Pharmacological Intervention Targeting FAF1 Restores Autophagic Flux for α-Synuclein Degradation in the Brain of a Parkinson's Disease Mouse Model.

α-Synuclein accumulation is implicated in the pathogenesis of neurodegenerative diseases, including Parkinson's disease (PD). Previously, we reported that Fas-associated factor 1 (FAF1), which plays a role in PD pathogenesis, potentiates α-synuclein accumulation through autophagy impairment in dopaminergic neurons. In this study, we show that KM-819, a FAF1-targeting compound, which has completed phase I clinical trials, interferes with α-synuclein accumulation in the mouse brain, as well as in human neuronal cells (SH-SY5Ys). KM-819 suppressed the accumulation of monomeric, oligomeric, and aggregated forms of α-synuclein in neuronal cells. Furthermore, KM-819 restored the turnover rate of α-synuclein in FAF1-overexpressing SH-SY5Y cells, implicating KM-819-mediated reconstitution of the α-synuclein degradative pathway. In addition, KM-819 reconstituted autophagic flux in FAF1-transfected SH-SY5Y cells, also suppressing α-synuclein-induced mitochondrial dysfunction. Moreover, oral administration of KM-819 also interfered with α-synuclein accumulation in the midbrain of mice overexpressing FAF1 via an adeno-associated virus system. Consistently, KM-819 reduced α-synuclein accumulation in both the hippocampus and the midbrain of human A53T α-synuclein transgenic mice. Collectively, these data imply that KM-819 may have therapeutic potential for patients with PD.

Fas-associated factor 1 induces the accumulation of α-synuclein through autophagic suppression in dopaminergic neurons.

Impairment of protein clearance mechanisms leads to α-synuclein accumulation in dopaminergic neurons, contributing to the pathogenesis of Parkinson's disease (PD). Based on the finding that Fas-associated factor 1 (FAF1), a positive modulator of PD, colocalizes with α-synuclein in PD patient brains, we investigated the existence of pathological interplay between FAF1 and α-synuclein. Monomeric and high-molecular-weight forms of α-synuclein were increased in FAF1-overexpressing SH-SY5Y cells. In particular, α-synuclein turnover was accelerated by genetic depletion of FAF1 in SH-SY5Y cells. Therefore, we questioned whether FAF1 is involved in the α-synuclein clearance process. Autophagy inhibitors, but not proteasome inhibitors, restored concurrent attenuation of α-synuclein expression by FAF1 depletion in SH-SY5Y cells. Moreover, we found alterations in autophagy markers in SH-SY5Y cells caused by FAF1 overexpression, indicating that FAF1 disturbed α-synuclein clearance through the autophagy-lysosome pathway. Indeed, FAF1 activated the mammalian target of rapamycin (mTOR) pathway, subsequently suppressing autophagosome formation. Consistently, α-synuclein-mediated mitochondrial dysfunction was observed in FAF1-overexpressing SH-SY5Y cells. Furthermore, FAF1 overexpression using stereotaxic injection of adeno-associated virus led to α-synuclein accumulation and autophagy dysregulation in the PD model mice. Taken together, our results reveal a novel role for FAF1: that of a negative regulator of autophagic α-synuclein clearance.

Neurorestorative Effects of a Novel Fas-Associated Factor 1 Inhibitor in the MPTP Model: An [18F]FE-PE2I Positron Emission Tomography Analysis Study.

Fas-associated factor 1 (FAF1), a Fas-binding protein, is implicated in neuronal cell death in Parkinson's disease (PD). We examined the effects of a novel FAF1 inhibitor, KM-819, in dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model using [18F]FE-PE2I positron emission tomography (PET). The MPTP model was generated with subacute MPTP treatment (20 mg/kg/day, i.p.) for 5 consecutive days in C57bl/6J mice. This study included three groups: the control group (treatment with saline only), the MPTP model group with KM-819 treatment (20 mg/kg/day p.o.) for 6 days, and the MPTP model group without KM-819 treatment. [18F]FE-PE2I PET studies were conducted in the same animals before and after MPTP with or without KM-819 treatment to monitor changes in striatal dopamine transporter activity indicated by non-displaceable binding potential (BPND) of [18F]FE-PE2I, and the expression levels of tyrosine hydroxylase were assessed using immunohistochemistry before and after KM-819 treatment. After MPTP injection, decreased striatal BPND was observed in the MPTP model group compared with the control group. Striatal BPND increased in the MPTP model group with KM-819 treatment, but not in the MPTP model group without KM-819 treatment. The tyrosine hydroxylase expression levels also significantly increased in the MPTP model group with KM-819 treatment compared with the control group. This study indicates that inhibition of the Fas-mediated cell death pathway by KM-819 has neurorestorative effects in striatal dopamine neurons in the MPTP model. Further studies would be needed to investigate the potential of KM-819 as a therapeutic drug for PD treatment.

