450 results on '"Ladds, Graham"'
Search Results
152. Extracellular degradation of agonists as an adaptive mechanism
- Author
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Ladds, Graham, primary, Hughes, Marcus, additional, and Davey, John, additional
- Published
- 1998
- Full Text
- View/download PDF
153. The processing of yeast pheromones
- Author
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Davey, John, primary, Davis, Kevin, additional, Hughes, Marcus, additional, Ladds, Graham, additional, and Powner, Dale, additional
- Published
- 1998
- Full Text
- View/download PDF
154. Proteolysis of Sxa2, a carboxypeptidase involved in pheromone adaptation in yeast
- Author
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LADDS, GRAHAM, primary and DAVEY, JOHN, additional
- Published
- 1997
- Full Text
- View/download PDF
155. Sxa2, a carboxypeptidase that degrades extracellular pheromone in fission yeast
- Author
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LADDS, GRAHAM, primary and DAVEY, JOHN, additional
- Published
- 1997
- Full Text
- View/download PDF
156. Defining the role of Sxa1 during pheromone adaptation in Schizosaccharomyces pombe
- Author
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HUGHES, MARCUS, primary, LADDS, GRAHAM, additional, and DAVEY, JOHN, additional
- Published
- 1996
- Full Text
- View/download PDF
157. Purification of Sxa2, a carboxypeptidase involved in pheromone recovery in fission yeast
- Author
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LADDS, GRAHAM, primary and DAVEY, JOHN, additional
- Published
- 1996
- Full Text
- View/download PDF
158. Characterisation of Sxa2, a carboxypeptidase involved in pheromone recovery in fission yeast
- Author
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LADDS, GRAHAM, primary and DAVEY, JOHN, additional
- Published
- 1996
- Full Text
- View/download PDF
159. Characterisation of Sxa2, a protease involved in pheromone communication in fission yeast
- Author
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LADDS, GRAHAM, primary, RASMUSSEN, MICHAEL, additional, and DAVEY, JOHN, additional
- Published
- 1995
- Full Text
- View/download PDF
160. Quantitative analysis of human ras localization and function in the fission yeast Schizosaccharomyces pombe.
- Author
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Bond, Michael, Croft, Wayne, Tyson, Richard, Bretschneider, Till, Davey, John, and Ladds, Graham
- Abstract
Ras signalling is central to fundamental and diverse cellular processes. In higher eukaryotes ras signalling is highly complex, involving multiple isoforms, regulatory proteins and effectors. As a consequence, the study of ras activity in mammalian systems presents a number of technical challenges. The model organism Schizosaccharomyces pombe has previously proved a key system for the study of human signalling components and provides an ideal model for the study of ras, as it contains just one ras protein (Ras1p), which is non-essential and controls a number of downstream processes. Here we present data demonstrating the quantitative analysis of three distinct Ras1-related signalling outputs, utilizing the three most abundant human ras isoforms, H-Ras, N-Ras and K-Ras4B, in Sz. pombe. Further, we have characterized the localization of these three human ras isoforms in Sz. pombe, utilizing quantitative image analysis techniques. These data indicate that all three human ras isoforms are functional in fission yeast, displaying differing localization patterns which correlate strongly with function in the regulation of pheromone response and cell shape. These data demonstrate that such yeast strains could provide powerful tools for the investigation of ras biology, and potentially in the development of cancer therapies. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]
- Published
- 2013
- Full Text
- View/download PDF
161. Differential effects of RGS proteins on Gαq and Gα11 activity
- Author
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Ladds, Graham, Goddard, Alan, Hill, Claire, Thornton, Steven, and Davey, John
- Subjects
- *
PROTEINS , *MEMBRANE proteins , *G proteins , *RAS proteins - Abstract
Abstract: Heterotrimeric G proteins play a pivotal role in GPCR signalling; they link receptors to intracellular effectors and their inactivation by RGS proteins is a key factor in resetting the pathway following stimulation. The precise GPCR:G protein:RGS combination determines the nature and duration of the response. Investigating the activity of particular combinations is difficult in cells which contain multiples of each component. We have therefore utilised a previously characterised yeast system to express mammalian proteins in isolation. Human Gαq and Gα11 spontaneously activated the yeast pheromone-response pathway by a mechanism which required the formation of Gα-GTP. This provided an assay for the specific activity of human RGS proteins. RGS1, RGS2, RGS3 and RGS4 inhibited the spontaneous activity of both Gαq and Gα11 but, in contrast, RGS5 and RGS16 were much less effective against Gα11 than Gαq. Interestingly, RGS2 and RGS3 were able to inhibit signalling from the constitutively active Gαq QL/Gα11 QL mutants, confirming the GAP-independent activity of these RGS proteins. To determine if the RGS-Gα specificity was maintained under conditions of GPCR stimulation, minor modifications to the C-terminus of Gαq/Gα11 enabled coupling to an endogenous receptor. RGS2 and RGS3 were effective inhibitors of both Gα subunits even at high levels of receptor stimulation, emphasising their GAP-independent activity. At low levels of stimulation RGS5 and RGS16 retained their differential Gα activity, further highlighting that RGS proteins can discriminate between two very closely related Gα subunits. [Copyright &y& Elsevier]
- Published
- 2007
- Full Text
- View/download PDF
162. Utilisation of the budding yeast Saccharomyces cerevisiae for the generation and isolation of non-lethal ricin A chain variants.
- Author
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Allen, Stuart C. H., Byron, Adam, Lord, J. Michael, Davey, John, Roberts, Lynne M., and Ladds, Graham
- Abstract
Knowledge of the uptake, membrane translocation, refolding and ribosome interaction of the ribosome-inactivating toxin ricin is incomplete at the present time. Ricin A chain (RTA) is the catalytic subunit of holotoxin and is also of particular interest as a vaccine candidate. For many studies into the uptake and immunological applications of ricin, it is essential to have inactive variants. Here, following error-prone polymerase chain reaction of the RTA open reading frame, we have used a modified gap-repair protocol in Saccharomyces cerevisiae to show that it is possible to rapidly generate a panel of inactive RTA mutants. Since yeast cells have ribosomes that are highly sensitive to RTA, we utilized a genetic selection based on the viability of transformants. This enabled the recovery of a number of mutations, some not previously identified, which permitted production of full-length but non-toxic RTA proteins. Such disarmed toxins may have utility as tools to study the cytosolic entry and action of RTA, and as potential vaccine candidates. Copyright © 2005 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
- View/download PDF
163. Development of a semi-quantitative plate-based α-galactosidase gene reporter for Schizosaccharomyces pombe and its use to isolate a constitutively active Mam2.
