A Double-Blind, Randomized, Placebo-Controlled, Phase II Clinical Study To Evaluate the Efficacy and Safety of Camostat Mesylate (DWJ1248) in Adult Patients with Mild to Moderate COVID-19.

Although several antiviral agents have become available for coronavirus disease 2019 (COVID-19) treatment, oral drugs are still limited. Camostat mesylate, an orally bioavailable serine protease inhibitor, has been used to treat chronic pancreatitis in South Korea, and it has an in vitro inhibitory potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study was a double-blind, randomized, placebo-controlled, multicenter, phase 2 clinical trial in mild to moderate COVID-19 patients. We randomly assigned patients to receive either camostat mesylate (DWJ1248) or placebo orally for 14 days. The primary endpoint was time to clinical improvement of subject symptoms within 14 days, measured using a subjective 4-point Likert scale. Three hundred forty-two patients were randomized. The primary endpoint was nonsignificant, where the median times to clinical improvement were 7 and 8 days in the camostat mesylate group and the placebo group, respectively (hazard ratio [HR] = 1.09; 95% confidence interval [CI], 0.84 to 1.43; P = 0.50). A post hoc analysis showed that the difference was greatest at day 7, without reaching significance. In the high-risk group, the proportions of patients with clinical improvement up to 7 days were 45.8% (50/109) in the camostat group and 38.4% (40/104) in the placebo group (odds ratio [OR] = 1.33; 95% CI, 0.77 to 2.31; P = 0.31); the ordinal scale score at day 7 improved in 20.0% (18/90) of the camostat group and 13.3% (12/90) of the placebo group (OR = 1.68; 95% CI, 0.75 to 3.78; P = 0.21). Adverse events were similar in the two groups. Camostat mesylate was safe in the treatment of COVID-19. Although this study did not show clinical benefit in patients with mild to moderate COVID-19, further clinical studies for high-risk patients are needed. (This trial was registered with ClinicalTrials.gov under registration no. NCT04521296).

MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc.

Potential of Pseudoshikonin I Isolated from Lithospermi Radix as Inhibitors of MMPs in IL-1ß-Induced SW1353 Cells.

Pseudoshikonin I, the new bioactive constituent of Lithospermi radix, was isolated from this methanol extract by employing reverse-phase medium-pressure liquid chromatography (MPLC) using acetonitrile/water solvent system as eluents. The chemical structure was determined based on spectroscopic techniques, including 1D NMR (¹H, (13)C, DEPT), 2D NMR (gCOSY, gHMBC, gHMQC), and QTOF/MS data. In this study, we demonstrated the effect of pseudoshikonin I on matrix-metalloproteinase (MMPs) activation and expression in interleukin (IL)-1ß-induced SW1353 chondrosarcoma cells. MMPs are considered important for the maintenance of the extracellular matrix. Following treatment with PS, active MMP-1, -2, -3, -9, -13 and TIMP-2 were quantified in the SW1353 cell culture supernatants using a commercially available ELISA kit. The mRNA expression of MMPs in SW1353 cells was measured by RT-PCR. Pseudoshikonin I treatment effectively protected the activation on all tested MMPs in a dose-dependent manner. TIMP-2 mRNA expression was significantly upregulated by pseudoshikonin I treatment. Overall, we elucidated the inhibitory effect of pseudoshikonin on MMPs, and we suggest its use as a potential novel anti-osteoarthritis agent.

Visualization of the physical and functional interaction between hMYH and hRad9 by Dronpa bimolecular fluorescence complementation.