Influences of cytochrome b5 expression and its genetic variant on the activity of CYP2C9, CYP2C19 and CYP3A4.

The objective of the present study was to investigate the effects of cytochrome b5 (cytb5) on the drug metabolism catalyzed by CYP2C9, CYP2C19 and CYP3A4. Activities of CYP2C9, CYP2C19, and CYP3A4 were determined by using the prototypical substrates tolbutamide, omeprazole and midazolam, respectively. Cytb5 protein and mRNA contents showed large inter-individual variations with 11- and 6-fold range, respectively. All of three P450s showed an increased activity in proportion to the amount of cytb5 expression. Particularly, CYP3A4 showed the strongest correlation between cytb5 protein amount and the activity, followed by CYP2C9 and CYP2C19. The putative splicing variant, c.288G>A (rs7238987) was identified and was screened in 36 liver tissues by direct DNA sequencing. Liver tissues having a splicing variant exhibited unexpected sizes of cytb5 mRNA and a decreased expression tendency of cytb5 protein compared to the wild-type. A decreased activity in the metabolism of the CYP2C19 substrate omeprazole was observed in liver tissues carrying the splicing variant when compared to the wild-type Cytb5 (P < 0.05). The present results propose that different expression of cytb5 can cause variations in CYP mediated drug metabolism, which may explain, at least in part, the inter-individual difference in drug responses in addition to the CYP genetic polymorphisms.

Novel colchicine derivatives enhance graft survival after transplantation via suppression of T-cell differentiation and activity.

Immunosuppressants are crucial in organ transplantation but their side effects are a trade-off for long-term use. Colchicine has been shown to be effective in various diseases, albeit with many side effects. To enhance the immunosuppressive effects of colchicine, in addition to minimizing its side effects, we attempted to synthesize new colchicine derivatives (KR compounds). In rat cardiac and pancreatic islet allograft, long-term graft survival was identified in KR compound-treated groups. The use of cyclosporine A (CsA) or colchicine inhibited the CD3+ and CD4+ T-cell proliferation, whereas KR compounds inhibited the CD8+ T cells as well as CD4+ T cells. KR compounds reduced the apoptosis, interleukin-2 receptor expression, and signal transducer and activator of transcription 3 phosphorylation more than CsA. These results indicate that KR compounds have a potential therapeutic value as novel agents for prevention of graft deterioration by allograft of rejection.

Physiologically Based Pharmacokinetic Modeling Approach to Predict Drug-Drug Interactions With Ethionamide Involving Impact of Genetic Polymorphism on FMO3.

The widely used second-line antituberculosis drug ethionamide shows wide interindividual variability in its disposition; however, the relevant factors affecting this phenomenon have not been characterized. We previously reported the major contribution of flavin-containing monooxygenase 3 (FMO3) in the reductive elimination pathway of ethionamide. In this study, ethionamide metabolism was potentially inhibited by methimazole in vitro. The drug-drug interaction leading to methimazole affecting the disposition of ethionamide mediated by FMO3 was then quantitated using a bottom-up approach with a physiologically based pharmacokinetic framework. The maximum concentration (Cmax ) and area under the curve (AUC) of ethionamide were estimated to increase by 13% and 16%, respectively, when coadministered with methimazole. Subsequently, we explored the effect of FMO3 genetic polymorphism on metabolic capacity, by constructing 2 common functional variants, Lys158 -FMO3 and Gly308 -FMO3. Compared to the wild type, recombinant Lys158 -FMO3 and Gly308 -FMO3 variants significantly decreased the intrinsic clearance of ethionamide by 2% and 24%, respectively. Two prevalent functional variants of FMO3 were predicted to affect ethionamide disposition, with mean ratios of Cmax and AUC of up to 1.5 and 1.7, respectively, in comparison with the wild type. In comparing single ethionamide administration with the wild type, simulations of the combined effects of comedications and FMO3 genetic polymorphism estimated that the Cmax and AUC ratios of ethionamide increased up to 1.7 and 2.0, respectively. These findings suggested that FMO3-mediated drug-drug interaction and genetic polymorphism could be important determinants of interindividual heterogeneity in ethionamide disposition that need to be considered comprehensively to optimize the personalized dosing of ethionamide.

Subacute toxicity evaluation of KR-33493, FAF1 inhibitor for a new anti-parkinson's disease agent, after oral administration in rats and dogs.