- Author
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Goddard, Alan, Ladds, Graham, and Davey, John
- Abstract
To extend the tools available for biochemical and genetical analysis in the fission yeast Schizosaccharomyces pombe we have investigated the development of gene reporter systems using the secreted α-galactosidase encoded by the Sz. pombe ORF SPAC869.07c (CAB60017), which we propose naming Mel1p to reflect its structural and functional similarity to MEL1p in Saccharomyces cerevisiae. The α-galactosidase activity can be monitored in liquid assays and converted the colourless substrate 5-bromo-4-chloro-3-indolyl-α-D-galactopyranoside (X-α-gal) into an insoluble blue product that was suitable for semi quantitative plate-based assays; colonies expressing the highest levels of α-galactosidase developed the most intense blue colour. Unlike assays based on β-galactosidase, the Sz. pombe colonies develop the blue colouration under normal growth conditions, avoiding the need to replicate colonies to fresh plates for analysis. It is therefore suitable for screening large numbers of colonies. To illustrate the use of mel1 as a reporter we linked expression to the sxa2 gene promoter to provide a convenient readout for signalling through the pheromone response pathway. The sxa2 > mel1 strain identified constitutively active Mam2 pheromone receptors from a randomly mutagenised library. There was an approximate correlation between the intensity of the blue colour developed by each mutant colony and its level of constitutive activity and we identified a subset of mutants with low constitutive activity that could not have been isolated by a previous screen using nutritional selection. The mel1 α-galactosidase activity identified and characterised in this study can be easily adapted to provide a gene reporter for many biological processes and is a new addition to the research tools available in Sz. pombe. Copyright © 2004 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]
- Published
- 2005
- Full Text
- View/download PDF
164. Discovery and Structure–Activity Relationship Studies of Novel Adenosine A1Receptor-Selective Agonists
- Author
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Preti, Barbara, Suchankova, Anna, Deganutti, Giuseppe, Leuenberger, Michele, Barkan, Kerry, Manulak, Iga, Huang, Xianglin, Carvalho, Sabrina, Ladds, Graham, and Lochner, Martin
- Abstract
A series of benzyloxy and phenoxy derivatives of the adenosine receptor agonists N6-cyclopentyl adenosine (CPA) and N6-cyclopentyl 5′-N-ethylcarboxamidoadenosine (CP-NECA) were synthesized, and their potency and selectivity were assessed. We observed that the most potent were the compounds with a halogen in the metaposition on the aromatic ring of the benzyloxy- or phenoxycyclopentyl substituent. In general, the NECA-based compounds displayed greater A1R selectivity than the adenosine-based compounds, with N6-2-(3-bromobenzyloxy)cyclopentyl-NECA and N6-2-(3-methoxyphenoxy)cyclopentyl-NECA showing ∼1500-fold improved A1R selectivity compared to NECA. In addition, we quantified the compounds’ affinity and kinetics of binding at both human and rat A1R using a NanoBRET binding assay and found that the halogen substituent in the benzyloxy- or phenoxycyclopentyl moiety seems to confer high affinity for the A1R. Molecular modeling studies suggested a hydrophobic subpocket as contributing to the A1R selectivity displayed. We believe that the identified selective potent A1R agonists are valuable tool compounds for adenosine receptor research.
- Published
- 2022
- Full Text
- View/download PDF
165. Correction to Insights to the Binding of a Selective Adenosine A3Receptor Antagonist using Molecular Dynamic Simulations, MM-PBSA and MM-GBSA Free Energy Calculations, and Mutagenesis
- Author
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Lagarias, Panagiotis, Barkan, Kerry, Tzortzini, Eva, Stampelou, Margarita, Vrontaki, Eleni, Ladds, Graham, and Kolocouris, Antonios
- Published
- 2020
- Full Text
- View/download PDF
166. Author Correction: Combinatorial expression of GPCR isoforms affects signalling and drug responses.
- Author
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Marti-Solano, Maria, Crilly, Stephanie E., Malinverni, Duccio, Munk, Christian, Harris, Matthew, Pearce, Abigail, Quon, Tezz, Mackenzie, Amanda E., Wang, Xusheng, Peng, Junmin, Tobin, Andrew B., Ladds, Graham, Milligan, Graeme, Gloriam, David E., Puthenveedu, Manojkumar A., and Babu, M. Madan