BACKGROUND: Human MutY glycosylase homolog (hMYH), a component of the base excision repair pathway, is responsible for the generation of apurinic/apyrimidinic sites. Rad9-Rad1-Hus1 (9-1-1) is a heterotrimeric protein complex that plays a role in cell cycle checkpoint control and DNA repair. In humans, hMYH and 9-1-1 interact through Hus1 and to a lesser degree with Rad1 in the presence of DNA damage. In Saccharomyces pombe, each component of the 9-1-1 complex interacts directly with SpMYH. The glycosylase activity of hMYH is stimulated by Hus1 and the 9-1-1 complex and enhanced by DNA damage treatment. Cells respond to different stress conditions in different manners. Therefore, we investigated whether Rad9 interacted with hMYH under different stresses. Here, we identified and visualized the interaction between hRad9 and hMYH and investigated the functional consequences of this interaction. RESULTS: Co-IP and BiFC indicates that hMYH interacts with hRad9. As shown by GST-pull down assay, this interaction is direct. Furthermore, BiFC with deletion mutants of hMYH showed that hRad9 interacts with N-terminal region of hMYH. The interaction was enhanced by hydroxyurea (HU) treatment. mRNA and protein levels of hMYH and hRad9 were increased following HU treatment. A marked increase in p-Chk1 (S345) and p-Cdk2 (T14, Y15) was observed. But this phosphorylation decreased in siMYH- or siRad9-transfected cells, and more pronounced decrease observed in co-transfected cells. CONCLUSIONS: Our data reveal that hRad9 interacts directly with N-terminal region of hMYH. This interaction is enhanced by HU treatment. Knockdown of one or both protein result in decreasing Chk1 and Cdk2 phosphorylation. Since both protein functions in the early detection of DNA damage, we suggest that this interaction occurs early in DNA damage pathway.

Efficacy, Safety, and Subject Satisfaction of PrabotulinumtoxinA for Moderate-to-Severe Crow's Feet: A Phase IV, Multicenter, Double-Blind, Randomized, Placebo-Controlled Trial.

PrabotulinumtoxinA has been identified as an effective agent against crow's feet. Our study, which included Korean patients with moderate to severe crow's feet, was undertaken to compare the efficacy and safety of PrabotulinumtoxinA and placebo treatments. Of the 90 study participants, 60 received prabotulinumtoxinA (24 U), whereas 30 received a placebo. The primary outcome assessment included facial wrinkle grading by investigators. At week 4, 69.64% of patients in the prabotulinumtoxinA group exhibited minimal crow's feet severity; in contrast, a 0% improvement was observed in the placebo group (p < 0.0001). At week 12, the improvement rates were 30.36% for prabotulinumtoxinA and 6.90% for the placebo, demonstrating a significant difference (p = 0.0152). Based on the independent review panel's assessment at week 4, the improvement rate was 39.29% in the prabotulinumtoxinA group and 3.45% in the placebo group during maximum smiling. Additionally, patient satisfaction was notably higher in the prabotulinumtoxinA group (32.14%) than in the placebo group (10.34%) at week 4 (p = 0.0289). Both treatments displayed comparable safety profiles, with only mild local reactions reported as ADRs for one patient from the prabotulinumtoxinA group. Thus, prabotulinumtoxinA demonstrates significant potential as a potent and safe remedy for crow's feet.

Evaluation of users' level of satisfaction for an artificial intelligence-based diagnostic program in pediatric rare genetic diseases.

The artificial intelligence (AI)-based genetic diagnostic program has been applied to genome sequencing to facilitate the diagnostic process. The objective of the current study was to evaluate the experience and level of satisfaction of participants using an AI-based diagnostic program for rare pediatric genetic diseases. The patients with neurodevelopmental disorders or hearing impairments, their guardians, and their physicians from 16 tertiary general hospitals were enrolled. The study period was from April 2020 to March 2021. A survey was designed to assess their experience and level of satisfaction. A total of 30 physicians and 243 patients and guardians (199 neurodevelopmental disorders and 44 hearing impairments) completed the survey. DNA samples of the subjects were collected through buccal swabs or blood collection: 211 subjects (86.8%) through buccal swab and 29 subjects (11.9%) through blood collection. Average turnaround time for result receipt was 57.54 ± 32.42 days. For the sampling method, 193 patients and guardians (81.1%) and 28 physicians (93.3%) preferred buccal swab. The level of satisfaction of the 2 groups participating in the AI-based diagnostic program was 8.31 ± 1.71 out of 10 in the patient and guardian group and 8.42 ± 1.23 in the physician group. Clinicians, patients, and guardians are satisfied with the AI-based diagnostic program in general. With an increase in AI-based precision medicine solutions, the evaluation of the user's satisfaction with appropriate provision will help improve personal health care.