KR33493, a newly developed FAS-associated factor 1 (FAF1) inhibitor for Parkinson's disease, is being evaluated in a Phase I clinical trial. In the present study, the subchronic toxicity of KR33493 in Sprague-Dawley (SD) rats and beagle dogs was investigated at various oral doses for 28 and 14 days, respectively. During the study, food consumption, body weights, organ weights, gross findings, and mortality were examined; and ophthalmoscopy, electrocardiography, hematology, serum biochemistry, urinalysis, histopathology, and toxicokinetics were performed. In rats, weight gain decreased in both sexes at 500 mg/kg/day, with no significant differences. In dogs, some significant differences compared with the control were found during the trial; however, at the end of recovery periods, these were no longer observed and there was no dose correlation. Some histopathological findings were observed, but these were considered as incidental changes. Since no other significant changes were observed, doses above 500 and 1000 mg/kg KR33493 in rat and dogs, respectively, caused no observed adverse effects. Therefore, based on these results, the Phase 1 clinical trial for KR33493 was approved by the Korean Food & Drug Administration.

Design and synthesis of fluorescent and biotin tagged probes for the study of molecular actions of FAF1 inhibitor.

To study the molecular action of ischemic Fas-mediated cell death inhibitor, we prepared fluorescent-tagged and biotin-tagged probes of the potent inhibitor, KR-33494, of ischemic cell death. We used the molecular modeling technique to find the proper position for attaching those probes with minimum interference in the binding process of probes with Fas-mediated cell death target, FAF1.

Quantitative determination of enzalutamide, an anti-prostate cancer drug, in rat plasma using liquid chromatography-tandem mass spectrometry, and its application to a pharmacokinetic study.

This report details a method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) that allows one to determine the concentration of an atypical anticancer drug, enzalutamide, in rat plasma. Specifically, this method involves the addition of an acetonitrile and bicalutamide (internal standard) solution to plasma samples. Following centrifugation of this mixture, an aliquot of the supernatant was directly injected into the LC-MS/MS system. Separation was achieved using a column packed with octadecylsilica (5 µm, 2.1 × 50 mm) with 10 mM ammonium acetate in acetonitrile as the mobile phase; detection was accomplished using MS/MS by multiple-reaction monitoring via an electrospray ionization source. This method demonstrated a linear standard curve (r = 0.997) over a concentration range of 0.001-1 µg/mL, as well as an intra- and inter-assay precision of 2.7 and 5.1%, respectively, and an accuracy range from 100.8 to 105.6%. The lower limit of quantification was 1.0 ng/mL in 50 µL of rat plasma sample. We also demonstrated that this analytical method could be successfully applied to the pharmacokinetic study of enzalutamide in rats.

Discovery of non-LBD inhibitor for androgen receptor by structure-guide design.

In this study, we synthesized the BF-3 binding small molecules, a series of pyridazinone-based compounds, as a novel class of non-LBP antiandrogens for treating prostate cancer by inhibiting androgen receptor. The new class compound was discovered to inhibitor the viability of AR-dependent human prostate LNCap cells and AR activity combining with the computational method. It showed a good physicochemical and PK property.

Accumulation of the parkin substrate, FAF1, plays a key role in the dopaminergic neurodegeneration.

This study reports the physical and functional interplay between Fas-associated factor 1 (FAF1), a death-promoting protein, and parkin, a key susceptibility protein for Parkinson's disease (PD). We found that parkin acts as an E3 ubiquitin ligase to ubiquitinate FAF1 both in vitro and at cellular level, identifying FAF1 as a direct substrate of parkin. The loss of parkin function due to PD-linked mutations was found to disrupt the ubiquitination and degradation of FAF1, resulting in elevated FAF1 expression in SH-SY5Y cells. Moreover, FAF1-mediated cell death was abolished by wild-type parkin, but not by PD-linked parkin mutants, implying that parkin antagonizes the death potential of FAF1. This led us to investigate whether FAF1 participates in the pathogenesis of PD. To address this, we used a gene trap mutagenesis approach to generate mutant mice with diminished levels of FAF1 (Faf1(gt/gt)). Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mouse model of PD, we found that FAF1 accumulated in the substantia nigra pars compacta (SNc) of MPTP-treated PD mice, and that MPTP-induced dopaminergic cell loss in the SNc was significantly attenuated in Faf1(gt/gt) mice versus Faf1(+/+) mice. MPTP-induced reduction of locomotor activity was also lessened in Faf1(gt/gt) mice versus Faf1(+/+) mice. Furthermore, we found that FAF1 deficiency blocked PD-linked biochemical events, including caspase activation, ROS generation, JNK activation and cell death. Taken together, these results suggest a new role for FAF1: that of a positive modulator for PD.

