5-Aminothiazoles Reveal a New Ligand-Binding Site on Prolyl Oligopeptidase Which is Important for Modulation of Its Protein-Protein Interaction-Derived Functions.

A series of novel 5-aminothiazole-based ligands for prolyl oligopeptidase (PREP) comprise selective, potent modulators of the protein-protein interaction (PPI)-mediated functions of PREP, although they are only weak inhibitors of the proteolytic activity of PREP. The disconnected structure-activity relationships are significantly more pronounced for the 5-aminothiazole-based ligands than for the earlier published 5-aminooxazole-based ligands. Furthermore, the stability of the 5-aminothiazole scaffold allowed exploration of wider substitution patterns than that was possible with the 5-aminooxazole scaffold. The intriguing structure-activity relationships for the modulation of the proteolytic activity and PPI-derived functions of PREP were elaborated by presenting a new binding site for PPI modulating PREP ligands, which was initially discovered using molecular modeling and later confirmed through point mutation studies. Our results suggest that this new binding site on PREP is clearly more important than the active site of PREP for the modulation of its PPI-mediated functions.

Nigrostriatal 6-hydroxydopamine lesions increase alpha-synuclein levels and permeability in rat colon.

Increasing evidence suggests that the gut-brain axis plays a crucial role in Parkinson's disease (PD). The abnormal accumulation of aggregated alpha-synuclein (aSyn) in the brain is a key pathological feature of PD. Intracerebral 6-hydroxydopamine (6-OHDA) is a widely used dopaminergic lesion model of PD. It exerts no aSyn pathology in the brain, but changes in the gut have not been assessed. Here, 6-OHDA was administered unilaterally either to the rat medial forebrain bundle (MFB) or striatum. Increased levels of glial fibrillary acidic protein in the ileum and colon were detected at 5 weeks postlesion. 6-OHDA decreased the Zonula occludens protein 1 barrier integrity score, suggesting increased colonic permeability. The total aSyn and Ser129 phosphorylated aSyn levels were elevated in the colon after the MFB lesion. Both lesions generally increased the total aSyn, pS129 aSyn, and ionized calcium-binding adapter molecule 1 (Iba1) levels in the lesioned striatum. In conclusion, 6-OHDA-induced nigrostriatal dopaminergic damage leads to increased aSyn levels and glial cell activation particularly in the colon, suggesting that the gut-brain axis interactions in PD are bidirectional and the detrimental process may start in the brain.

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

Prolyl oligopeptidase (PREP) is a widely distributed serine protease in the human body cleaving proline-containing peptides; however, recent studies suggest that its effects on pathogenic processes underlying neurodegeneration 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, which deviate from the known structure-activity relationship for PREP inhibitors. These new compounds are effective modulators of the PPIs of PREP, reducing α-synuclein (αSyn) dimerization and enhancing protein phosphatase 2A activity in a concentration-response manner, as well as reducing reactive oxygen species production. From the best performing oxazoles, HUP-55 was selected for in vivo studies. Its brain penetration was evaluated, and it was tested in αSyn virus vector-based and αSyn transgenic mouse models of Parkinson's disease, where it restored motor impairment and reduced levels of oligomerized αSyn in the striatum and substantia nigra.

A prolyl oligopeptidase inhibitor reduces tau pathology in cellular models and in mice with tauopathy.

Tauopathies are neurodegenerative diseases that are characterized by accumulation of hyperphosphorylated tau protein, higher-order aggregates, and tau filaments. Protein phosphatase 2A (PP2A) is a major tau dephosphorylating phosphatase, and a decrease in its activity has been demonstrated in tauopathies, including Alzheimer's disease. Prolyl oligopeptidase is a serine protease that is associated with neurodegeneration, and its inhibition normalizes PP2A activity without toxicity under pathological conditions. Here, we assessed whether prolyl oligopeptidase inhibition could protect against tau-mediated toxicity in cellular models in vitro and in the PS19 transgenic mouse model of tauopathy carrying the human tau-P301S mutation. We show that inhibition of prolyl oligopeptidase with the inhibitor KYP-2047 reduced tau aggregation in tau-transfected HEK-293 cells and N2A cells as well as in human iPSC-derived neurons carrying either the P301L or tau-A152T mutation. Treatment with KYP-2047 resulted in increased PP2A activity and activation of autophagic flux in HEK-293 cells and N2A cells and in patient-derived iNeurons, as indicated by changes in autophagosome and autophagy receptor markers; this contributed to clearance of insoluble tau. Furthermore, treatment of PS19 transgenic mice for 1 month with KYP-2047 reduced tau burden in the brain and cerebrospinal fluid and slowed cognitive decline according to several behavioral tests. In addition, a reduction in an oxidative stress marker was seen in mouse brains after KYP-2047 treatment. This study suggests that inhibition of prolyl oligopeptidase could help to ameliorate tau-dependent neurodegeneration.