Analgesic Efficacy of α2 Adrenergic Receptor Agonists Depends on the Chronic State of Neuropathic Pain: Role of Regulator of G Protein Signaling 4.

The analgesic effect of alpha-2 adrenergic receptor (α2AR) agonists, which relieve chronic neuropathic pain, is highly variable among individuals. Here, we used a mouse model of spared nerve injury (SNI) to show that treatment time after the establishment of neuropathic pain was important for the variability in the analgesic efficacy of α2AR agonists, which was related to the activity of regulator of G-protein signaling protein 4 (RGS4). Intrathecal treatment with α2AR agonists, clonidine (0.1-1 nmol) or dexmedetomidine (0.3-1 nmol), relieved mechanical allodynia and thermal hyperalgesia on postoperative day (POD) 14, but their efficacy was weaker on POD28 and absent on POD56. The RGS4 level of plasma membrane was increased on POD56 compared to that on POD14. Moreover, in RGS4-deficient or RGS4 inhibitor (CCG50014)-treated mice, the analgesic effect of the α2AR agonists was conserved even on POD56. The increased plasma membrane RGS4 expression and the reduced level of active Gαi after clonidine injection on POD56 were completely restored by CCG50014. Higher doses of clonidine (10 nmol) and dexmedetomidine (3 nmol) relieved neuropathic pain on POD56 but were accompanied with serious side effects. Whereas, the coadministration of CCG50014 with clonidine (1 nmol) or dexmedetomidine (1 nmol) did not cause side effects. These findings demonstrated that SNI-induced increase in plasma membrane RGS4 expression was associated with low efficacy of α2AR agonists in a model of persistent, chronic neuropathic pain. Furthermore, α2AR agonist administration together with RGS4-targeted intervention represents a novel strategy for the treatment of neuropathic pain to overcome dose-limiting side effects.

A novel pyrazolo [3,4-d] pyrimidine, KKC080106, activates the Nrf2 pathway and protects nigral dopaminergic neurons.

The transcription factor nuclear factor-erythroid 2-related factor-2 (Nrf2) is known to induce neuroprotective and anti-inflammatory effects and is considered to be an excellent molecular target for drugs related to neurodegenerative disease therapy. Nrf2 activators previously tested in clinical trials were electrophilic, causing adverse effects due to non-selective and covalent modification of cellular thiols. In order to circumvent this issue, we constructed and screened a chemical library consisting of 241 pyrazolo [3,4-d] pyrimidine derivatives and discovered a novel, non-electrophilic compound: 1-benzyl-6-(methylthio)-N-(1-phenylethyl)-1H-pyrazolo[3,4-d]pyrimidine-4-amine (KKC080106). KKC080106 was able to activate Nrf2 signaling as it increases the cellular levels of Nrf2, binds to the Nrf2 inhibitor protein Keap1, and causes the accumulation of nuclear Nrf2. We also observed an increase in the expression levels of Nrf2-dependent genes for antioxidative/neuroprotective enzymes in dopaminergic neuronal cells. In addition, in lipopolysaccharide-activated microglia, KKC080106 suppressed the generation of the proinflammatory markers, such as IL-1ß, TNF-α, cyclooxygenase-2, inducible nitric oxide synthase, and nitric oxide, and inhibited the phosphorylation of kinases known to be involved in inflammatory signaling, such as IκB kinase, p38, JNK, and ERK. As a drug, KKC080106 exhibited excellent stability against plasma enzymes and a good safety profile, evidenced by no mortality after the administration of 2000 mg/kg body weight, and minimal inhibition of the hERG channel activity. Pharmacokinetic analysis revealed that KKC080106 has good bioavailability and enters the brain after oral and intravenous administration, in both rats and mice. In MPTP-treated mice that received KKC080106 orally, the compound blocked microglial activation, protected the nigral dopaminergic neurons from degeneration, and prevented development of the dopamine deficiency-related motor deficits. These results suggest that KKC080106 has therapeutic potential for neurodegenerative disorders such as Parkinson's disease.

Activation of the Nrf2 signaling pathway and neuroprotection of nigral dopaminergic neurons by a novel synthetic compound KMS99220.

