Publications by authors named "Zining Wu"

41 Publications

Corrigendum to "Changing of haemostatic system in a pig model during different types of hypothermic circulatory arrest" [95, 2021, 102817].

J Therm Biol 2021 Jul 13;99:102974. Epub 2021 May 13.

Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing, China; Beijing Aortic Disease Center, Cardiovascular Surgery Center, Beijing, China; Beijing Engineering Research Center for Vascular Prostheses, Beijing, China. Electronic address:

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http://dx.doi.org/10.1016/j.jtherbio.2021.102974DOI Listing
July 2021

Changing of haemostatic system in a pig model during different types of hypothermic circulatory arrest.

J Therm Biol 2021 Jan 4;95:102817. Epub 2021 Jan 4.

Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing, China; Beijing Aortic Disease Center, Cardiovascular Surgery Center, Beijing, China; Beijing Engineering Research Center for Vascular Prostheses, Beijing, China. Electronic address:

Background: Hypothermic circulatory arrest is usually used in aortic surgery, congenital heart defect repairs and other complex surgeries. It is frequently associated with excessive postoperative bleeding and the transfusion of allogeneic blood products. The physiopathology of hypothermic circulatory arrest-induced coagulopathy has never been systematically studied. The aim of the study was to investigate this phenomenon in a pig model.

Methods: Ten pigs were randomly assigned to 30 min of hypothermic circulatory arrest at either 15 °C (n = 5) or 25 °C (n = 5). Detection of apoptosis and haemostatic system assays were performed in this experiment. Enzyme-linked immunosorbent assays were performed at ten time points in each group to study the changes in the coagulation system in hypothermic circulatory arrest. All of the statistical analyses were performed in SPSS software, version 18.0, and as bilateral tests, and p < 0.05 was considered statistically significant.

Results: There was no significant difference in the effect of different types of hypothermic circulatory arrest on routine laboratory tests and tissue sample analysis (p > 0.05, for all). Our results demonstrated that more severe systemic activation of the coagulation system (TAT and F1+2) was applied in the deep hypothermic circulatory arrest group but not in the moderate hypothermic circulatory arrest group (TAT/p = 0.01, F1+2/p = 0.03). However, this activation of the coagulation system (AT III and PC) was not associated with changes in the anticoagulation pathway (AT III/p = 0.24, PC/p = 0.33). In addition, analysis of biomarkers of the haemostatic system revealed that the consumption of coagulation is more concentrated on extrinsic coagulation factors (FVII/p = 0.01).

Conclusions: Moderate hypothermic circulatory arrest is more suitable for patients with coagulation dysfunction. We believe the application of deep hypothermic circulatory arrest should pay more attention to changes in coagulation rather than the anticoagulation pathway. Extrinsic coagulation factor supplementation is more effective after deep hypothermic circulatory arrest.
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http://dx.doi.org/10.1016/j.jtherbio.2020.102817DOI Listing
January 2021

Are dilated ascending aortas of Chinese patients more likely to dissect?

Cardiovasc Diagn Ther 2020 Aug;10(4):786-795

Beijing Lab for Cardiovascular Precision Medicine, Beijing, China.

Background: Ascending aortic aneurysm is a disease requiring surgical intervention. However, the timing of operation is still controversial. The purpose of this study is to compare the ascending aortic diameter and postoperative outcomes in hospital between patients with simple ascending aortic dissection and patients with simple ascending aortic dilation in China, and to investigate the accuracy of the timing of operation determined by ascending aortic diameter alone.

Methods: We reviewed the data from 2,520 hospitalized patients of aortic aneurysm and aortic dissection who underwent surgical treatment from January 2010 to June 2017 in our hospital. A total of 139 simple ascending aortic dissection and simple ascending aortic aneurysm hospitalized patients excluding Marfan syndrome and heart valve diseases etc. (56 in the aortic dilatation group and 83 in the aortic dissection group) were enrolled. The t-test and univariable analysis were used to compare the differences between two groups.

Results: For the aortic diameter, the group of aneurysm has greater ascending aortic diameter and the index of ascending aortic diameter compared with the group of dissection (P<0.001, P<0.001). For male patients, the result is the same (P<0.001, P<0.001). But for female patients, there was no significant statistical significance between the two groups (P=0.631, P=0.288). For the postoperative outcomes, the dissection group had higher mortality, incidence of tracheotomy and postoperative re-exploration for hemorrhage (P=0.040, P=0.011, P=0.028).

Conclusions: The majority of patients with simple ascending aortic dissection present with aortic diameters <5.5 cm and this is not consistent with the current operation indications of aortic aneurysm. It is far from enough to predict aortic dissection with aortic diameter alone. More indicators are needed to do this.
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http://dx.doi.org/10.21037/cdt-20-313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487387PMC
August 2020

and induction of fetal hemoglobin with a reversible and selective DNMT1 inhibitor.

Haematologica 2021 07 1;106(7):1979-1987. Epub 2021 Jul 1.

GlaxoSmithKline, Collegeville, Pennsylvania, USA.