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.

Deletion or inhibition of prolyl oligopeptidase blocks lithium-induced phosphorylation of GSK3b and Akt by activation of protein phosphatase 2A.

Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5-triphosphate (IP3 ) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP-related lithium insensitivity is dependent on PP2A. To assess this, HEK-293 and SH-SY5Y cells with PREP deletion or PREP inhibition (KYP-2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium-induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP-2047 did not have effect on lithium-induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation.

Prolyl oligopeptidase inhibition by KYP-2407 increases alpha-synuclein fibril degradation in neuron-like cells.

Growing evidence emphasizes insufficient clearance of pathological alpha-synuclein (αSYN) aggregates in the progression of Parkinson's disease (PD). Consequently, cellular degradation pathways represent a potential therapeutic target. Prolyl oligopeptidase (PREP) is highly expressed in the brain and has been suggested to increase αSYN aggregation and negatively regulate the autophagy pathway. Inhibition of PREP with a small molecule inhibitor, KYP-2407, stimulates autophagy and reduces the oligomeric species of αSYN aggregates in PD mouse models. However, whether PREP inhibition has any effects on intracellular αSYN fibrils has not been studied before. In this study, the effect of KYP2407 on αSYN preformed fibrils (PFFs) was tested in SH-SY5Y cells and human astrocytes. Immunostaining analysis revealed that both cell types accumulated αSYN PFFs intracellularly but KYP-2047 decreased intracellular αSYN deposits only in SH-SY5Y cells, as astrocytes did not show any PREP activity. Western blot analysis confirmed the reduction of high molecular weight αSYN species in SH-SY5Y cell lysates, and secretion of αSYN from SH-SY5Y cells also decreased in the presence of KYP-2407. Accumulation of αSYN inside the SH-SY5Y cells resulted in an increase of the auto-lysosomal proteins p62 and LC3BII, as well as calpain 1 and 2, which have been shown to be associated with PD pathology. Notably, treatment with KYP-2407 significantly reduced p62 and LC3BII levels, indicating an increased autophagic flux, and calpain 1 and 2 levels returned to normal in the presence of KYP-2407. Our findings indicate that PREP inhibition can potentially be used as therapy to reduce the insoluble intracellular αSYN aggregates.

N-stearoyltyrosine protects primary cortical neurons against oxygen-glucose deprivation-induced apoptosis through inhibiting anandamide inactivation system.

N-stearoylthrosine (NST), a synthesized anandamide (AEA) analogue, plays a neuroprotective role in neurodegenerative diseases and cerebrovascular diseases. Several studies have demonstrated that the endocannabinoids systems (ECS) are involved in the neuroprotective effects against cerebral ischemic injury. Oxygen-glucose deprivation (OGD)-induced neuronal injury elevated the levels of endocannabinoids and activated ECS. This research was conducted to investigate the neuroprotective effect of NST against OGD-induced neuronal injury in cultured primary cortical neurons and the potential mechanism involved. Cortical neurons were treated with NST at indicate concentrations for 30min prior to injury and OGD injured neurons were incubated with normal conditions for 0-24h. The best neuroprotective effect of NST against OGD-induced injury occurred at 10µM. All data indicated that the neuroprotective effect of NST against OGD-induced injury resulted from blocking anandamide membrane transporter (AMT) (IC50=11.74nM) and inhibiting fatty acid amide hydrolase activity (FAAH) (IC50=16.54nM). Our findings demonstrated that NST has an important role in cerebral ischemic injury pathological progression through activating cannabinoid receptors by inhibiting AEA inactivation system. These data suggested a potential role for NST in the therapeutic consideration of cerebral ischemic injury. However, inhibition of AEA inactivation system may provide a neuroprotective effect during cerebral ischemic injury.

Dipotassium N-stearoyltyrosinate ameliorated pathological injuries in triple-transgenic mouse model of Alzheimer's disease.

Recently, anandamide (AEA) analogues have been well recognized for its potent neuroprotective effects in counteracting the deterioration of Alzheimer's disease (AD) brains through multiple pathological processes. In our previous studies, dipotassium N-stearoyltyrosinate (NSTK), an AEA analogue synthesized by our laboratory was reported to exert significant efficacy through multiple interventions. Within this study, the amyloid precursor protein (APP)SWE/presenilin-1 (PS1)M146V/TauP301L mouse (3×Tg-AD) model was used to explore further the neuroprotective effects of NSTK and its underlying mechanisms. NSTK could increase spontaneous locomotor activity in the open field and low anxiety-like behavior in the elevated plus maze, and improve the spatial memory deficits in the Morris water maze. The biochemical analysis suggested that NSTK could decrease Aß42 deposition, abnormal tau aggregation, and the expressions of p-APP Thr668, PS1 and p-tau Ser202/Thr205 in the hippocampus of 3×Tg-AD mice. Consistently, NSTK could reduce the level of malondialdehyde, increase the activity of superoxide dismutase and catalase. Up-regulation of Bcl-2, and down-regulation of BAX, caspase-3 and inflammatory cytokines also occurred in the hippocampus of 3×Tg-AD mice after treatment with NSTK. Thus, NSTK could intervene in multiple pathological processes of AD and would be a drug candidate against AD.