The transcription factor Nrf2 is known to induce gene expression of antioxidant enzymes and proteasome subunits. Because both oxidative stress and protein aggregation have damaging effects on neurons, activation of the Nrf2 signaling should be beneficial against neurodegeneration. In this study, we report a novel synthetic morpholine-containing chalcone KMS99220 that confers neuroprotection. It showed high binding affinity to the Nrf2 inhibitory protein Keap-1 and increased nuclear translocation of Nrf2 and gene expression of the antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase-1, and the catalytic and modifier subunits of glutamate-cysteine ligase in dopaminergic CATH.a cells. KMS99220 also increased expression of the proteasome subunits PSMB5, PSMB7, PSMB8 and PSMA1, and the respective chymotrypsin and trypsin-like proteasomal enzyme activities, and reduced α-synuclein aggregate in GFP-α-syn A53T-overexpressing cells. KMS99220 exhibited a favorable pharmacokinetic profile with excellent bioavailability and metabolic stability, did not interfere with activities of the cytochrome p450 isotypes, and showed no apparent in vivo toxicity when administered up to 2000 mg/kg. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mice, oral administration of KMS99220 prevented degeneration of the nigral dopaminergic neurons, induced the Nrf2 target genes, and effectively prevented the associated motor deficits. These results suggest KMS99220 as a potential candidate for therapy against Parkinson's disease.

Observations of Forsythia koreana methanol extract on mast cell-mediated allergic reactions in experimental models.

To explore effects of Forsythia koreana methanol extract (FKME) on mast cell-mediated allergic and inflammatory properties, the effect of FKME was evaluated on compound 48/80-induced systemic anaphylaxis, ear swelling, and anti-dinitrophenyl (DNP) immunoglobulin E (IgE)-induced passive cutaneous anaphylaxis (PCA). In addition, the effect of FKME was investigated on the histamine release from rat peritoneal mast cells (RPMCs) stimulated by compound 48/80, which promotes histamine release. The human mast cell line HMC-1 was stimulated by phorbol 12-myristate 13-acetate plus calcium ionophore A23187. Activated HMC-1 can produce several proinflammatory and chemotactic cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-8. Cytokine levels in the culture supernatant were measured by an enzyme-linked immunosorbent assay. Cytotoxicity by FKME was determined by a 3-(4,5-dimethylthiazol-2-yl)-diphenyl-tetrazolium bromide (MTT) assay. FKME inhibited compound 48/80-induced systemic anaphylactic shock and ear swelling in mice. When 1 g/kg FKME was pretreated or posttreated with mice, compound 48/80-induced mice morality was 50 and 66.7%, respectively. One gram per kilogram of FKME pretreatment inhibited ear-swelling responses derived from compound 48/80 by 29.75%. A PCA reaction was inhibited by 17.9%. In an in vitro model, FKME (1 mg/ml) inhibited histamine release from the RPMCs by 13.8% and TNF-alpha, IL-6, and IL-8 production from HMC-1 cells by 71.16% (P < 0.001), 86.72% (P < 0.001), and 44.6%, respectively. However, FKME had no cytotoxic effects on cell viability. In conclusion, FKME inhibited not only systemic anaphylaxis and ear swelling induced by compound 48/80 but also inhibited a PCA reaction induced by anti-DNP IgE in vivo. Treatment with FKME showed significant inhibitory effects on histamine, TNF-alpha, IL-6, and IL-8 release from mast cells.

New role of human ribosomal protein S3: Regulation of cell cycle via phosphorylation by cyclin-dependent kinase 2.

Human ribosomal protein S3 (hRpS3) is a component of the 40S ribosomal subunit that associated in protein synthesis. hRpS3 has additional ribosomal functions such as DNA repair, transcription, metastasis, and apoptosis via interaction with numerous signaling molecules and has different modifications. Cyclin-dependent kinases (CDKs) are heterodimeric serine/threonine protein kinases that regulate cell cycle progression. Among its members, the Cdk1-cyclin B complex is known to control cell progression in the G2/M phase, while Cdk2-cyclin E/A complexes function in G1/S and S/G2 transition. In our previous study, we observed interaction between hRpS3 and Cdk1. The present study investigated the interaction between hRpS3 and Cdk2. Cdk2 phosphorylated hRps3 at amino acid residues S6 and T221 during the S-phase. Furthermore, hRpS3 knockdown delayed cell cycle progression by modulating the expression of cell cycle-related proteins, including cyclin B1 and cyclin E1. These findings suggest that hRpS3 is involved in Cdk2-mediated cell cycle regulation.