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
167. Pharmacological characterisation of novel adenosine A3 receptor antagonists.
- Author
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Barkan, Kerry, Lagarias, Panagiotis, Stampelou, Margarita, Stamatis, Dimitrios, Hoare, Sam, Safitri, Dewi, Klotz, Karl-Norbert, Vrontaki, Eleni, Kolocouris, Antonios, and Ladds, Graham
- Subjects
ADENOSINES ,ASTHMA ,BINDING sites ,FUNCTIONAL groups ,MOLECULAR dynamics - Abstract
The adenosine A
3 receptor (A3 R) belongs to a family of four adenosine receptor (AR) subtypes which all play distinct roles throughout the body. A3 R antagonists have been described as potential treatments for numerous diseases including asthma. Given the similarity between (adenosine receptors) orthosteric binding sites, obtaining highly selective antagonists is a challenging but critical task. Here we screen 39 potential A3 R, antagonists using agonist-induced inhibition of cAMP. Positive hits were assessed for AR subtype selectivity through cAMP accumulation assays. The antagonist affinity was determined using Schild analysis (pA2 values) and fluorescent ligand binding. Structure–activity relationship investigations revealed that loss of the 3-(dichlorophenyl)-isoxazolyl moiety or the aromatic nitrogen heterocycle with nitrogen at α-position to the carbon of carboximidamide group significantly attenuated K18 antagonistic potency. Mutagenic studies supported by molecular dynamic simulations combined with Molecular Mechanics—Poisson Boltzmann Surface Area calculations identified the residues important for binding in the A3 R orthosteric site. We demonstrate that K18, which contains a 3-(dichlorophenyl)-isoxazole group connected through carbonyloxycarboximidamide fragment with a 1,3-thiazole ring, is a specific A3 R (< 1 µM) competitive antagonist. Finally, we introduce a model that enables estimates of the equilibrium binding affinity for rapidly disassociating compounds from real-time fluorescent ligand-binding studies. These results demonstrate the pharmacological characterisation of a selective competitive A3 R antagonist and the description of its orthosteric binding mode. Our findings may provide new insights for drug discovery. [ABSTRACT FROM AUTHOR]- Published
- 2020
- Full Text
- View/download PDF
168. BCL11A interacts with SOX2 to control the expression of epigenetic regulators in lung squamous carcinoma
- Author
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Lazarus, Kyren A, Hadi, Fazal, Zambon, Elisabetta, Bach, Karsten, Santolla, Maria-Francesca, Watson, Julie K, Correia, Lucia L, Das, Madhumita, Ugur, Rosemary, Pensa, Sara, Becker, Lukas, Campos, Lia S, Ladds, Graham, Liu, Pentao, Evan, Gerard I, McCaughan, Frank M, Le Quesne, John, Lee, Joo-Hyeon, Calado, Dinis, and Khaled, Walid T
- Subjects
Lung Neoplasms ,SOXB1 Transcription Factors ,Nuclear Proteins ,Histone-Lysine N-Methyltransferase ,Oncogenes ,3. Good health ,Epigenesis, Genetic ,Gene Expression Regulation, Neoplastic ,Organoids ,Repressor Proteins ,Mice ,Genetic Loci ,Cell Line, Tumor ,Gene Knockdown Techniques ,Carcinoma, Squamous Cell ,Animals ,Humans ,Carrier Proteins ,Lung ,Cell Proliferation ,Protein Binding - Abstract
Patients diagnosed with lung squamous cell carcinoma (LUSC) have limited targeted therapies. We report here the identification and characterisation of BCL11A, as a LUSC oncogene. Analysis of cancer genomics datasets revealed BCL11A to be upregulated in LUSC but not in lung adenocarcinoma (LUAD). Experimentally we demonstrate that non-physiological levels of BCL11A in vitro and in vivo promote squamous-like phenotypes, while its knockdown abolishes xenograft tumour formation. At the molecular level we found that BCL11A is transcriptionally regulated by SOX2 and is required for its oncogenic functions. Furthermore, we show that BCL11A and SOX2 regulate the expression of several transcription factors, including SETD8. We demonstrate that shRNA-mediated or pharmacological inhibition of SETD8 selectively inhibits LUSC growth. Collectively, our study indicates that BCL11A is integral to LUSC pathology and highlights the disruption of the BCL11A-SOX2 transcriptional programme as a novel candidate for drug development.
169. Additional file 1 of Structure‐based identification of dual ligands at the A2AR and PDE10A with anti‐proliferative effects in lung cancer cell‐lines
- Author
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Kalash, Leen, Winfield, Ian, Safitri, Dewi, Bermudez, Marcel, Carvalho, Sabrina, Glen, Robert, Ladds, Graham, and Bender, Andreas
- Subjects
Data_FILES ,3. Good health - Abstract
Additional file 1. Discussion. Additional figures and tables
170. Transcriptomics-Based Phenotypic Screening Supports Drug Discovery in Human Glioblastoma Cells
- Author
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Shapovalov, Vladimir, Kopanitsa, Liliya, Pruteanu, Lavinia-Lorena, Ladds, Graham, and Bailey, David S.
- Subjects
Regorafenib ,tumor antigens ,Mardepodect ,drug targets ,glioblastoma ,drug-inducible gene expression ,3. Good health - Abstract
We have used three established human glioblastoma (GBM) cell lines—U87MG, A172, and T98G—as cellular systems to examine the plasticity of the drug-induced GBM cell phenotype, focusing on two clinical drugs, the phosphodiesterase PDE10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib, using genome-wide drug-induced gene expression (DIGEX) to examine the drug response. Both drugs upregulate genes encoding specific growth factors, transcription factors, cellular signaling molecules, and cell surface proteins, while downregulating a broad range of targetable cell cycle and apoptosis-associated genes. A few upregulated genes encode therapeutic targets already addressed by FDA approved drugs, but the majority encode targets for which there are no approved drugs. Amongst the latter, we identify many novel druggable targets that could qualify for chemistry-led drug discovery campaigns. We also observe several highly upregulated transmembrane proteins suitable for combined drug, immunotherapy, and RNA vaccine approaches. DIGEX is a powerful way of visualizing the complex drug response networks emerging during GBM drug treatment, defining a phenotypic landscape which offers many new diagnostic and therapeutic opportunities. Nevertheless, the extreme heterogeneity we observe within drug-treated cells using this technique suggests that effective pan-GBM drug treatment will remain a significant challenge for many years to come.
171. Additional file 1 of Structure‐based identification of dual ligands at the A2AR and PDE10A with anti‐proliferative effects in lung cancer cell‐lines
- Author
-
Kalash, Leen, Winfield, Ian, Safitri, Dewi, Bermudez, Marcel, Carvalho, Sabrina, Glen, Robert, Ladds, Graham, and Bender, Andreas
- Subjects
Data_FILES ,3. Good health - Abstract
Additional file 1. Discussion. Additional figures and tables
172. MOESM1 of Computer-aided design of multi-target ligands at A1R, A2AR and PDE10A, key proteins in neurodegenerative diseases
- Author
-
Kalash, Leen, Val, Cristina, Jhonny Azuaje, Loza, María, Svensson, Fredrik, Azedine Zoufir, Mervin, Lewis, Ladds, Graham, Brea, José, Glen, Robert, Sotelo, Eddy, and Bender, Andreas
- Subjects
3. Good health - Abstract
Additional file 1. Supplementary data describing substructural analysis of extracted ChEMBL compounds, statistical analysis of enriched target prediction of RECAP compounds, separation in medians of active/inactive docking score distributions for the docking models, computed logP and tPSA values and selectivity profiling data for compounds 1–25, docking parameters used, scripts for compound extraction from the ChEMBL database, computation of Mann–Whitney test and F1 scores.
