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

Adenosine analogs bearing phosphate isosteres as human MDO1 ligands

The human O-acetyl-ADP-ribose deacetylase MDO1 is a mono-ADP-ribosylhydrolase involved in the reversal of post-translational modifications. Until now MDO1 has been poorly characterized, partly since no ligand is known besides adenosine nucleotides. Here, we synthesized thirteen compounds retaining the adenosine moiety and bearing bioisosteric replacements of the phosphate at the ribose 50-oxygen. These compounds are composed of either a squaryldiamide or an amide group as the bioisosteric replacement and/or as a linker. To these groups a variety of substituents were attached such as phenyl, benzyl, pyridyl, carboxyl, hydroxy and tetrazolyl. Biochemical evaluation showed that two compounds, one from both series, inhibited ADP-ribosyl hydrolysis mediated by MDO1 in high concentrations. (C) 2018 Elsevier Ltd. All rights reserved.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

Synthesis, identification and structure-activity relationship analysis of GATA4 and NKX2-5 protein-protein interaction modulators

Transcription factors GATA4 and NKX2-5 directly interact and synergistically activate several cardiac genes and stretch-induced cardiomyocyte hypertrophy. Previously, we identified phenylisoxazole carboxamide 1 as a hit compound, which inhibited the GATA4-NKX2-5 transcriptional synergy. Here, the chemical space around the molecular structure of 1 was explored by synthesizing and characterizing 220 derivatives and structurally related compounds. In addition to the synergistic transcriptional activation, selected compounds were evaluated for their effects on transcriptional activities of GATA4 and NKX2-5 individually as well as potential cytotoxicity. The structure-activity relationship (SAR) analysis revealed that the aromatic isoxazole substituent in the southern part regulates the inhibition of GATA4-NKX2-5 transcriptional synergy. Moreover, inhibition of GATA4 transcriptional activity correlated with the reduced cell viability. In summary, comprehensive SAR analysis accompanied by data analysis successfully identified potent and selective inhibitors of GATA4-NKX2-5 transcriptional synergy and revealed structural features important for it.Peer reviewe

GATA4:än kohdennettujen yhdisteiden rakenteen optimointi kantasolutoksisuuden vähentämiseksi