Daxx is a key downstream component of receptor interacting protein kinase 3 mediating retinal ischemic cell death.

Receptor-interacting protein 3 (RIP3) has been implicated in ischemic necrosis of retinal cells. An in silico analysis followed by experimental validation identified death associated protein (Daxx) as a novel substrate of RIP3. In vitro binding studies revealed that RIP3 binds to the serine/proline/threonine-rich domain (amino acid 625-740) of Daxx. Upon ischemic insult, RIP3 phosphorylated Daxx at Ser-668 in the retinal ganglion cells, triggering nuclear export of Daxx. Depletion of RIP3 significantly inhibited nuclear export of Daxx and attenuated cell death to a great extent. Collectively, the findings of this study demonstrate that phosphorylation of Daxx by RIP3 comprises an important part of ischemic necrosis in rat retinal ganglion cells.

KR-31543 reduces the production of proinflammatory molecules in human endothelial cells and monocytes and attenuates atherosclerosis in mouse model.

KR-31543, (2S, 3R, 4S)-6-amino-4-[N-(4-chlorophenyl)- N-(2-methyl-2H-tetrazol-5-ylmethyl) amino]-3,4-dihydro- 2-dimethyoxymethyl-3-hydroxy-2-methyl-2H-1-benz opyran is a new neuroprotective agent for ischemiareperfusion damage. It has also been reported that KR-31543 has protective effects on lipid peroxidation and H2O2-induced reactive oxygen species production. In this study, we investigated the anti-inflammatory and anti-atherogenic properties of KR-31543. We observed that KR-31543 treatment reduced the production of MCP-1, IL-8, and VCAM-1 in HUVECs, and of MCP-1 and IL-6 in THP-1 human monocytes. We also examined the effect of KR-31543 on monocytes migration in vitro. KR-31543 treatment effectively reduced the migration of THP-1 human monocytes to the HUVEC monolayer in a dose-dependent manner. We next examined the effects of this compound on atherogenesis in LDL receptor deficient (Ldlr ⁻/⁻) mice. After 10 weeks of western diet, the formation of atherosclerotic lesion in aorta was reduced in the KR-31543-treated group compared to the control group. The accumulation of macrophages in lesion was also reduced in KR-31543 treated group. However, the plasma levels of total cholesterol, HDL, LDL, and triglyceride were not affected by KR-31543 treatment. Taken together, these results show that KR-31543 has anti-inflammatory properties on human monocytes and endothelial cells, and inhibits fatty streak lesion formation in mouse model of atherosclerosis, suggesting the potential of KR-31543 for the treatment for atherosclerosis.

Ascorbic acid deficiency accelerates aging of hepatic stellate cells with up-regulation of PPARγ.

Senescent cells have been observed in certain aged or damaged tissues. However, the information about the effects of aging on liver cells is limited. In the present study, we have examined age-related histological changes in the livers of senescence marker protein knockout (SMP30-/-) mice, which are considered as a murine aging model due to the more sensitive response to apoptotic reagents and due to their shorter life span. In livers of old SMP30-/- mice, numerous hepatic stellate cells (HSCs) were hypertrophic and contained abundant microvesicular lipid droplets in cytoplasm. We have found that the expression of peroxisome proliferators-activated receptor γ (PPARγ), which is a protein related to lipid metabolism and HSC quiescence, was increased in hypertrophic HSCs by aging and vitamin C (VC) deficiency, whereas these phenomena were dramatically reduced by antioxidant treatment. Therefore, these prominent phenotypic changes can be considered as aging markers in the livers of animals which are subjected to antioxidant property evaluation.

Determination of a dipeptidyl peptidase IV agonist, ß-aminoacyl containing thiazolidine derivatives (KR-66223) in rat plasma by liquid chromatography-tandem mass spectrometry.

A sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method was developed for a novel dipeptidyl peptidase IV agonist (DDP-IV) agonist, KR-66223, in rat plasma. It involves liquid-liquid extraction (LLE) followed by HPLC separation and electrospray ionization tandem mass spectrometry. KR-66223 and imipramine (IS) was separated on Gemini-NX C18 column with mixture of acetonitrile-ammonium formate (10mM) (90:10, v/v) as mobile phase. The ion transitions monitored were m/z 553.2→206.2 for KR-66223, m/z 281.3→86.1 for imipramine in multiple reaction monitoring (MRM) mode. The linear ranges of the assay were 0.003-10µg/ml with a correlation coefficient (R(2)) greater than 0.99 and the lower limit of quantification was 3ng/ml. The average recovery was 78.9% and 87.1% from rat plasma for KR-66223 and imipramine, respectively. The coefficients of variation of intra- and inter-assay were 3.9-14.4% and the relative error was 0.8-11.5%. The method was validated and successfully applied to the pharmacokinetic study of KR-66223 in rat.