173. Transcriptomics-Based Phenotypic Screening Supports Drug Discovery in Human Glioblastoma Cells
- Author
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Shapovalov, Vladimir, Kopanitsa, Liliya, Pruteanu, Lavinia-Lorena, Ladds, Graham, and Bailey, David S
- Subjects
Regorafenib ,tumor antigens ,Mardepodect ,drug targets ,glioblastoma ,drug-inducible gene expression ,3. Good health - Abstract
We have used three established human glioblastoma (GBM) cell lines-U87MG, A172, and T98G-as cellular systems to examine the plasticity of the drug-induced GBM cell phenotype, focusing on two clinical drugs, the phosphodiesterase PDE10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib, using genome-wide drug-induced gene expression (DIGEX) to examine the drug response. Both drugs upregulate genes encoding specific growth factors, transcription factors, cellular signaling molecules, and cell surface proteins, while downregulating a broad range of targetable cell cycle and apoptosis-associated genes. A few upregulated genes encode therapeutic targets already addressed by FDA approved drugs, but the majority encode targets for which there are no approved drugs. Amongst the latter, we identify many novel druggable targets that could qualify for chemistry-led drug discovery campaigns. We also observe several highly upregulated transmembrane proteins suitable for combined drug, immunotherapy, and RNA vaccine approaches. DIGEX is a powerful way of visualizing the complex drug response networks emerging during GBM drug treatment, defining a phenotypic landscape which offers many new diagnostic and therapeutic opportunities. Nevertheless, the extreme heterogeneity we observe within drug-treated cells using this technique suggests that effective pan-GBM drug treatment will remain a significant challenge for many years to come.
174. Receptor component protein, an endogenous allosteric modulator of family B G protein coupled receptors
- Author
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Routledge, Sarah J, Simms, John, Clark, Ashley, Yeung, Ho Yan, Wigglesworth, Mark J, Dickerson, Ian M, Kitchen, Philip, Ladds, Graham, and Poyner, David R
- Subjects
Accessory protein ,Allosteric modulator ,Calcitonin Gene-Related Peptide ,Peptide Hormones ,Calcitonin Receptor-Like Protein ,Membrane Proteins ,Ligands ,3. Good health ,Receptor Activity-Modifying Protein 1 ,Receptors, G-Protein-Coupled ,Adrenomedullin ,HEK293 Cells ,G protein coupling ,Cyclic AMP ,Animals ,Humans ,CGRP ,hormones, hormone substitutes, and hormone antagonists ,GPCR signalling ,Adaptor Proteins, Signal Transducing ,Signal Transduction - Abstract
Receptor component protein (RCP) is a 148 amino acid intracellular peripheral membrane protein, previously identified as promoting the coupling of CGRP to cAMP production at the CGRP receptor, a heterodimer of calcitonin receptor like-receptor (CLR), a family B G protein-coupled receptor (GPCR) and receptor activity modifying protein 1 (RAMP1). We extend these observations to show that it selectively enhances CGRP receptor coupling to Gs but not Gq or pERK activation. At other family B GPCRs, it enhances cAMP production at the calcitonin, corticotrophin releasing factor type 1a and glucagon-like peptide type 2 receptors with their cognate ligands but not at the adrenomedullin type 1 (AM1), gastric inhibitory peptide and glucagon-like peptide type 1 receptors, all expressed in transfected HEK293S cells. However, there is also cell-line variability as RCP did not enhance cAMP production at the endogenous calcitonin receptor in HEK293T cells and it has previously been reported that it is active on the AM1 receptor expressed on NIH3T3 cells. RCP appears to behave as a positive allosteric modulator at coupling a number of family B GPCRs to Gs, albeit in a manner that is regulated by cell-specific factors. It may exert its effects at the interface between the 2nd intracellular loop of the GPCR and Gs, although there is likely to be some overlap between this location and that occupied by the C-terminus of RAMPs if they bind to the GPCRs.
175. MOESM1 of Computer-aided design of multi-target ligands at A1R, A2AR and PDE10A, key proteins in neurodegenerative diseases
- Author
-
Kalash, Leen, Val, Cristina, Jhonny Azuaje, Loza, María, Svensson, Fredrik, Azedine Zoufir, Mervin, Lewis, Ladds, Graham, Brea, José, Glen, Robert, Sotelo, Eddy, and Bender, Andreas
- Subjects
3. Good health - Abstract
Additional file 1. Supplementary data describing substructural analysis of extracted ChEMBL compounds, statistical analysis of enriched target prediction of RECAP compounds, separation in medians of active/inactive docking score distributions for the docking models, computed logP and tPSA values and selectivity profiling data for compounds 1–25, docking parameters used, scripts for compound extraction from the ChEMBL database, computation of Mann–Whitney test and F1 scores.
176. Transcriptomics-Based Phenotypic Screening Supports Drug Discovery in Human Glioblastoma Cells
- Author
-
Shapovalov, Vladimir, Kopanitsa, Liliya, Pruteanu, Lavinia-Lorena, Ladds, Graham, and Bailey, David S
- Subjects
Regorafenib ,Mardepodect ,drug targets ,Drug-inducible Gene Expression ,Glioblastoma ,Tumor antigens ,3. Good health - Abstract
We have used three established human glioblastoma (GBM) cell lines-U87MG, A172, and T98G-as cellular systems to examine the plasticity of the drug-induced GBM cell phenotype, focusing on two clinical drugs, the phosphodiesterase PDE10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib, using genome-wide drug-induced gene expression (DIGEX) to examine the drug response. Both drugs upregulate genes encoding specific growth factors, transcription factors, cellular signaling molecules, and cell surface proteins, while downregulating a broad range of targetable cell cycle and apoptosis-associated genes. A few upregulated genes encode therapeutic targets already addressed by FDA approved drugs, but the majority encode targets for which there are no approved drugs. Amongst the latter, we identify many novel druggable targets that could qualify for chemistry-led drug discovery campaigns. We also observe several highly upregulated transmembrane proteins suitable for combined drug, immunotherapy, and RNA vaccine approaches. DIGEX is a powerful way of visualizing the complex drug response networks emerging during GBM drug treatment, defining a phenotypic landscape which offers many new diagnostic and therapeutic opportunities. Nevertheless, the extreme heterogeneity we observe within drug-treated cells using this technique suggests that effective pan-GBM drug treatment will remain a significant challenge for many years to come.
