to alpha Syn aggregation. However, the KYP-2047 treatment that was initiated at the time of symptom onset, failed to protect the nigrostriatal dopaminergic neurons. Our results emphasize the importance of whole alpha Syn aggregation process in the pathology of PD and raise an important question about the forms of alpha Syn that are reasonable targets for PD drug therapy.Peer reviewe

The effect of prolyl oligopeptidase inhibitors on alpha-synuclein aggregation and autophagy cannot be predicted by their inhibitory efficacy

Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (alpha Syn), linking it to the pathophysiology of Parkinson's disease. Our earlier results have revealed that the potent small molecular PREP inhibitor KYP-2047 is able to increase autophagy and decrease dimerization of alpha Syn but other PREP inhibitors have not been systematically studied for these two protein-protein interaction mediated biological functions of PREP. In this study, we characterized these effects for 12 known PREP inhibitors with IC50-values ranging from 0.2 nM to 1010 nM. We used protein-fragment complementation assay (PCA) to assess alpha Syn dimerization and Western Blot of microtubule-associated protein light chain 3B II (LC3B-II) and a GFP-LC3-RFP expressing cell line to study autophagy. In addition, we tested selected compounds in a cell-free alpha Syn aggregation assay, native gel electrophoresis, and determined the compound concentration inside the cell by LC-MS. We found that inhibition of the proteolytic activity of PREP did not predict decreased alpha Syn dimerization or increased autophagy, and we also confirmed that this result did not simply reflect concentration differences of the compounds inside the cell. Thus, PREP ligands regulate the effect of PREP on autophagy and alpha Syn aggregation through a conformational stabilization of the enzyme that is not equivalent to inhibiting its proteolytic activity.Peer reviewe

Prolyl oligopeptidase inhibition reduces alpha-synuclein aggregation in a cellular model of multiple system atrophy

Multiple system atrophy (MSA) is a fatal neurodegenerative disease where the histopathological hallmark is glial cytoplasmic inclusions in oligodendrocytes, rich of aggregated alpha-synuclein (aSyn). Therefore, therapies targeting aSyn aggregation and toxicity have been studied as a possible disease-modifying therapy for MSA. Our earlier studies show that inhibition of prolyl oligopeptidase (PREP) with KYP-2047 reduces aSyn aggregates in several models. Here, we tested the effects of KYP-2047 on a MSA cellular models, using rat OLN-AS7 and human MO3.13 oligodendrocyte cells. As translocation of p25 alpha to cell cytosol has been identified as an inducer of aSyn aggregation in MSA models, the cells were transiently transfected with p25 alpha. Similar to earlier studies, p25 alpha increased aSyn phosphorylation and aggregation, and caused tubulin retraction and impaired autophagy in OLN-AS7 cells. In both cellular models, p25 alpha transfection increased significantly aSyn mRNA levels and also increased the levels of inactive protein phosphatase 2A (PP2A). However, aSyn or p25 alpha did not cause any cellular death in MO3.13 cells, questioning their use as a MSA model. Simultaneous administration of 10 mu M KYP-2047 improved cell viability, decreased insoluble phosphorylated aSyn and normalized autophagy in OLN-AS7 cells but similar impact was not seen in MO3.13 cells

2-Imidazole as a Substitute for the Electrophilic Group Gives Highly Potent Prolyl Oligopeptidase Inhibitors

Different five-membered nitrogen-containing heteroaromatics in the position of the typical electrophilic group in prolyl oligopeptidase (PREP) inhibitors were investigated and compared to tetrazole. The 2-imidazoles were highly potent inhibitors of the proteolytic activity. The binding mode for the basic imidazole was studied by molecular docking as it was expected to differ from the acidic tetrazole. A new putative noncovalent binding mode with an interaction to His680 was found for the 2-imidazoles. Inhibition of the proteolytic activity did not correlate with the modulating effect on protein-protein-interaction-derived functions of PREP (i.e., dimerization of alpha-synuclein and autophagy). Among the highly potent PREP inhibiting 2-imidazoles, only one was also a potent modulator of PREP-catalyzed alpha-synuclein dimerization, indicating that the linker length on the opposite side of the molecule from the five-membered heteroaromatic is critical for the disconnected structure-activity relationships