Potential repositioning of exemestane as a neuroprotective agent for Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterised by selective degeneration of the nigral dopaminergic neurons, and neuroinflammation and oxidative stress are believed to be involved in its pathogenesis. In the present study, we provide data that the synthetic steroid exemestane, which is currently being used to treat breast cancer, may be useful for PD therapy. In BV-2 microglial cells, exemestane activated the transcription factor Nrf2 and induced expression of the Nrf2-dependent genes that encode the antioxidant enzymes NAD(P)H: quinone oxidoreductase 1, haem oxygenase-1, and glutamylcysteine ligase. It also downregulated gene expression of inducible nitric oxide (NO) synthase, lowered the levels of NO and reactive oxygen species, interleukin-1ß and tumour necrosis factor-α in lipopolysaccharide-activated microglial cells. In CATH.a dopaminergic neuronal cells, exemestane also induced the same set of Nrf2-dependent antioxidant enzyme genes and provided neuroprotection against oxidative damage. In vivo, the drug protected the nigral dopaminergic neurons, decreased microglial activation, and prevented motor deficits in C57Bl/6 male mice that had been administered with the dopaminergic neurotoxin MPTP. Taken together, the results suggested a utility of repositioning exemestane towards disease-modifying therapy for PD.

2-Acetyl-7-hydroxy-6-methoxy-1-methyl-1,2,3,4,-tetrahydroisoquinoline exhibits anti-inflammatory properties and protects the nigral dopaminergic neurons.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by degeneration of dopamine(DA)ergic neurons. Neuroinflammation caused by microglial activation is believed to be involved in the pathogenesis of neurodegenerative diseases including PD. In the present study, we tested the effects of a novel compound 2-acetyl-7-hydroxy-6-methoxy-1-methyl-1,2,3,4,-tetarhydroisoquinoline (AMTIQ) on neuroinflammatory response and DAergic neurodegeneration. In lipopolysaccharide-activated BV-2 microglial cells, AMTIQ lowered nitric oxide and tetrahydrobiopterin levels and downregulated gene expression of inducible nitric oxide synthase and GTP cyclohydrolase I. AMTIQ also repressed gene expression of the proinflammatory cytokines IL-1ß and TNF-α, and attenuated nuclear translocation of NF-κB. AMTIQ was stable against liver microsomal enzymes from human and mouse and did not interfere with activities of the cytochrome p450 enzymes 1A2, 2D6, 2C9, 2C19 and 3A4. Pharmacokinetic studies revealed the brain to plasma ratio of AMTIQ to be 45%, suggesting it can penetrate the blood brain barrier. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse PD model, AMTIQ led to decreased microglial activation, increased survival of DAergic neurons and their fibers, and improved behavioral scores on rotarod and vertical grid tests. Taken together, these results suggest that AMTIQ might serve as a candidate preventive-therapeutic agent for neurodegenerative diseases such as PD.

Antiobesity Effects of Salvia plebeia R. Br. Extract in High-Fat Diet-Induced Obese Mice.