177. Endothelial Differentiation G Protein-Coupled Receptor 5 Plays an Important Role in Induction and Maintenance of Pluripotency
- Author
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Neganova, Irina, Cotts, Lewis, Banks, Peter, Gassner, Katja, Shukurov, Anvar, Armstrong, Lyle, Ladds, Graham, and Lako, Majlinda
- Subjects
Human somatic cell reprogramming ,EDG5 ,Human Embryonic Stem Cells ,Induced Pluripotent Stem Cells ,Down-Regulation ,Genome-wide RNAi screen ,Cellular Reprogramming ,hESCs/hiPSCs ,3. Good health ,Cell Line ,GPCR ,Humans ,Sphingosine-1-Phosphate Receptors ,Cytoskeleton ,Signal Transduction - Abstract
Direct reprogramming of human somatic cells toward induced pluripotent stem cells holds great promise for regenerative medicine and basic biology. We used a high-throughput small interfering RNA screening assay in the initiation phase of reprogramming for 784 genes belonging to kinase and phosphatase families and identified 68 repressors and 22 effectors. Six new candidates belonging to the family of the G protein-coupled receptors (GPCRs) were identified, suggesting an important role for this key signaling pathway during somatic cell-induced reprogramming. Downregulation of one of the key GPCR effectors, endothelial differentiation GPCR5 (EDG5), impacted the maintenance of pluripotency, actin cytoskeleton organization, colony integrity, and focal adhesions in human embryonic stem cells, which were associated with the alteration in the RhoA-ROCK-Cofilin-PAXILLIN-actin signaling pathway. Similarly, downregulation of EDG5 during the initiation stage of somatic cell-induced reprogramming resulted in alteration of cytoskeleton, loss of human-induced pluripotent stem cell colony integrity, and a significant reduction in partially and fully reprogrammed cells as well as the number of alkaline phosphatase positive colonies at the end of the reprogramming process. Together, these data point to an important role of EDG5 in the maintenance and acquisition of pluripotency. Stem Cells 2019;37:318-331.
178. Investigating RGS proteins in yeast
- Author
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Hill, Claire, Goddard, Alan, Davey, John, Ladds, Graham, Hill, Claire, Goddard, Alan, Davey, John, and Ladds, Graham
- Abstract
Regulator of G protein signalling (RGS) proteins are vital in the adaptation of cells to stimulation via G protein-coupled receptors. Yeast have been integral in elucidating the important role that RGS proteins play within cellular processes. In addition to extensive characterisation of the endogenous RGS proteins, these organisms have enabled the identification and analysis of numerous mammalian homologues. The simplicity and plasticity of the yeast pheromone-response pathway has facilitated studies which would have been impossible in mammalian systems and it is certain that yeast will continue to have a great impact on this field of research in the future.
179. Functional analysis of heterologous GPCR signalling pathways in yeast
- Author
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Ladds, Graham, Goddard, Alan, Davey, John, Ladds, Graham, Goddard, Alan, and Davey, John
- Abstract
G protein-coupled receptors (GPCRs) regulate diverse biological processes in eukaryotes and such conservation allows an almost unrestricted interchange of signalling components between different cell types. Yeasts are attractive hosts in which to study GPCRs - they are amenable to both genetic and biochemical manipulation and their robustness, low cost and our ability to create strains that lack endogenous GPCRs make them ideal starting points for the development of assays suitable for high-throughput screening. Here we introduce readers to the possibilities of using yeast to analyse GPCRs describing the endogenous signalling pathways, the development of assays for heterologous GPCRs and the technology to elucidate GPCR structure and activity, focusing on the budding yeast Saccharomyces cerevisiae and recent developments using the fission yeast Schizosaccharomyces pombe.
180. BCL11A interacts with SOX2 to control the expression of epigenetic regulators in lung squamous carcinoma.
- Author
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Lazarus, Kyren A., Hadi, Fazal, Zambon, Elisabetta, Bach, Karsten, Santolla, Maria-Francesca, Watson, Julie K., Correia, Lucia L., Das, Madhumita, Ugur, Rosemary, Pensa, Sara, Becker, Lukas, Campos, Lia S., Ladds, Graham, Liu, Pentao, Evan, Gerard I., McCaughan, Frank M., Le Quesne, John, Lee, Joo-Hyeon, Calado, Dinis, and Khaled, Walid T.
- Abstract
Patients diagnosed with lung squamous cell carcinoma (LUSC) have limited targeted therapies. We report here the identification and characterisation of BCL11A, as a LUSC oncogene. Analysis of cancer genomics datasets revealed BCL11A to be upregulated in LUSC but not in lung adenocarcinoma (LUAD). Experimentally we demonstrate that non-physiological levels of BCL11A in vitro and in vivo promote squamous-like phenotypes, while its knockdown abolishes xenograft tumour formation. At the molecular level we found that BCL11A is transcriptionally regulated by SOX2 and is required for its oncogenic functions. Furthermore, we show that BCL11A and SOX2 regulate the expression of several transcription factors, including SETD8. We demonstrate that shRNA-mediated or pharmacological inhibition of SETD8 selectively inhibits LUSC growth. Collectively, our study indicates that BCL11A is integral to LUSC pathology and highlights the disruption of the BCL11A-SOX2 transcriptional programme as a novel candidate for drug development. Amongst the non-small cell lung cancers, to date, lung squamous cell carcinoma remains the most challenging to treat. Here the authors report BCL11A as an important factor which together with SOX2 can drive lung squamous cell carcinoma development and highlight a potential novel therapeutic candidate for this devastating disease. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
181. Influence of peptide allosteric modulators on agonist bias at Class B1 G protein-coupled receptors
- Author
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Pearce, Abigail and Ladds, Graham
- Subjects
Agonist Bias ,Diabetes Mellitus ,G protein-Coupled Receptor ,Insulin - Abstract
Class B1 G Protein-Coupled Receptors (GPCRs) are a small family within the GPCR superfamily. However, they are implicated in the pathologies of some of the most prevalent diseases, such as type 2 diabetes and heart disease. Despite being such a small family, their signalling is very diverse; each receptor responds to multiple endogenous agonists and couples to different G proteins, displaying pleiotropy. There is added variation in receptor desensitisation and internalisation, with intracellular signalling and β-arrestin-mediated pathways adding spatial and temporal complexity. How this crosstalk regulates intracellular signalling was investigated at the Glucagon-Like Peptide-1 Receptor (GLP1R), a Class B1 GPCR with implications in glucose homeostasis. Its G protein-dependent signalling was measured, activating a wide range of G proteins, not confined to a certain subfamily. β-arrestin recruitment and internalisation were examined, with GLP1R undergoing rapid internalisation, with a complicated dependency on β-arrestins. Insulin secretion was also measured, and the role of receptor desensitisation examined in this downstream response. Reducing internalisation correlated with a reduction in insulin secretion. Genetic variation in Class B1 GPCRs can lead to differences in signalling. Single Nucleotide Polymorphisms (SNPs) resulting in missense mutations can directly alter agonist or G protein binding, or alter stabilisation of active and inactive receptor conformations through allosteric mechanisms. The consequences of SNPs in N-terminal and C-terminal regions of GLP1R, glucagon receptor (GCGR), secretin receptor (SCTR), and corticotropin-releasing factor receptor type 1 (CRF1) were therefore investigated. Whilst some effects were observed when GLP1R was mutated, in many cases these SNPs had little effect on signalling. However, mutation of a conserved residue, arginine3.30, was severely detrimental to GLP1R signalling. In addition to SNPs, large genetic variation is found in the form of splice isoforms. A GCGR splice isoform found in human cells was shown to express poorly, displaying little signalling. However, its co-expression altered signalling of the reference GCGR, reducing G protein signalling but increasing β-arrestin recruitment, showing how dimer formation alters agonist binding. In addition to internal variation, the expression of peptide modulators such as receptor activity-modifying proteins (RAMPs) can greatly influence Class B1 GPCR pharmacology. The effect of RAMP3 expression on GLP1R signalling and desensitisation was measured, with increases in Gαq coupling and