Prominent bone loss mediated by RANKL and IL-17 produced by CD4+ T cells in TallyHo/JngJ mice.

Increasing evidence that decreased bone density and increased rates of bone fracture are associated with abnormal metabolic states such as hyperglycemia and insulin resistance indicates that diabetes is a risk factor for osteoporosis. In this study, we observed that TallyHo/JngJ (TH) mice, a polygenic model of type II diabetes, spontaneously developed bone deformities with osteoporotic features. Female and male TH mice significantly gained more body weight than control C57BL/6 mice upon aging. Interestingly, bone density was considerably decreased in male TH mice, which displayed hyperglycemia. The osteoblast-specific bone forming markers osteocalcin and osteoprotegerin were decreased in TH mice, whereas osteoclast-driven bone resorption markers such as IL-6 and RANKL were significantly elevated in the bone marrow and blood of TH mice. In addition, RANKL expression was prominently increased in CD4+ T cells of TH mice upon T cell receptor stimulation, which was in accordance with enhanced IL-17 production. IL-17 production in CD4+ T cells was directly promoted by treatment with leptin while IFN-γ production was not. Moreover, blockade of IFN-γ further increased RANKL expression and IL-17 production in TH-CD4+ T cells. In addition, the osteoporotic phenotype of TH mice was improved by treatment with alendronate. These results strongly indicate that increased leptin in TH mice may act in conjunction with IL-6 to preferentially stimulate IL-17 production in CD4+ T cells and induce RANKL-mediated osteoclastogenesis. Accordingly, we propose that TH mice could constitute a beneficial model for osteoporosis.

Synthesis and SAR investigations of novel 2-arylbenzimidazole derivatives as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists.

Compounds containing 2-arybenzimidazole ring systems linked to arylpiperidines were synthesized and evaluated as MCH-R1 antagonists. The results of structure-activity relationship studies led to the identification of compound 4c as a potent MCH-R1 antagonist (IC(50)=1 nM). This compound also has good metabolic stability, and favorable pharmacokinetic and brain penetration properties. However 4c was found to be potent inhibitor of the hERG potassium channel.

Discovery of ß-aminoacyl containing thiazolidine derivatives as potent and selective dipeptidyl peptidase IV inhibitors.

A series of ß-aminoacyl containing thiazolidine derivatives was synthesized and evaluated for their ability to inhibit DPP-IV. Several thiazolidine derivatives with an acid moiety were found to be potent DPP-IV inhibitors. Among them, compound 2da is the most active in this series with an IC(50) value of 1 nM, and it showed excellent selectivity over DPP-IV related enzymes including DPP-2, DPP-8, and DPP-9. Compound 2da is chemically and metabolically stable, and showed no CYP inhibition, hERG binding or cytotoxicity. Compound 2db, an ester prodrug of 2da, showed good in vivo DPP-IV inhibition after oral administration in rat and dog models.

Discovery of new inhibitor for PDE3 by virtual screening.

In this work, we tried to find a new scaffold for a PDE3 using virtual screening for the obesity treatment. We first analyzed structural features for the known PDE3 inhibitors based on the PDE3B-ligand complex structure, and then carried out a docking study based on PDE3B 3D structure. We obtained a compound as potent PDE3 inhibitor stimulating lipolysis in murine adipocytes and human adipocytes.

Synthesis and biological evaluation of 3-substituted-benzofuran-2-carboxylic esters as a novel class of ischemic cell death inhibitors.

A series of 3-substituted-benzofuran-2-carboxylic esters was synthesized and evaluated for biological activity as ischemic cell death inhibitors in H9c2 cells and rat primary cardiac myocytes under conditions of oxygen and glucose deprivation. The introduction of a sulfur atom at the three-position substituent of the benzofuran ring markedly improved ischemic cell death inhibitory potency. In particular, 3-[2-(4-nitro-phenylsulfanyl)-acetylamino]-benzofuran-2-carboxylic acid ester (10) (EC(50)=0.532 µM, cell death=6.18%) and 4-chloro-3-[3-(pyridin-2-ylsulfanyl)-propionylamino]-benzofuran-2-carboxylic ester (18) (EC(50)=0.557 µM, cell death=7.02%) were shown to be the most potent in this series of benzofuran analogs.