Pharmacological induction of fetal hemoglobin (HbF) expression is an effective therapeutic strategy for the management of beta-hemoglobinopathies such as sickle cell disease. DNA methyltransferase (DNMT) inhibitors 5-azacytidine (5-aza) and 5-aza-2'-deoxycytidine (decitabine) have been shown to induce fetal hemoglobin expression in both preclinical models and clinical studies, but are not currently approved for the management of hemoglobinopathies. We report here the discovery of a novel class of orally bioavailable DNMT1-selective inhibitors as exemplified by GSK3482364. This molecule potently inhibits the methyltransferase activity of DNMT1, but not DNMT family members DNMT3A or DNMT3B. In contrast with cytidine analog DNMT inhibitors, the DNMT1 inhibitory mechanism of GSK3482364 does not require DNA incorporation and is reversible. In cultured human erythroid progenitor cells (EPCs), GSK3482364 decreased overall DNA methylation resulting in de-repression of the gamma globin genes HBG1 and HBG2 and increased HbF expression. In a transgenic mouse model of sickle cell disease, orally administered GSK3482364 caused significant increases in both HbF levels and in the percentage HbF-expressing erythrocytes, with good overall tolerability. We conclude that in these preclinical models, selective, reversible inhibition of DNMT1 is sufficient for the induction of HbF, and is well-tolerated. We anticipate that GSK3482364 will be a useful tool molecule for the further study of selective DNMT1 inhibition both in vitro and in vivo.
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http://dx.doi.org/10.3324/haematol.2020.248658DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8252945PMC
July 2021

Protocol for creation of a risk scoring system for acute type A aortic dissection surgery.

Int J Surg Protoc 2019 25;14:19-23. Epub 2019 Feb 25.

Department of Cardiac Surgery, Beijing Aortic Disease Center, Beijing Anzhen Hospital, Capital Medical University, Beijing 100069, China.

Stanford type A aortic dissection is a kind of cardiovascular disease which seriously threatens human life and health. It has the characteristics of rapid onset, rapid progress and high mortality. Surgical treatment is a recognized treatment for type A aortic dissection. There are many disputed places in the actual clinical work about the timing, prognosis and methods of the operation. This study aims to establish an early mortality risk scoring system for acute Stanford A aortic dissection surgery patients.

Methods And Analysis: The structured data of patients with acute type A aortic dissection were collected. The primary outcome is death during hospitalization. Secondary outcomes will include re-operation and related complications. A risk scoring system of patients with acute type A aortic dissection undergoing surgical treatment will be established. Prospective data will be used to validate the risk stratification ability and accuracy of the model in operative risk prediction.
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http://dx.doi.org/10.1016/j.isjp.2019.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6913569PMC
February 2019

MrgX2 is a promiscuous receptor for basic peptides causing mast cell pseudo-allergic and anaphylactoid reactions.

Pharmacol Res Perspect 2019 12 2;7(6):e00547. Epub 2019 Dec 2.

Medicines Research Centre GlaxoSmithKline R&D Ltd Stevenage United Kingdom.

Activation of MrgX2, an orphan G protein-coupled receptor expressed on mast cells, leads to degranulation and histamine release. Human MrgX2 binds promiscuously to structurally diverse peptides and small molecules that tend to have basic properties (basic secretagogues), resulting in acute histamine-like adverse drug reactions of injected therapeutic agents. We set out to identify MrgX2 orthologues from other mammalian species used in nonclinical stages of drug development. Previously, the only known orthologue of human MrgX2 was from mouse, encoded by . MrgX2 genes of rat, dog (beagle), minipig, pig, and Rhesus and cynomolgus monkey were identified by bioinformatic approaches and verified by their ability to mediate calcium mobilization in transfected cells in response to the classical MrgX2 agonist, compound 48/80. The peptide GSK3212448 is an inhibitor of the PRC2 epigenetic regulator that caused profound anaphylactoid reactions upon intravenous infusion to rat. We showed GSK3212448 to be a potent MrgX2 agonist particularly at rat MrgX2. We screened sets of drug-like molecules and peptides to confirm the highly promiscuous nature of MrgX2. Approximately 20% of drug-like molecules activated MrgX2 (pEC ranging from 4.5 to 6), with the principle determinant being basicity. All peptides tested of net charge +3 or greater exhibited agonist activity, including the cell penetrating peptides polyarginine (acetyl-Arg-amide) and TAT (49-60), a fragment of HIV-1 TAT protein. Finally, we showed that the glycopeptide antibiotic vancomycin, which is associated with clinical pseudo-allergic reactions known as red man syndrome, is an agonist of MrgX2.
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http://dx.doi.org/10.1002/prp2.547DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6887720PMC
December 2019

Increased frequency of FBN1 frameshift and nonsense mutations in Marfan syndrome patients with aortic dissection.

Mol Genet Genomic Med 2020 01 12;8(1):e1041. Epub 2019 Dec 12.

Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing, China.

Background: Marfan syndrome (MFS) is an inherited connective tissue disease that mainly involves Fibrillin-1 (FBN1) mutations and aortic manifestations. In this study, we investigated the correlations between the FBN1 genotype-phenotype and aortic events (aortic dissection and aortic aneurysm) in patients with Marfan syndrome.

Methods: Genotype and phenotype information was evaluated in 180 patients with MFS. DNA sequencing was performed on each patient. According to the clinical manifestation, these patients were split into two groups: the aortic dissection group and the aortic aneurysm group. Aortic wall tissue was obtained from Marfan patients who underwent surgery and was used for staining.

Results: A total of 180 patients with FBN1 mutations were grouped into four categories: 90 with missense mutations, 32 with splicing mutations, 29 with frameshift mutations, and 29 with nonsense mutations. There was a significantly higher frequency of frameshift and nonsense mutations observed in aortic dissection than in aortic aneurysm (25.58% vs. 4.35%, p = .005; 25.58% vs. 8.70%, p = .033, respectively;), while missense mutations showed a higher frequency in aortic aneurysm than in aortic dissection (69.57% vs. 32.56%, respectively; p < .001) and a higher rate of lens dislocation (34.78% vs. 13.95%, respectively; p = .008). Pathological staining showed that elastic fibers were sparser in patients with a frameshift and nonsense mutations, and the smooth muscle cells were sparser and more disorganized than those observed in patients with missense mutations.