intracellular Ca2+ (Ca2+)i mobilisation mediated by GLP1R transiently expressed in HEK293T cells. (Ca2+)I mobilisation was also increased by RAMP3 overexpression in INS-1 832/3 cells, which endogenously express the receptor. Increased Gαq/11 signalling increased insulin secretion in response to GLP1. The calcitonin-like receptor (CLR) is known to interact with all three RAMPs to generate functionally distinct receptors. Biased G protein-mediated signalling of CLR has been well-studied, but the role of RAMPs in CLR desensitisation and internalisation has been relatively unexamined. A global characterisation of CLR-RAMP internalisation in response to the three primary endogenous agonists was therefore achieved. The mechanism of internalisation was elucidated, and its role in cAMP signalling tested. GPCR-kinases (GRKs) are important in GPCR β-arrestin recruitment and subsequent internalisation. Attempts to identify which GRKs are responsible for phosphorylation of CLR instead identified constitutive phosphorylation and internalisation of the receptor. This study includes several different allosteric means to regulate Class B1 GPCR signalling. Mutation of residues outside the orthosteric binding site can change G protein coupling, even without interference of the interacting residues. However, more common peptide allosteric modulators are those co-expressed with the receptor, such as RAMPs and GRKs.
- Published
- 2022
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182. Drug discovery at class A and class B GPCRs
- Author
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Hilšer, Anna and Ladds, Graham
- Subjects
GPCR ,pharmacology ,diabetes ,obesity ,allosteric modulators ,AR agonists ,AR antagonists ,GIPR - Abstract
G protein-coupled receptors (GPCRs) are a big family of membrane receptors encoded by more than 800 genes in humans. The vast number and diversity of GPCRs enables them to interact with an equally great number of ligands enabling them to regulate many physiological functions such as senses, metabolism, neurotransmission or cell growth. Given GPCRs' involvement in the regulation of many physiological functions, it then comes as no surprise that their malfunction often leads to pathological states such as cancer, diabetes mellitus, inflammation or central nervous system disorders. This makes GPCRs the focus of drug discovery with roughly 34% of all FDA (Food and drug administration) approved drugs targeting them. This thesis presents the drug discovery at adenosine receptors, class A GPCRs, and gastric inhibitory polypeptide receptor (GIPR), a class B GPCR. Given the possible therapeutic effects of modulating GIPR signalling pathway in diabetes and obesity, the primary objective of this thesis was to discover and improve GIPR allosteric modulators using both in silico and in vitro techniques. This resulted in successful identification of potent and selective GIPR negative allosteric modulators like compound C25, while also investigating the bias of the compounds at different pathways and their selectivity. Combinational approach of in silico blind docking and in vitro mutagenesis was then used to successfully identify the GIPR allosteric binding site of the compounds located around at the top of transmembrane domain 2/3 and extracellular loop 1. The second part of this thesis is then focused on drug discovery at adenosine receptors with the aim of developing more selective and more potent compounds. Firstly, compounds were screened for more potent adenosine 1 agonists that would retain or improve upon BnOCPA compound, which is a powerful analgesic lacking the common side effects. This was successfully achieved and some really potent and selective adenosine 1 agonists like compound 27 were identified. Secondly, potent adenosine 1 and adenosine 3 antagonists were discovered, and their potency, selectivity and binding were measured. This led to the identification of several potent dual adenosine 1 and 3 antagonists like compounds A17 and A47, which hold potential in the treatment of asthma, lowering intraocular pressure or in several central nervous system disorders. Ultimately, these findings show how a combinational approach of in silico and in vitro pharmacology can be successfully used to identify new small molecule GPCR allosteric modulators and identify new potent adenosine receptor agonists and antagonists with potential therapeutic benefits.
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- 2022
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183. Modulation of cyclic adenosine monophosphate (cAMP) signalling and its therapeutic potential : pharmacological characterisation of PDE inhibitors
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Safitri, Dewi and Ladds, Graham
- Subjects
cAMP ,phosphodiesterase ,glioblastoma ,GPCR ,proliferation - Abstract
The alteration of intracellular cyclic adenosine monophosphate (cAMP) levels plays important regulatory roles in both physiological and pathological conditions, such as cancer. As the most aggressive form of brain tumour, glioblastoma is currently incurable due to limited treatment modalities. Low level of intracellular cAMP levels has been reported to be a feature of brain tumours. Thus, it is hypothesised that increasing cAMP concentrations by targeting regulatory proteins involved in the cAMP signalling pathways may offer advantages in preventing or treating glioblastoma. The overarching goal of this study, therefore, is to determine the dynamic effects of cAMP modulation on glioblastoma cell proliferation. The efficacies of compounds targeting various proteins involved in cAMP pathways were first investigated. The mechanisms explored were elevation of cAMP level through phosphodiesterases (PDEs) inhibition, adenylyl cyclase (AC) activation, as well as modulation via β-adrenoceptor (β-AR) and G proteins. A series of compounds were evaluated applying various assaying techniques and pharmacological tools using cAMP accumulation assays, cell proliferation, caspase-3/7 activation, and flow cytometry to determine cell cycle. It was demonstrated that increasing cAMP levels by multiple PDE inhibition or AC stimulation resulted in cell growth suppression on both rat and human glioblastoma models. The study was also extended to identify the role of possible crosstalk between calcium through SOCE (store-operated calcium entry) and cAMP pathways, which both were found to contribute to cell growth modulation. The effect of the elevation of intracellular cAMP on cell proliferation was further explored through the direct activation of adenosine A2A receptor (A2AR) and inhibition of cAMP degradation via PDE10A. Previous computational studies revealed that the triazoloquinazoline-based compounds (compound 1-6), initially known as PDE10A inhibitors, are bound at the orthosteric site of A2AR. To validate the computational results, these compounds were characterised using NanoBRET-based ligand binding studies with HEK293T expressing Nluc-A2AR and functional assays in lung cancer cell lines and glioma/glioblastoma cell models, which both cell models expressed endogenous levels of PDE10A and A2AR. The study highlighted that compounds 1 and 5 were dual-target ligands to A2AR and PDE10A, whereas compound 3 appeared to be a pan-agonist of adenosine receptors (ARs), and compound 4 was more potent when A2BR was expressed. Compound 2 seems to possess toxic effects that may be independent of action to A2AR or PDE10A. Lastly, preliminary studies were conducted to investigate the possibility of biased signalling by RAMPs on protease-activated receptor 4 (PAR4) and calcitonin-like receptor (CLR). Using PAR4 transiently transfected HEK293T cells, both cognate ligand and agonist peptide were used to profile PAR4 signalling including RAMPs-trafficking to the plasma membrane, promoting intracellular calcium release and recruiting β-arrestins. The effects of RAMPs were also investigated in HUVECs and cardiomyocytes focusing on the effect of endogenous ligands on cell growth. Whilst RAMPs altered PAR4 initial signalling events in promoting β-arrestin recruitment, the study on heterodimer complex of RAMPs and CLR on cell growth further corroborated that signalling bias can be translated into physiological responses in HUVECs and cardiomyocytes. To conclude, these studies provided evidence on how the alterations of intracellular cAMP levels affected cell proliferation in numerous cancer models, and that the cAMPmediated anti-proliferative effect was cell-line dependent. Targeting multiple PDEs suppressed cell growth in cancer-derived cells, therefore providing a viable target to reduce tumour progression. Given the critical role of PAR4 in platelet aggregation and pro-proliferative of calcitonin peptide family, this research may have important implications for the role of RAMPs in cardiovascular pathologies.