N-Stearoyltyrosine protects primary cortical neurons against Aß(1-40)-induced injury through inhibiting endocannabinoid degradation.

AIMS: N-stearoyltyrosine (NsTyr) as an anandamide (AEA) analog has close relationships with AEA not only in structure but also in terms of biological actions of endocannabinoids. Since ß-amyloid (Aß)-induced primary neuronal injury involves the activation of the endocannabinoid systems (ECS), the protective effects of NsTyr against Aß(1-40)-induced neuronal injury and the mechanism were studied systematically in this paper. MAIN METHODS: Cortical neurons were incubated with Aß(1-40) for 24 h. NsTyr was added to indicated concentrations 30 min prior to injury. KEY FINDINGS: The best effects of NsTyr on Aß(1-40)-induced primary neuronal injury occurred at 10 µM. The mechanism of NsTyr on neuroprotective effects against Aß(1-40)-induced cellular death first involved anti-apoptosis resulting from the activation of cannabinoid receptors, then the pre-receptor regulation of AEA by the inhibition of endocannabinoid inactivation. These data demonstrated that the protective effects of NsTyr on Aß(1-40)-induced primary neuronal injury resulted from the inhibition of fatty acid amide hydrolase (FAAH) (IC50=16.54 nM) and blocked AEA uptake mediated by anandamide membrane transporter (AMT) (IC50=11.74 nM). SIGNIFICANCE: The activation of ECS by inhibiting the degradation of AEA is an effective pharmacological approach to suppress Aß-induced neuropathic injury. Our research could result in a more realistic alternative for AD treatment.

N-stearoyltyrosine protects against glutamate-induced oxidative toxicity by an apoptosis-inducing factor (AIF)-mediated caspase-independent cell death pathway.

N-stearoyltyrosine (NsTyr), a synthesized anandamide (AEA) analogue, could exert potent neuroprotective effects on cerebral ischemia models both in vivo and in vitro via intervening in multiple injuries. Glutamate, a major excitatory neurotransmitter, plays a critical role during stroke/cerebral ischemia. In this study, we explored the protective effects of NsTyr on glutamate neurotoxicity in PC12 cells and investigated its underlying mechanisms. NsTyr treatment attenuated glutamate-induced oxidative toxicity in a dose-dependent manner and the best performance was observed at 10 µΜ. NsTyr treatment suppressed glutamate-induced upregulation of lipoxygenase 12/15 (LOX 12/15) activity and reactive oxygen species (ROS) elevation, attenuated the increase of BH3-interacting domain death agonist (Bid) in the mitochondria, prevented the loss of mitochondria membrane potential and consequently inhibited apoptosis-inducing factor (AIF) translocation into the nucleus. The results demonstrated that NsTyr could protect cells against AIF-mediated caspase-independent cell death induced by glutamate, which may be due to the blockage of Bid-mediated mitochondrial damage via reducing LOX 12/15 activity and ROS accumulation.

Gastrin inhibits a novel, pathological colon cancer signaling pathway involving EGR1, AE2, and P-ERK.

Human anion exchanger 2 (AE2) is a plasma membrane protein that regulates intracellular pH and cell volume. AE2 contributes to transepithelial transport of chloride and bicarbonate in normal colon and other epithelial tissues. We now report that AE2 overexpression in colon cancer cells is correlated with expression of the nuclear proliferation marker, Ki67. Survival analysis of 24 patients with colon cancer in early stage or 33 patients with tubular adenocarcinoma demonstrated that expression of AE2 is correlated with poor prognosis. Cellular and molecular experiments indicated that AE2 expression promoted proliferation of colon cancer cells. In addition, we found that transcription factor EGR1 underlies AE2 upregulation and the AE2 sequester p16INK4a (P16) in the cytoplasm of colon cancer cells. Cytoplasmic P16 enhanced ERK phosphorylation and promoted proliferation of colon cancer cells. Gastrin inhibited proliferation of colon cancer cells by suppressing expression of EGR1 and AE2 and by blocking ERK phosphorylation. Taken together, our data describe a novel EGR1/AE2/P16/P-ERK signaling pathway in colon carcinogenesis, with implications for pathologic prognosis and for novel therapeutic approaches.