Conclusion: This study showed that FBN1 gene frameshift and nonsense mutations are more common in patients with aortic dissection and may have meaningful guidance for the treatment of Marfan syndrome patients.
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http://dx.doi.org/10.1002/mgg3.1041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978253PMC
January 2020

Comparison of prognostic ability of perioperative myocardial biomarkers in acute type A aortic dissection.

Medicine (Baltimore) 2019 Oct;98(43):e17023

Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University.

Stanford type A aortic dissection (AD) is a lethal disease requiring surgery. Evidence regarding the prognostic ability of perioperative myocardiac markers on long-term outcome is limited.In this cohort study, we measured perioperative myocardiac markers level in 583 surgical patients with type A AD in our hospital between 2015 and 2017. All patients were followed up after surgery for a median period of 864 days to determine short- and long-term mortality.About one-fifth of patients has a positive preoperative myocardial markers, which was increased significantly after operation. Increase log10 post-creatine kinase MB isoenzyme (CK-MB) (hazard ratio [HR], 4.64; 95% confidence interval [CI] 1.89-11.43; P = .0008), log10 post-TnI (HR, 3.11; 95% CI 1.56-6.21; P = .0013), log10 post-Mb (HR, 3.00; 95% CI 1.40-6.43; P = .0048), log10 pre-CK-MB (HR,1.82; 95% CI 1.03-3.21; P = .0377), and upper tertile of post-CK-MB (HR,1.52; 95% CI 1.05-2.20; P = .0261) were the independent risk factor for 30 days mortality adjusted for potential confounders. None of cardiac markers was significantly associated with long-term outcome independent of other factors.Perioperative myocardiac predicts early outcome in type A AD patients undergoing surgery. Increasing perioperative myocardial markers do not appear to be a predictor for long-term all-cause mortality.
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http://dx.doi.org/10.1097/MD.0000000000017023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824686PMC
October 2019

Bioinformatics analysis of the regulatory lncRNA‑miRNA‑mRNA network and drug prediction in patients with hypertrophic cardiomyopathy.

Mol Med Rep 2019 Jul 23;20(1):549-558. Epub 2019 May 23.

Department of Epidemiology and Biostatistics, School of Public Health, Capital Medical University, Beijing 100069, P.R. China.

Hypertrophic cardiomyopathy (HCM) is a complex inherited cardiovascular disease. The present study investigated the long noncoding (lnc)RNA/microRNA (mi)RNA/mRNA expression pattern of patients with HCM and aimed to identify key molecules involved in the development of this condition. An integrated strategy was conducted to identify differentially expressed miRNAs (DEmiRs), differentially expressed lncRNAs (DElncs) and differentially expressed genes (DEGs) based on the GSE36961 (mRNA), GSE36946 (miRNA), GSE68316 (lncRNA/mRNA) and GSE32453 (mRNA) expression profiles downloaded from the Gene Expression Omnibus datasets. Bioinformatics tools were employed to perform function and pathway enrichment analysis, protein‑protein interaction, lncRNA‑miRNA‑mRNA and hub gene networks. Subsequently, DEGs were used as targets to predict drugs. The results indicated that a total of 2,234 DElncs (1,120 upregulated and 1,114 downregulated), 5 DEmiRs (2 upregulated and 3 downregulated) and 42 DEGs (35 upregulated and 7 downregulated) were identified in 4 microarray profiles. Gene ontology analysis revealed that DEGs were mainly involved in actin filament and stress fiber formation and in calcium ion binding, whereas Kyoto Encyclopedia of Genes and Genomes pathway analysis identified the hypoxia inducible factor‑1, transforming growth factor‑β and tumor necrosis factor signaling pathways as the main pathways involved in these processes. The hub genes were screened using cytoHubba. A total of 1,086 lncRNA‑miRNA‑mRNA interactions including 67 lncRNAs, 5 miRNAs and 25 mRNAs were mined in the present study based on prediction websites. Drug prediction indicated that the targeted drugs mainly included angiotensin converting enzyme inhibitors or β‑blockers. A comprehensive bioinformatics analysis of the molecular regulatory lncRNA‑miRNA‑mRNA network was performed and potential therapeutic applications of drugs were predicted in HCM patients. The data may unravel the future molecular mechanism of HCM.
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http://dx.doi.org/10.3892/mmr.2019.10289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6579968PMC
July 2019

Deep Hypothermic Circulatory Arrest Does Not Show Better Protection for Vital Organs Compared with Moderate Hypothermic Circulatory Arrest in Pig Model.

Biomed Res Int 2019 17;2019:1420216. Epub 2019 Apr 17.

Department of Cardiac Surgery, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing Lab for Cardiovascular Precision Medicine, Beijing Aortic Disease Center, Cardiovascular Surgery Center, Beijing Engineering Research Center for Vascular Prostheses, Beijing, China.

Background: Continued debates exist regarding the optimal temperature during hypothermic circulatory arrest in aortic arch repair for patients with type A aortic dissection. This study seeks to examine whether the use of moderate hypothermic circulatory arrest in a pig model provides comparable vital organ protection outcomes to the use of deep hypothermic circulatory arrest.

Methods: Thirteen pigs were randomly assigned to 30 minutes of hypothermic circulatory arrest without cerebral perfusion at 15°C (n = 5), 25°C (n = 5), and a control group (n = 3). The changes in standard laboratory tests and capacity for protection against apoptosis in different vital organs were monitored with different temperatures of hypothermic circulatory arrest management in pig model to determine which temperature was optimal for hypothermic circulatory arrest.

Results: There were no significant differences in the capacity for protection against apoptosis in vital organs between 2 groups (p > 0.05, respectively). Compared with the moderate hypothermic circulatory arrest group, the deep hypothermic circulatory arrest group had no significant advantages in terms of the biologic parameters of any other organs (p > 0.05).