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- 2021
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184. CGRP family of G protein-coupled receptors and their signalling in human cardiovascular cells
- Author
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Clark, Ashley and Ladds, Graham
- Subjects
615.1 ,doctor of philosophy ,pharmacology - Abstract
The aim of this body of work has been to further understanding of the signalling and functional properties of a unique family of cell surface receptors known as G protein-coupled receptors (GPCRs), and one of their subclasses: The CGRP receptor family. These receptors are crucial for transducing information from the extracellular to intracellular space. While much pharmacological research has gone into understanding the signalling and function of GPCRs in recombinant systems, there is a very little knowledge of these receptors in their native environment and at endogenous expression levels. Nor is there direct evidence for potentially controversial phenomena such as signalling bias in endogenous cells. This provided the impetus to address the need for a better understanding of the pharmacology of endogenous human calcitonin receptor-like receptor (CLR) in its native cellular environment. Therefore, primary human cardiovascular cells, gene editing techniques, and a host of intracellular assays were used to study the signalling properties of this GPCR family, revolving around the CLR, to attempt to uncover how these receptors function endogenously. Through the research presented here, it is shown that the CLR, when stimulated by endogenous agonists activates a whole host of signalling pathways to bring about differing physiological effects. In doing so it has revealed unique roles for calcitonin-gene related peptide (CGRP), adrenomedullin (AM), and the little-understood peptide adrenomedullin 2 (AM2). All of which are dependent on the presence of a group of GPCR accessory proteins known as receptor activity-modifying proteins (RAMPs). These proteins are not only crucial for CLR function, but this dissertation demonstrates the remarkable way in which they govern and dictate the intracellular signalling of the CGRP family of peptides endogenously. Beyond this, the G protein and accessory protein involvement in certain signalling cascades, the spatiotemporal aspects to CGRP peptide signalling, and the functional outcomes of signalling in cell organoid models are all explored. It is the author's belief that this work adds a great deal to the understanding of the CLR, and more generally takes a step forward in the understanding of endogenous GPCR signalling bias.
- Published
- 2020
185. Characterisation and pharmacological regulation of GLP-1-mediated glucose homeostasis
- Author
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Yeung, Ho Yan and Ladds, Graham
- Subjects
616.4 ,G protein-coupled receptors ,Incretins ,Drug discovery ,Type 2 diabetes mellitus ,GLP-1 - Abstract
Type 2 diabetes mellitus (T2DM) is characterised by the hormonal imbalance of insulin and glucagon, leading to dysfunctional glucose homeostasis. Glucagon-like peptide 1 (GLP-1), which is an incretin hormone, activates the predominantly Gαs-coupled glucagon-like peptide 1 receptor (GLP-1R), which is a class B G protein-coupled receptor (GPCR), to mediate glucose homeostasis. It does so by promoting glucose stimulated insulin secretion (GSIS) in the pancreatic β cells and inhibiting glucagon secretion in the pancreatic α cells. Given its proven clinical efficacy in reducing long term blood glucose level, GLP-1-based treatments, such as exenatide and liraglutide, have been widely used in T2DM patients. However, in contrast to the well-studied phenomenon of how GLP-1 enhances GSIS, the mechanism of how GLP-1 regulates glucagon secretion is still unclear. Therefore, the aim of this work is to shed new lights on how GLP-1 mediates its glucagonostatic action. To do so, the signalling properties of GLP-1 and its closely-related peptide hormones, namely oxyntomodulin (OXM), glucagon (GCG), glucose-dependent insulinotropic polypeptide (GIP), and its metabolite, GLP-1(9-36)NH2, were examined in recombinant cell lines and rodent clonal α and β cell lines using cAMP functional assaying technique. It was demonstrated that these glucagon-like peptides, including GLP-1(9-36)NH2 yet except GIP, can activate both GLP-1R and glucagon receptor (GCGR), which is struc- turally analogous to GLP-1R. Furthermore, GLP-1R, despite its very low expression in the mouse αTC1.6 cell line detected through semi-quantitative RT-PCR studies, is found to play a critical role in directly inhibiting glucagon secretion upon GLP-1 activation through performing glucagon secretion antagonism studies. More importantly, the physiologically abundant GLP-1 metabolite is discovered to play a glucagonostatic role in the mouse glucagonoma cell line via the direct actions of GLP-1R and GCGR, an observation that has not yet been documented. Therefore, this thesis provides evidence of how GLP-1 and its metabolite are actively involved in their glucagonostatic actions via direct activations of GLP-1R and GCGR. Another aim of this work is to identify viable pharmacological regulator of GLP-1- mediated glucose homeostasis through the action of positive allosteric modulator (PAM). Here, compound 249, which was identified previously as a small molecule GLP-1R PAM, was further pharmacologically validated using various signal transduction assaying techniques in recombinant cell lines. It was also demonstrated that compound 249 works independent of the cysteine-347 residue on the GLP-1R, an amino acid residue which has been previously shown to be instrumental for the actions of another GLP-1R agonist-PAMs. More importantly, compound 249 demonstrates robust potentiation of GLP-1 and OXM-augmented GSIS in the rat INS-1 832/3 insulinoma cell line and ex vivo isolated mouse islets, substantiating the potential of compound 249 to be further developed as a novel T2DM treatment. Overall this thesis presents new evidence on the direct involvement of GLP-1R on GLP-1-regulated glucagon secretion in the pancreatic α cells and illustrates compound 249 as a PAM to promote GLP-1 mediated GSIS. The findings in this thesis will be used for future design of safer and more efficacious T2DM treatments.