Sydämen vajaatoiminta on merkittävä sairaus, joka syntyy useimmin sydäninfarktin aiheuttaman sydänsolujen kuoleman seurauksena. Sairaus on vakava; sen viisivuotiskuolleisuus on suurempi kuin monilla syövillä. Sydänlihassolukon kuolema johtaa patologiseen vasemman kammion seinämän paksuuntumiseen ja fibroosiin, jotka edelleen heikentävät sydänlihaksen toimintaa. Sydänlihassolut eivät juurikaan uusiudu, mikä pahentaa ongelmaa. Useat soluviestintäreitit ohjaavat sydämen patologista uudelleenohjelmointia, eikä reittien merkitystä sydämen vajaatoiminnan syntyyn tunneta täysin. Erilaiset sydänlihassolujen uusiutumiseen perustuvat strategiat, kuten kantasolujen suora injektointi infarktiarven alueelle, ovat menestyneet kliinisissä tutkimuksissa heikosti. Sen sijaan sydänlihaksen patologisen uudelleenohjelmoimisen estäminen pienmolekyyleillä on yhä mahdollinen keino sydämen vajaatoiminnan synnyn ehkäisemiseksi. GATA4, eli GATAan sitoutuva proteiini 4, on sydämessä, keuhkoissa, suolistossa, sukuelimissä ja maksassa ilmentyvä transkriptiotekijä, joka aktivoi useita keskeisiä kudosten uusiutumiseen ja solunjakautumiseen vaikuttavia viestireittejä säätelemällä proteiinisynteesiä. Se sitoutuu DNA:ssa sijaitseviin GATA-jaksoihin kahden sinkkisormilenkin avulla ja käynnistää kohdegeenien luennan. GATA4:n merkittävin vaikutus on sydämessä, jossa sen vuorovaikutukset useiden muiden transkriptiotekijöiden kanssa ohjaavat sydämen kehitystä sekä sydänlihaksen paksuuntumista ja fibroosia. NKX2-5 on eräs GATA4:n kanssa synergisesti vuorovaikuttavista transkriptiotekijöistä ja tämän vuorovaikutuksen estämisen on havaittu rotan sydäninfarktimallissa ehkäisevän sydämen vajaatoiminnan syntyä. GATA4:n ja NKX2-5:n välistä proteiini-proteiini-interaktiota kohtaan on yhdisteseulontatutkimuksessa löydetty inhibiittoreita, mutta nämä yhdisteet ovat toksisia kantasoluille. Tutkimuksen tarkoituksena oli vähentää GATA4-NKX2-5 -vuorovaikutusta estävien molekyylien kantasolutoksisuutta syntetisoimalla hit-yhdisteen johdannaisia. Hit-yhdisteen fenyyliseen renkaaseen suunniteltiin muutoksia, jotka lisäävät fenyylirenkaan elektronitiheyttä tai mahdollisesti vaikuttavat fenyyli- ja isoksatsolirenkaan välisen kiertokulman suuruuteen. Yhdisteiden aktiivisuus testattiin COS-1 -soluihin transfektoidun lusiferaasireportterijärjestelmän ja toksisuus ihmisen indusoitujen pluripotenttien kantasolujen (IMR90) avulla. Toksisuuskokeiksi valittiin 3-(4,5-dimetyylitiatsol-2-yyli)-2,5-difenyylitetratsoliumbromidi- (MTT) ja laktaattidehydrogenaasikokeet (LDH). Uusien johdannaisten lisäksi tutkittiin viiden aiemmin syntetisoidun johdannaisen kantasolutoksisuutta samoissa kokeissa. Kymmenen uutta johdannaista syntetisoitiin ja testattiin solumalleissa. Yhdisteistä neljä, mono- ja di-orto-metyyli-, di-meta-metoksi- sekä sykloheksyylijohdannainen, estivät reportterigeenin aktiivisuutta hit-yhdistettä vastaavalla voimakkuudella. Lisäksi mono- ja di-orto-metyylijohdannaiset sekä di-meta-metoksijohdannainen olivat hit-yhdistettä vähemmän toksisia kantasoluille MTT-kokeessa. Myös muiden yhdisteiden joukossa oli heikosti toksisia johdannaisia, mutta niiden vaikutus GATA4-NKX2-5-synergiaan oli vähäinen. LDH-kokeiden mukaan yksikään yhdisteistä ei aiheuttanut kantasoluissa merkittävää nekroottista toksisuutta. Tutkimuksen perusteella voidaan todeta, että hit-yhdisteen fenyylirenkaan substituentteihin vaikuttamalla voidaan vähentää yhdisteen kantasolutoksisuutta. Osa muutoksista, erityisesti para-asemaan tehdyt muutokset ja fenyylirenkaan korvaaminen pienemmällä tyydyttyneellä hiilivetyrenkaalla, johti yhdisteaktiivisuuden vähenemiseen. Orto-asemaan tehtyjen muutosten vähäinen toksisuus vahvistaa hypoteesia, jonka mukaan fenyylirenkaan kiertokulma isoksatsolirenkaaseen nähden on merkittävä toksisuuden kannalta. Toisaalta, metoksiryhmän liittämisen fenyylirenkaan molempiin meta-asemoihin ei luultavasti vaikuta oleellisesti renkaan kiertokulmaan, mikä tarkoittaisi paremman siedettävyyden johtuvan muusta vaikutuksesta yhdisteen sitoutumiseen. Yhdisteiden aktiivisuus ja parantunut turvallisuusprofiili on vahvistettava eläinkokeilla, ennen kuin merkittäviä johtopäätöksiä yhdisteen rakennemuutosten vaikutuksista voidaan tehdä. Lisäksi muita toksisuuden mekanismeja tulisi tutkia solumalleilla.Heart failure is a disease of major social and economic impact. The disease is most commonly onset by extensive cardiomyocyte death following a myocardial infarction. Five-year mortality of heart failure is higher than some cancers. Loss of cardiac muscle tissue leads to pathological thickening and fibrosis of the left ventricular wall, which eventually further diminish cardiac function. Cardiomyocytes hardly proliferate, which also exacerbates the problem. Several cell signalling pathways are indicated in pathological reprogramming of the heart and the exact significance of these pathways remains to be demonstrated. Treatment strategies based on renewing cardiac muscle, such as direct injection of stem cells into the myocardium, have failed to reach clinically significant effects on heart failure patients. Direct inhibition of pathological cardiac reprogramming by using small molecule modulators remains as an auspicious strategy to treat heart failure. GATA4, or GATA binding protein 4, is a transcription factor expressed mainly in heart, lung, intestine, gonad and liver tissues, which regulates tissue renewal and cell proliferation by controlling protein transcription. GATA4 binds to GATA sequences in DNA with two zinc finger moieties and enables transcription of target genes. Interactions of GATA4 and several other transcription factors are in central role of guiding heart development, hypertrophy and fibrosis. One of these transcription factors is NKX2-5, which synergistically interacts with GATA4. Inhibition of this interaction in rat myocardial infarction model has been shown to protect against development of heart failure. A screening campaign against the transcriptional synergy of GATA4 and NKX2-5 found potent small molecule inhibitors of this interaction, but these compounds are characterised with stem cell toxicity. The aim of the study was to design and synthesise novel derivatives of GATA4-NKX2-5 synergy inhibitor hit molecule with reduced stem cell toxicity. Modifications on the phenyl ring of the hit molecule were designed, which either increase electron density of the ring or possibly alter the torsional angle between the phenyl and isoxazole ring moieties. Activity of the compounds was studied on a luciferase reporter gene system in COS-1 cells and toxicity was analysed on IMR90 human induced pluripotent stem cell line. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide and lactate dehydrogenase (LDH) assays were selected to measure toxicity on stem cells. Stem cell toxicity of several previously synthesised compounds was assayed in parallel with the novel derivatives. Ten novel derivatives were designed, synthesised and assayed. Four of the new compounds, a mono-ortho-methyl, a di-ortho-methyl, a di-meta-methoxy and cyclohexyl derivatives were found to be equipotent inhibitors of reporter gene activity compared to the hit compound. Additionally, the mono-ortho-methyl, di-ortho-methyl and di-meta-methoxy derivatives were less toxic to stem cells than the hit molecule in the MTT assay. Several other derivatives were found to be less toxic, but also non-active in the luciferase assay. None of the studied compounds exhibited notable necrotic toxicity on stem cells, as examined by the LDH assay. According to this study it may be concluded that substituents of the hit molecule phenyl ring may be altered to decrease stem cell toxicity of the compound. Some of the alterations, most notably substituents in the para-position of the phenyl ring and substitution of the phenyl ring with smaller saturated hydrocarbon rings, diminish the activity of the hit compound. Remarkable toleration of ortho-substitution reinforces the hypothesis of phenyl-isoxazole torsional angle significance for toxicity. On the other hand, addition of two methoxy groups to both meta positions most likely lacks any substantial effect on the torsional angle, which implies another mechanism of toxicity avoidance. Activity and improved safety of the novel inhibitors should be confirmed in animal models before any decisive conclusions on the effects of structural modifications on the hit molecule can be made. In addition, other mechanisms of toxicity should be studied with relevant cell-based assays