Conclusions: Compared with deep hypothermic circulatory arrest, moderate hypothermic circulatory arrest is a moderate technique that has similar advantages with regard to the levels of biomarkers of injury and capacity for protection against apoptosis in vital organs.
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http://dx.doi.org/10.1155/2019/1420216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6500684PMC
November 2019

Cardiopulmonary bypass time is an independent risk factor for acute kidney injury in emergent thoracic aortic surgery: a retrospective cohort study.

J Cardiothorac Surg 2019 May 7;14(1):90. Epub 2019 May 7.

Department of Cardiac Surgery, Beijing Aortic Disease Center, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing Lab for Cardiovascular Precision Medicine, and Beijing Engineering Research Center of Vascular Prostheses, No.2 Anzhen Street, Beijing, 100029, China.

Background: Thoracic aortic surgery and cardiopulmonary bypass are both associated with development of postoperative acute kidney injury. In this study, we undertook to investigate the relationship between cardiopulmonary bypass time and postoperative acute kidney injury in patients undergoing thoracic aortic surgery for acute DeBakey Type I aortic dissection.

Methods: All patients receiving thoracic aortic surgery for acute DeBakey Type I aortic dissection in Beijing Anzhen hospital from December 2015 to April 2017 were included. Cardiopulmonary bypass time was recorded during surgery. Acute kidney injury was defined based on the Kidney Disease Improving Global Outcomes criteria. A total of 115 consecutive patients were eventually analyzed.

Results: The overall incidence of acute kidney injury was 53.0% (n = 61). The average age was 47.8 ± 10.7 years; 74.8% were male. Mean cardiopulmonary bypass time was 211 ± 56 min. In-hospital mortality was 7.8%. Multivariate logistic regression revealed that cardiopulmonary bypass time was independently associated with the occurrence of postoperative acute kidney injury after adjust confounding factors (odds ratio = 1.171; 95% confidence interval: 1.002-1.368; P = 0.047).

Conclusions: Cardiopulmonary bypass time is independently associated with an increased hazard of acute kidney injury after thoracic aortic surgery for acute DeBakey Type I aortic dissection. Further understanding of the mechanism of this association is crucial to the design of preventative strategies.
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http://dx.doi.org/10.1186/s13019-019-0907-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6505293PMC
May 2019

Increased risk for the development of postoperative severe hypoxemia in obese women with acute type a aortic dissection.

J Cardiothorac Surg 2019 Apr 25;14(1):81. Epub 2019 Apr 25.

Department of Cardiac Surgery, Beijing Aortic Disease Center, Beijing Anzhen Hospital, Capital Medical University, Beijing Institute of Heart Lung and Blood Vessel Diseases, Beijing Laboratory for Cardiovascular Precision Medicine, and Beijing Engineering Research Center of Vascular Prostheses, No.2 Anzhen Street, Beijing, 100029, China.

Background: The purpose of this study is to identify the risk factors for postoperative severe hypoxemia after surgery for acute type A aortic dissection.

Methods: This was a single-center retrospective study including 112 consecutive patients undergoing urgent aortic arch surgery for acute type A aortic dissection between December 2016 and April 2017 at Beijing Anzhen Hospital.

Results: Multivariate logistic regression analysis identified female (OR, 12.978; 95% CI, 3.332 to 50.546; p < 0.001) and increased body mass index (OR, 1.473; 95% CI, 1.213 to 1.789; p < 0.001) as independent predictors of postoperative severe hypoxemia in patients with acute type A aortic dissection.

Conclusions: Obesity and female were independent risk factors for postoperative severe hypoxemia in patients with acute type A aortic dissection. More attention should be paid to preventing postoperative severe hypoxemia in obese women with acute type A aortic dissection.
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http://dx.doi.org/10.1186/s13019-019-0888-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6482483PMC
April 2019

Structure-Activity and Structure-Conformation Relationships of Aryl Propionic Acid Inhibitors of the Kelch-like ECH-Associated Protein 1/Nuclear Factor Erythroid 2-Related Factor 2 (KEAP1/NRF2) Protein-Protein Interaction.

J Med Chem 2019 05 25;62(9):4683-4702. Epub 2019 Apr 25.

Astex Pharmaceuticals , 436 Cambridge Science Park , Cambridge CB4 0QA , U.K.

The KEAP1-NRF2-mediated cytoprotective response plays a key role in cellular homoeostasis. Insufficient NRF2 signaling during chronic oxidative stress may be associated with the pathophysiology of several diseases with an inflammatory component, and pathway activation through direct modulation of the KEAP1-NRF2 protein-protein interaction is being increasingly explored as a potential therapeutic strategy. Nevertheless, the physicochemical nature of the KEAP1-NRF2 interface suggests that achieving high affinity for a cell-penetrant druglike inhibitor might be challenging. We recently reported the discovery of a highly potent tool compound which was used to probe the biology associated with directly disrupting the interaction of NRF2 with the KEAP1 Kelch domain. We now present a detailed account of the medicinal chemistry campaign leading to this molecule, which included exploration and optimization of protein-ligand interactions in three energetic "hot spots" identified by fragment screening. In particular, we also discuss how consideration of ligand conformational stabilization was important to its development and present evidence for preorganization of the lead compound which may contribute to its high affinity and cellular activity.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00279DOI Listing
May 2019

A hit deconstruction approach for the discovery of fetal hemoglobin inducers.

Bioorg Med Chem Lett 2018 12 22;28(23-24):3676-3680. Epub 2018 Oct 22.

GlaxoSmithKline, 1250 South Collegeville Rd., Collegeville, PA 19426, USA.