This study was designed to investigate the antiobesity effects of Salvia plebeia R. Br. ethanolic extracts (SPE) in mice fed high-fat diets (HFD). Male C57BL/6J mice were randomly assigned to four groups: normal diet (Chow), high-fat diet (HFD, 45% fat), HFD+SPE 200 (200 mg/kg b.w.), and HFD+SPE 400 (400 mg/kg b.w.). Extracts were administered orally every day for 8 weeks. Increases in body/fat weight and feed efficiency ratio were monitored in all mice. In addition, obesity resulting from feeding HFD to the mice was confirmed by the increase of glucose level, aspartate transaminase, alanine transaminase, triglyceride (TG), high-density lipoprotein cholesterol, very low-density lipoprotein-c, leptin, and adiponectin in blood. The SPE-treated mice gained less body and mesenteric/subcutaneous adipose tissues weights and had lower TG, very low-density lipoprotein cholesterol, leptin, and glucose level in serum, compared to the HFD group. Moreover, histopathological examinations revealed that the size of adipocytes in liver and adipose tissue was significantly decreased by SPE, compared to the HFD group. The expression of adipogenesis transcription factors (e.g., peroxisome proliferator activated receptor γ and CCAAT/enhancer binding protein α) and lipogenesis-related target genes (adipocyte fatty acid-binding protein 2, lipoprotein lipase, fatty acid synthase, and sterol regulatory element-binding transcription factor 1c) in HFD-induced obese mice was decreased by SPE treatment. These results suggest that SPE attenuates the fat accumulation in HFD-induced obese mice by suppressing the expressions of genes related to adipogenesis and lipogenesis activity. Therefore, SPE could be developed as a potential therapy for reduction of body weight and antiobesity intervention.

Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells.

UNLABELLED: The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD-hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1-TRADD complex. Thus, this study identified a novel mechanism through which the hMYH-hTRADD interaction may affect the TNF-α signaling pathway. IMPLICATIONS: In HeLa cells, the hTRADD-hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation.

A physical association between the human mutY homolog (hMYH) and DNA topoisomerase II-binding protein 1 (hTopBP1) regulates Chk1-induced cell cycle arrest in HEK293 cells.

BACKGROUND: Human DNA topoisomerase II-binding protein 1 (hTopBP1) plays an important role in DNA replication and the DNA damage checkpoint pathway. The human mutY homolog (hMYH) is a base excision repair DNA glycosylase that excises adenines or 2-hydroxyadenines that are mispaired with guanine or 7,8-dihydro-8-oxoguanine (8-oxoG). hTopBP1 and hMYH were involved in ATR-mediated Chk1 activation, moreover, both of them were associated with ATR and hRad9 which known as checkpoint-involved proteins. Therefore, we investigated whether hTopBP1 interacted with hMYH, and what the function of their interaction is. RESULTS: We documented the interaction between hTopBP1 and hMYH and showed that this interaction increased in a hydroxyurea-dependent manner. We also mapped the hMYH-interacting region of hTopBP1 (residues 444-991). In addition, we investigated several cell cycle-related proteins and found that co-knockdown of hTopBP1 and hMYH significantly diminished cell cycle arrest due to compromised checkpoint kinase 1 (Chk1) activation. Moreover, we observed that hMYH was essential for the accumulation of hTopBP1 on damaged DNA, where hTopBP1 interacts with hRad9, a component of the Rad9-Hus1-Rad1 complex. The accumulation of hTopBP1 on chromatin and its subsequent interaction with hRad9 lead to cell cycle arrest, a process mediated by Chk1 phosphorylation and ataxia telangiectasia and Rad3-related protein (ATR) activation. CONCLUSIONS: Our results suggested that hMYH is necessary for the accumulation of hTopBP1 to DNA damage lesion to induce the association of hTopBP1 with 9-1-1 and that the interaction between hMYH and hTopBP1 is essential for Chk1 activation. Therefore, we suggest that the interaction between hMYH and hTopBP1 is crucial for activation of the ATR-mediated cell cycle checkpoint.

A Novel Compound ITC-3 Activates the Nrf2 Signaling and Provides Neuroprotection in Parkinson's Disease Models.

Parkinson's disease (PD) is a progressive neurodegenerative disorder accompanied by a selective loss of the dopamine(DA)ergic neurons residing in the substantia nigra. There is ample evidence that neuroinflammation and oxidative stress are involved in the pathogenesis of PD. In the present study, we aimed at protecting the DAergic neurons by suppressing these cellular events and generated a novel synthetic isothiocyanate ITC-3. The compound led to elevation of nuclear and total levels of the transcription factor Nrf2 and interacted with its binding protein Keap1 with high affinity, suggesting Nrf2 activation. ITC-3 was able to suppress production of the proinflammatory mediators in lipopolysaccharide-activated BV-2 microglial cells. It also increased mRNA and protein levels of the Nrf2-dependent antioxidant enzymes NAD(P)H quinone oxidoreductase, heme oxygenase-1, and glutamylcysteine ligase in both BV-2 and DAergic neuronal CATH.a cells. The compound protected the DAergic cells against oxidative stress. In vivo, ITC-3 attenuated the loss of tyrosine hydroxylase-immunopositive nigrostriatal DAergic neurons, suppressed microglial activation, and abolished PD-associated motor deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-elicited animal model of PD. Taken together, ITC-3 may be useful toward development of neuroprotective therapy for PD.