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.Peer reviewe

Stem cells are the most sensitive screening tool to identify toxicity of GATA4-targeted novel small-molecule compounds

Safety assessment of drug candidates in numerous in vitro and experimental animal models is expensive, time consuming and animal intensive. More thorough toxicity profiling already in the early drug discovery projects using human cell models, which more closely resemble the physiological cell types, would help to decrease drug development costs. In this study we aimed to compare different cardiac and stem cell models for in vitro toxicity testing and to elucidate structure-toxicity relationships of novel compounds targeting the cardiac transcription factor GATA4. By screening the effects of eight compounds at concentrations ranging from 10 nM up to 30 A mu M on the viability of eight different cell types, we identified significant cell type- and structure-dependent toxicity profiles. We further characterized two compounds in more detail using high-content analysis. The results highlight the importance of cell type selection for toxicity screening and indicate that stem cells represent the most sensitive screening model, which can detect toxicity that may otherwise remain unnoticed. Furthermore, our structure-toxicity analysis reveals a characteristic dihedral angle in the GATA4-targeted compounds that causes stem cell toxicity and thus helps to direct further drug development efforts towards non-toxic derivatives.Peer reviewe

Nonpeptidic Oxazole-Based Prolyl Oligopeptidase Ligands with Disease-Modifying Effects on alpha-Synuclein Mouse Models of Parkinson's Disease

Prolyl oligopeptidase (PREP) is a widely distributedserine proteasein the human body cleaving proline-containing peptides; however, recentstudies suggest that its effects on pathogenic processes underlyingneurodegeneration are derived from direct protein-protein interactions(PPIs) and not from its regulation of certain neuropeptide levels.We discovered novel nonpeptidic oxazole-based PREP inhibitors, whichdeviate from the known structure-activity relationship forPREP inhibitors. These new compounds are effective modulators of thePPIs of PREP, reducing alpha-synuclein (alpha Syn) dimerizationand enhancing protein phosphatase 2A activity in a concentration-responsemanner, as well as reducing reactive oxygen species production. Fromthe best performing oxazoles, HUP-55 was selected for in vivo studies. Its brain penetration was evaluated, andit was tested in alpha Syn virus vector-based and alpha Syn transgenicmouse models of Parkinson's disease, where it restored motorimpairment and reduced levels of oligomerized alpha Syn in the striatumand substantia nigra.Peer reviewe