Beta-hemoglobinopathies such as sickle cell disease represent a major global unmet medical need. De-repression of fetal hemoglobin in erythrocytes is a clinically validated approach for the management of sickle cell disease, but the only FDA-approved medicine for this purpose has limitations to its use. We conducted a phenotypic screen in human erythroid progenitor cells to identify molecules with the ability to de-repress fetal hemoglobin, which resulted in the identification of the benzoxaborole-containing hit compound 1. This compound was found to have modest cellular potency and lead-like pharmacokinetics, but no identifiable SAR to enable optimization. Systematic deconstruction of a closely related analog of 1 revealed the fragment-like carboxylic acid 12, which was then optimized to provide tetrazole 31, which had approximately 100-fold improved cellular potency compared to 1, high levels of oral exposure in rats, and excellent solubility.
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http://dx.doi.org/10.1016/j.bmcl.2018.10.032DOI Listing
December 2018

Screen for modulators of atonal homolog 1 gene expression using notch pathway-relevant gene transcription based cellular assays.

PLoS One 2018 12;13(12):e0207140. Epub 2018 Dec 12.

R&D Alternative Discovery and Development, GlaxoSmithKline, Stevenage, Hertfordshire, United Kingdom.

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix 9 (bHLH) transcription factor acting downstream of Notch and is required for the differentiation of sensory hair cells in the inner ear and the specification of secretory cells during the intestinal crypt cell regeneration. Motivated by the observations that the upregulation of Atoh1 gene expression, through genetic manipulation or pharmacological inhibition of Notch signaling (e.g. γ-secretase inhibitors, GSIs), induces ectopic hair cell growth in the cochlea of the inner ear and partially restores hearing after injuries in experimental models, we decided to identify small molecule modulators of the Notch-Atoh1 pathway, which could potentially regenerate hair cells. However, the lack of cellular models of the inner ear has precluded the screening and characterization of such modulators. Here we report using a colon cancer cell line LS-174T, which displays Notch inhibition-dependent Atoh1 expression as a surrogate cellular model to screen for inducers of Atoh1 expression. We designed an Atoh1 promoter-driven luciferase assay to screen a target-annotated library of ~6000 compounds. We further developed a medium throughput, real-time quantitative RT-PCR assay measuring the endogenous Atoh1 gene expression to confirm the hits and eliminate false positives from the reporter-based screen. This strategy allowed us to successfully recover GSIs of known chemotypes. This LS-174T cell-based assay directly measures Atoh1 gene expression induced through Notch-Hes1 inhibition, and therefore offers an opportunity to identify novel cellular modulators along the Notch-Atoh1 pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0207140PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291236PMC
April 2019

Development of a High-Throughput Cul3-Keap1 Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) Assay for Identifying Nrf2 Activators.

SLAS Discov 2019 02 1;24(2):175-189. Epub 2018 Nov 1.

1 Screening, Profiling and Mechanistic Biology, Platform Technology and Science, GlaxoSmithKline, Collegeville, PA, USA.

Nrf2, a master regulator of the phase II gene response to stress, is kept at low concentrations in the cell through binding to Keap1, an adaptor protein for the Cul3 ubiquitin ligase complex. To identify Nrf2 activators, two separate time-resolved fluorescence resonance energy transfer (TR-FRET) assays were developed to monitor the binding of Nrf2-Keap1 and Cul3-Keap1, respectively. The triterpenoid, 1-[2-cyano-3-,12-dioxooleana-1,9(11)-dien-28-oyl] imidazole (CDDO-Im) and its analogs, exhibited approximately 100-fold better potency in the Cul3-Keap1 assay than in the Nrf2-Keap1 assay, and this difference was more profound at 37 °C than at room temperature in the Nrf2-Keap1 assay, but this phenomenon was not observed in the Cul3-Keap1 assay. A full diversity screen of approximately 2,200,000 GSK compounds was run with the Cul3-Keap1 TR-FRET assay and multiple chemical series were identified and characterized.
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http://dx.doi.org/10.1177/2472555218807698DOI Listing
February 2019

A Luciferase Reporter Gene System for High-Throughput Screening of γ-Globin Gene Activators.

Methods Mol Biol 2016 ;1439:207-26

Department of Biological Sciences, Platform Technology and Science, GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, PA, USA.

Luciferase reporter gene assays have long been used for drug discovery due to their high sensitivity and robust signal. A dual reporter gene system contains a gene of interest and a control gene to monitor non-specific effects on gene expression. In our dual luciferase reporter gene system, a synthetic promoter of γ-globin gene was constructed immediately upstream of the firefly luciferase gene, followed downstream by a synthetic β-globin gene promoter in front of the Renilla luciferase gene. A stable cell line with the dual reporter gene was cloned and used for all assay development and HTS work. Due to the low activity of the control Renilla luciferase, only the firefly luciferase activity was further optimized for HTS. Several critical factors, such as cell density, serum concentration, and miniaturization, were optimized using tool compounds to achieve maximum robustness and sensitivity. Using the optimized reporter assay, the HTS campaign was successfully completed and approximately 1000 hits were identified. In this chapter, we also describe strategies to triage hits that non-specifically interfere with firefly luciferase.
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http://dx.doi.org/10.1007/978-1-4939-3673-1_14DOI Listing
December 2017

A High-Content Imaging Screen for Cellular Regulators of β-Catenin Protein Abundance.

J Biomol Screen 2016 Mar 9;21(3):260-8. Epub 2015 Dec 9.

R&D Platform Technology Sciences, GlaxoSmithKine, Upper Providence, Collegeville, PA, USA.