A novel compound VSC2 has anti-inflammatory and antioxidant properties in microglia and in Parkinson's disease animal model.

BACKGROUND AND PURPOSE: Neuroinflammation through microglial activation is involved in the pathogenesis of neurodegenerative diseases including Parkinson's disease (PD), a major neurodegenerative disorder characterized by dopaminergic neuronal death in the substantia nigra. We examined our novel synthetic compound VSC2 for its anti-inflammatory properties towards development of a PD therapy. EXPERIMENTAL APPROACH: We tested the effects of VSC2 on production of various NF-κB-dependent proinflammatory molecules and Nrf2-dependent antioxidant enzymes in BV-2 microglia and in vivo. KEY RESULTS: The vinyl sulfone compound, VSC2, most effectively suppressed the production of NO in LPS-activated microglia. It also down-regulated expression of inducible NOS (iNOS), COX-2, IL-1ß and TNF-α and inhibited nuclear translocalization and transcriptional activity of NF-κB. VSC2 increased total and nuclear Nrf2 levels, induced Nrf2 transcriptional activity and was bound to Keap1 with high affinity. Expression of the Nrf2-regulated antioxidant enzyme genes NAD(P)H quinone oxidoreducase-1 (NQO-1), haem oxygenase-1 (HO-1) and glutamylcysteine ligase (GCL) were up-regulated by VSC2. In the MPTP mouse model of PD, oral administration of VSC2 decreased the number of activated microglia in the substantia nigra, lowered the levels of iNOS, COX-2 and IL-1ß, and protected the dopaminergic neurons. VSC2 also elevated the levels of NQO1, HO-1, GCL and Nrf2 in the nigrostriatal area. CONCLUSIONS AND IMPLICATIONS: VSC2 has both anti-inflammatory and antioxidant properties and prevented neuroinflammation in microglia and in an animal model of PD. This suggests VSC2 as a potential candidate for PD therapy.

Discovery of vinyl sulfones as a novel class of neuroprotective agents toward Parkinson's disease therapy.

Although the etiology of Parkinson's disease (PD) remains elusive, recent studies suggest that oxidative stress contributes to the cascade leading to dopaminergic (DAergic) neurodegeneration. The Nrf2 signaling is the main pathway responsible for cellular defense system against oxidative stress. Nrf2 is a transcription factor that regulates environmental stress response by inducing expression of antioxidant enzyme genes. We have synthesized novel vinyl sulfone derivatives. They exhibited a broad range of activities in inducing HO-1, whose gene expression is under the control of Nrf2. Among them, compound 12g was confirmed to activate Nrf2 and induce expression of the Nrf2-dependent antioxidant enzymes NQO1, GCLC, GLCM, and HO-1, at both mRNA and protein levels in DAergic neuronal cells. This was accompanied by protection of DAergic neurons in both in vitro and MPTP-induced in vivo models of PD. In addition, compound 12g effectively resulted in attenuation of the PD-associated behavioral deficits in the mouse model.

Human MutY homolog induces apoptosis in etoposide-treated HEK293 cells.

Etoposide (ETP) treatment of ataxia telangiectasia mutated (ATM) and Rad3-related protein (ATR)-, topoisomerase-binding protein-1 (TopBP1) and human MutY homolog (hMYH)-depleted cells results in a significant reduction in apoptotic signaling. The association between ATR or TopBP1 and hMYH increased following ETP treatment. In hMYH knockdown cells, the interaction between ATR and TopBP1 decreased following ETP treatment. We suggest that hMYH functions as a sensor of ETP-induced apoptosis. The results suggest that in the absence of hMYH, cells are unable to recognize the damage signal and the ATR pathway is not activated.