Abnormal accumulation of β-catenin protein, a key transcriptional activator required for Wnt signaling, is the hallmark of many tumor types, including colon cancer. In normal cells, β-catenin protein level is tightly controlled by a multiprotein complex through the proteosome pathway. Mutations in the components of the β-catenin degradation complex, such as adenomatous polyposis coli (APC) and Axin, lead to β-catenin stabilization and the constitutive activation of target genes. Since the signal transduction of Wnt/β-catenin is mainly mediated by protein-protein interactions, this pathway has been particularly refractory to conventional target-based small-molecule screening. Here we designed a cellular high-content imaging assay to detect β-catenin protein through immunofluorescent staining in the SW480 colon cancer cell line, which has elevated β-catenin endogenously. We demonstrate that the assay is robust and specific to screen a focused biologically diverse chemical library set against known targets that play diverse cellular functions. We identified a number of hits that reduce β-catenin levels without causing cell death. These hits may serve as tools to understand the dynamics of β-catenin degradation. This study demonstrates that detecting cell-based β-catenin protein stability is a viable approach to identifying novel mechanisms of β-catenin regulation as well as small molecules of therapeutic potential.
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http://dx.doi.org/10.1177/1087057115620169DOI Listing
March 2016

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

ACS Comb Sci 2015 Dec 3;17(12):722-31. Epub 2015 Dec 3.

Molecular Discovery Research, GlaxoSmithKline , Waltham, Massachusetts 02451, United States.

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.
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http://dx.doi.org/10.1021/acscombsci.5b00124DOI Listing
December 2015

High throughput screening identifies ATP-competitive inhibitors of the NLRP1 inflammasome.

Bioorg Med Chem Lett 2015 Jul 19;25(14):2739-43. Epub 2015 May 19.

Pattern Recognition Receptor DPU, GlaxoSmithKline, Collegeville Road, Collegeville, PA 19426, USA.

Nod-like receptors (NLRs) are cytoplasmic pattern recognition receptors that are promising targets for the development of anti-inflammatory therapeutics. Drug discovery efforts targeting NLRs have been hampered by their inherent tendency to form aggregates making protein generation and the development of screening assays very challenging. Herein we report the results of an HTS screen of NLR family member NLRP1 (NLR family, pyrin domain-containing 1) which was achieved through the large scale generation of recombinant GST-His-Thrombin-NLRP1 protein. The screen led to the identification of a diverse set of ATP competitive inhibitors with micromolar potencies. Activity of these hits was confirmed in a FP binding assay, and two homology models were employed to predict the possible binding mode of the leading series and facilitate further lead-optimization. These results highlight a promising strategy for the identification of inhibitors of NLR family members which are rapidly emerging as key drivers of inflammation in human disease.
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http://dx.doi.org/10.1016/j.bmcl.2015.05.032DOI Listing
July 2015

Identification of selective small molecule inhibitors of the nucleotide-binding oligomerization domain 1 (NOD1) signaling pathway.

PLoS One 2014 7;9(5):e96737. Epub 2014 May 7.

Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, United States of America.

NOD1 is an intracellular pattern recognition receptor that recognizes diaminopimelic acid (DAP), a peptidoglycan component in gram negative bacteria. Upon ligand binding, NOD1 assembles with receptor-interacting protein (RIP)-2 kinase and initiates a signaling cascade leading to the production of pro-inflammatory cytokines. Increased NOD1 signaling has been associated with a variety of inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. We utilized a cell-based screening approach with extensive selectivity profiling to search for small molecule inhibitors of the NOD1 signaling pathway. Via this process we identified three distinct chemical series, xanthines (SB711), quinazolininones (GSK223) and aminobenzothiazoles (GSK966) that selectively inhibited iE-DAP-stimulated IL-8 release via the NOD1 signaling pathway. All three of the newly identified compound series failed to block IL-8 secretion in cells following stimulation with ligands for TNF receptor, TLR2 or NOD2 and, in addition, none of the compound series directly inhibited RIP2 kinase activity. Our initial exploration of the structure-activity relationship and physicochemical properties of the three series directed our focus to the quinazolininone biarylsulfonamides (GSK223). Further investigation allowed for the identification of significantly more potent analogs with the largest boost in activity achieved by fluoro to chloro replacement on the central aryl ring. These results indicate that the NOD1 signaling pathway, similarly to activation of NOD2, is amenable to modulation by small molecules that do not target RIP2 kinase. These compounds should prove useful tools to investigate the importance of NOD1 activation in various inflammatory processes and have potential clinical utility in diseases driven by hyperactive NOD1 signaling.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096737PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4013053PMC
October 2015

A novel approach applying a chemical biology strategy in phenotypic screening reveals pathway-selective regulators of histone 3 K27 tri-methylation.

Mol Biosyst 2014 Feb;10(2):251-7

Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA, USA.

Epigenetic regulation by histone methylation is crucial for proper programming of the genome during development. Homeostasis of histone methylation is balanced by the activities of histone methyltransferases and demethylases. Although these methyltransferases and demethylases represent logical targets for potential drug discovery, the activities of methyltransferases and demethylases regulated in response to a complex biological stimulus are also important and not yet clear. To manipulate and study histone methylation in biological systems, we screened a Biologically Diverse Compound Set (BDCS) utilizing a phenotypic assay system that directly measures the Histone 3 K27 tri-methylation (H3K27me3) level in cells. The BDCS is a unique set of target-annotated chemical probes, containing a total of 5853 compounds targeting 736 unique proteins with multiple maximally selective compounds for each target. A number of targets, with multiple hits against each target, were identified in the screen. This gave us confidence that these targets and pathways may be relevant, and included the identification of non-methyltransferase/demethylase targets as potential upstream regulators of H3K27me3. Our study suggests that a systematically designed chemical probe library can serve as a powerful drug discovery tool when combined with phenotypic screening. Follow-up studies using these findings may reveal novel therapeutically useful pathways and targets of H3K27me3 regulation.
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http://dx.doi.org/10.1039/c3mb70413kDOI Listing
February 2014

Development of phenotypic screening assays for γ-globin induction using primary human bone marrow day 7 erythroid progenitor cells.

J Biomol Screen 2013 Dec 25;18(10):1212-22. Epub 2013 Oct 25.

1Molecular Discovery Research, GlaxoSmithKline, Collegeville, PA, USA.

Sickle cell anemia (SCA) is a genetic disorder of the β-globin gene. SCA results in chronic ischemia with pain and tissue injury. The extent of SCA symptoms can be ameliorated by treatment with drugs, which result in increasing the levels of γ-globin in patient red blood cells. Hydroxyurea (HU) is a Food and Drug Administration-approved drug for SCA, but it has dose-limiting toxicity, and patients exhibit highly variable treatment responses. To identify compounds that may lead to the development of better and safer medicines, we have established a method using primary human bone marrow day 7 erythroid progenitor cells (EPCs) to screen for compounds that induce γ-globin production. First, human marrow CD34(+) cells were cultured and expanded for 7 days and characterized for the expression of erythroid differentiation markers (CD71, CD36, and CD235a). Second, fresh or cryopreserved EPCs were treated with compounds for 3 days in 384-well plates followed by γ-globin quantification by an enzyme-linked immunosorbent assay (ELISA), which was validated using HU and decitabine. From the 7408 compounds screened, we identified at least one new compound with confirmed γ-globin-inducing activity. Hits are undergoing analysis in secondary assays. In this article, we describe the method of generating fit-for-purpose EPCs; the development, optimization, and validation of the ELISA and secondary assays for γ-globin detection; and screening results.
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http://dx.doi.org/10.1177/1087057113499776DOI Listing
December 2013

Identification of benzimidazole diamides as selective inhibitors of the nucleotide-binding oligomerization domain 2 (NOD2) signaling pathway.

PLoS One 2013 1;8(8):e69619. Epub 2013 Aug 1.

Pattern Recognition Receptor Discovery Performance Unit, Immuno-Inflammation Therapeutic Area, GlaxoSmithKline, Collegeville, Pennsylvania, USA.

NOD2 is an intracellular pattern recognition receptor that assembles with receptor-interacting protein (RIP)-2 kinase in response to the presence of bacterial muramyl dipeptide (MDP) in the host cell cytoplasm, thereby inducing signals leading to the production of pro-inflammatory cytokines. The dysregulation of NOD2 signaling has been associated with various inflammatory disorders suggesting that small-molecule inhibitors of this signaling complex may have therapeutic utility. To identify inhibitors of the NOD2 signaling pathway, we utilized a cell-based screening approach and identified a benzimidazole diamide compound designated GSK669 that selectively inhibited an MDP-stimulated, NOD2-mediated IL-8 response without directly inhibiting RIP2 kinase activity. Moreover, GSK669 failed to inhibit cytokine production in response to the activation of Toll-like receptor (TLR)-2, tumor necrosis factor receptor (TNFR)-1 and closely related NOD1, all of which share common downstream components with the NOD2 signaling pathway. While the inhibitors blocked MDP-induced NOD2 responses, they failed to block signaling induced by NOD2 over-expression or single stranded RNA, suggesting specificity for the MDP-induced signaling complex and activator-dependent differences in NOD2 signaling. Investigation of structure-activity relationship allowed the identification of more potent analogs that maintained NOD2 selectivity. The largest boost in activity was achieved by N-methylation of the C2-ethyl amide group. These findings demonstrate that the NOD2 signaling pathway is amenable to modulation by small molecules that do not target RIP2 kinase activity. The compounds we identified should prove useful tools to investigate the importance of NOD2 in various inflammatory processes and may have potential clinical utility.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069619PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3731320PMC
March 2014

Selective class IIa histone deacetylase inhibition via a nonchelating zinc-binding group.

Nat Chem Biol 2013 May 24;9(5):319-25. Epub 2013 Mar 24.

Tempero Pharmaceuticals, Cambridge, Massachusetts, USA.

In contrast to studies on class I histone deacetylase (HDAC) inhibitors, the elucidation of the molecular mechanisms and therapeutic potential of class IIa HDACs (HDAC4, HDAC5, HDAC7 and HDAC9) is impaired by the lack of potent and selective chemical probes. Here we report the discovery of inhibitors that fill this void with an unprecedented metal-binding group, trifluoromethyloxadiazole (TFMO), which circumvents the selectivity and pharmacologic liabilities of hydroxamates. We confirm direct metal binding of the TFMO through crystallographic approaches and use chemoproteomics to demonstrate the superior selectivity of the TFMO series relative to a hydroxamate-substituted analog. We further apply these tool compounds to reveal gene regulation dependent on the catalytic active site of class IIa HDACs. The discovery of these inhibitors challenges the design process for targeting metalloenzymes through a chelating metal-binding group and suggests therapeutic potential for class IIa HDAC enzyme blockers distinct in mechanism and application compared to current HDAC inhibitors.
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http://dx.doi.org/10.1038/nchembio.1223DOI Listing
May 2013

Development and validation of reagents and assays for EZH2 peptide and nucleosome high-throughput screens.

J Biomol Screen 2012 Dec 17;17(10):1279-92. Epub 2012 Aug 17.

Platform Technology and Science, GlaxoSmithKline, Collegeville, PA 19426, USA.

Histone methyltransferases (HMT) catalyze the methylation of histone tail lysines, resulting in changes in gene transcription. Misregulation of these enzymes has been associated with various forms of cancer, making this target class a potential new area for the development of novel chemotherapeutics. EZH2 is the catalytic component of the polycomb group repressive complex (PRC2), which selectively methylates histone H3 lysine 27 (H3K27). EZH2 is overexpressed in prostate, breast, bladder, brain, and other tumor types and is recognized as a molecular marker for cancer progression and aggressiveness. Several new reagents and assays were developed to aid in the identification of EZH2 inhibitors, and these were used to execute two high-throughput screening campaigns. Activity assays using either an H3K27 peptide or nucleosomes as substrates for methylation are described. The strategy to screen EZH2 with either a surrogate peptide or a natural substrate led to the identification of the same tractable series. Compounds from this series are reversible, are [(3)H]-S-adenosyl-L-methionine competitive, and display biochemical inhibition of H3K27 methylation.
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http://dx.doi.org/10.1177/1087057112453765DOI Listing
December 2012

Novel N-Substituted Benzimidazolones as Potent, Selective, CNS-Penetrant, and Orally Active M1 mAChR Agonists.

ACS Med Chem Lett 2010 Sep 8;1(6):244-8. Epub 2010 Jun 8.

GlaxoSmithKline, 1250 South Collegeville Road, Collegeville, Pennsylvania 19426.

Virtual screening of the corporate compound collection yielded compound 1 as a subtype selective muscarinic M1 receptor agonist hit. Initial optimization of the N-capping group of the central piperidine ring resulted in compounds 2 and 3 with significantly improved potency and selectivity. Subsequent optimization of substituents on the phenyl ring of the benzimidazolone moiety led to the discovery of novel muscarinic M1 receptor agonists 4 and 5 with excellent potency, general and subtype selectivity, and pharmacokinetic (PK) properties including good central nervous system (CNS) penetration and oral bioavailability. Compound 5 showed robust in vivo activities in animal models of cognition enhancement. The combination of high potency, excellent selectivity, and good PK properties makes compounds 4 and 5 valuable tool compounds for investigating and validating potential therapeutic benefits resulting from selective M1 activation.
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http://dx.doi.org/10.1021/ml100105xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4007837PMC
September 2010

2' biaryl amides as novel and subtype selective M1 agonists. Part I: Identification, synthesis, and initial SAR.

Bioorg Med Chem Lett 2010 Jun 17;20(12):3540-4. Epub 2010 May 17.

Discovery Research and Centers of Excellence for Drug Discovery, GlaxoSmithKline, Collegeville, PA 19426, USA.

Biaryl amides were discovered as novel and subtype selective M(1) muscarinic acetylcholine receptor agonists. The identification, synthesis, and initial structure-activity relationships that led to compounds 3j and 4c, possessing good M(1) agonist potency and intrinsic activity, and subtype selectivity for M(1) over M(2-5), are described.
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http://dx.doi.org/10.1016/j.bmcl.2010.04.128DOI Listing
June 2010

2' biaryl amides as novel and subtype selective M1 agonists. Part II: Further optimization and profiling.

Bioorg Med Chem Lett 2010 Jun 17;20(12):3545-9. Epub 2010 May 17.

Discovery Research and Centers of Excellence for Drug Discovery, GlaxoSmithKline, Collegeville, PA 19426, USA.

Further optimization of the biaryl amide series via extensively exploring structure-activity relationships resulted in potent and subtype selective M(1) agonists exemplified by compounds 9a and 9j with good rat PK properties including CNS penetration. Synthesis, structure-activity relationships, subtype selectivity for M(1) over M(2-5), and DMPK properties of these novel compounds are described.
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http://dx.doi.org/10.1016/j.bmcl.2010.04.127DOI Listing
June 2010

In vitro and in vivo comparison of two non-peptide tachykinin NK3 receptor antagonists: Improvements in efficacy achieved through enhanced brain penetration or altered pharmacological characteristics.

Eur J Pharmacol 2010 Feb 30;627(1-3):106-14. Epub 2009 Oct 30.

Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, UK.

Clinical evaluation of tachykinin NK(3) receptor antagonists has provided support for the therapeutic utility of this target in schizophrenia. However, these studies have not been entirely conclusive, possibly because of the pharmacokinetic limitations of these molecules. In the search for tachykinin NK(3) receptor antagonists with improved properties, we have discovered GSK172981 and GSK256471. Both compounds demonstrated high affinity for recombinant human (pK(i) values 7.7 and 8.9, respectively) and native guinea pig (pK(i) values 7.8 and 8.4, respectively) tachykinin NK(3) receptors. In vitro functional evaluations revealed GSK172981 to be a competitive antagonist (pA(2)=7.2) at cloned human tachykinin NK(3) receptor whereas GSK256471 diminished the neurokinin B-induced E(max) response, indicative of non-surmountable antagonist pharmacology (pA(2)=9.2). GSK172981 also exhibited a competitive profile in antagonizing neurokinin B-stimulated neuronal activity recorded from the guinea pig medial habenula slices (apparent pK(B)=8.1), whilst GSK256471 abolished the agonist-induced response. Central nervous system penetration by GSK172981 and GSK256471 was indicated by dose-dependent ex vivo tachykinin NK(3) receptor occupancy in medial prefrontal cortex (ED(50) values of 0.8 and 0.9 mg/kg, i.p., respectively) and the dose-dependent attenuation of agonist-induced "wet dog shake" behaviours in guinea pigs. Finally, in vivo microdialysis studies demonstrated that acute GSK172981 (30 mg/kg, i.p.) and GSK256471 (1mg/kg, i.p.) attenuated haloperidol-induced increases in extracellular dopamine in the guinea pig nucleus accumbens. Taken together, these in vitro and in vivo characterisations of the tachykinin NK(3) receptor antagonists GSK172981 and GSK256471 support their potential utility in the treatment of schizophrenia.
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http://dx.doi.org/10.1016/j.ejphar.2009.10.054DOI Listing
February 2010
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