- Published
- 2020
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186. Receptor activity-modifying protein and small molecule modulation of the gastric inhibitory polypeptide receptor
- Author
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Harris, Matthew and Ladds, Graham
- Subjects
572 ,GIPR ,Pharmacology ,Receptor activity-modifying proteins ,Allosteric modulators - Published
- 2020
- Full Text
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187. Adaptation with the fission yeast Schizosaccharomyces pombe : the characterisation of the secreted serine carboxypeptidase Sxa2
- Author
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Ladds, Graham Robert
- Subjects
- 572.8, Pheromone
- Published
- 1998
188. Parameter identification problems in the modelling of cell motility.
- Author
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Croft, Wayne, Elliott, Charles, Ladds, Graham, Stinner, Björn, Venkataraman, Chandrasekhar, and Weston, Cathryn
- Subjects
- *
PARAMETER identification , *CELL motility , *COMPUTER algorithms , *LEAST squares , *REACTION-diffusion equations , *MATHEMATICAL models - Abstract
We present a novel parameter identification algorithm for the estimation of parameters in models of cell motility using imaging data of migrating cells. Two alternative formulations of the objective functional that measures the difference between the computed and observed data are proposed and the parameter identification problem is formulated as a minimisation problem of nonlinear least squares type. A Levenberg-Marquardt based optimisation method is applied to the solution of the minimisation problem and the details of the implementation are discussed. A number of numerical experiments are presented which illustrate the robustness of the algorithm to parameter identification in the presence of large deformations and noisy data and parameter identification in three dimensional models of cell motility. An application to experimental data is also presented in which we seek to identify parameters in a model for the monopolar growth of fission yeast cells using experimental imaging data. Our numerical tests allow us to compare the method with the two different formulations of the objective functional and we conclude that the results with both objective functionals seem to agree. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
189. Emerging roles of adhesion G protein-coupled receptors.
- Author
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Rosa, Matthew, Noel, Timothy, Harris, Matthew, and Ladds, Graham
- Subjects
- *
G protein coupled receptors , *EXTRACELLULAR matrix proteins , *LIGAND binding (Biochemistry) , *BINDING sites , *LIGANDS (Biochemistry) - Abstract
Adhesion G protein-coupled receptors (aGPCRs) form a sub-group within the GPCR superfamily. Their distinctive structure contains an abnormally large N-terminal, extracellular region with a GPCR autoproteolysis-inducing (GAIN) domain. In most aGPCRs, the GAIN domain constitutively cleaves the receptor into two fragments. This process is often required for aGPCR signalling. Over the last two decades, much research has focussed on aGPCR-ligand interactions, in an attempt to deorphanize the family. Most ligands have been found to bind to regions N-terminal to the GAIN domain. These receptors may bind a variety of ligands, ranging across membrane-bound proteins and extracellular matrix components. Recent advancements have revealed a conserved method of aGPCR activation involving a tethered ligand within the GAIN domain. Evidence for this comes from increased activity in receptor mutants exposing the tethered ligand. As a result, G protein-coupling partners of aGPCRs have been more extensively characterised, making use of their tethered ligand to create constitutively active mutants. This has led to demonstrations of aGPCR function in, for example, neurodevelopment and tumour growth. However, questions remain around the ligands that may bind many aGPCRs, how this binding is translated into changes in the GAIN domain, and the exact mechanism of aGPCR activation following GAIN domain conformational changes. This review aims to examine the current knowledge around aGPCR activation, including ligand binding sites, the mechanism of GAIN domain-mediated receptor activation and how aGPCR transmembrane domains may relate to activation. Other aspects of aGPCR signalling will be touched upon, such as downstream effectors and physiological roles. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
190. CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells.
- Author
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Clark, Ashley J., Mullooly, Niamh, Safitri, Dewi, Harris, Matthew, de Vries, Tessa, MaassenVanDenBrink, Antoinette, Poyner, David R., Gianni, Davide, Wigglesworth, Mark, and Ladds, Graham
- Subjects
- *
ADRENOMEDULLIN , *G protein coupled receptors , *GENOME editing , *NITRIC oxide , *CELLULAR signal transduction - Abstract
Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can "re-route" the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling. Clark et al. explore the ability of ligands to activate the calcitonin-like receptor (CLR) in primary endothelial cells, and the influence of co-expressed receptor-activity modifying proteins (RAMPs). Their study reveals that GPCR agonist bias occurs naturally in human cells and plays a fundamental role in providing unique functions to endogenous agonists. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
191. Structure‐based identification of dual ligands at the A2AR and PDE10A with anti‐proliferative effects in lung cancer cell‐lines.
- Author
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Kalash, Leen, Winfield, Ian, Safitri, Dewi, Bermudez, Marcel, Carvalho, Sabrina, Glen, Robert, Ladds, Graham, and Bender, Andreas
- Subjects
- *
LUNG cancer , *LIGANDS (Chemistry) , *BINDING site assay , *NON-small-cell lung carcinoma , *VISUAL fields - Abstract
Enhanced/prolonged cAMP signalling has been suggested as a suppressor of cancer proliferation. Interestingly, two key modulators that elevate cAMP, the A2A receptor (A2AR) and phosphodiesterase 10A (PDE10A), are differentially co-expressed in various types of non-small lung cancer (NSCLC) cell-lines. Thus, finding dual-target compounds, which are simultaneously agonists at the A2AR whilst also inhibiting PDE10A, could be a novel anti-proliferative approach. Using ligand- and structure-based modelling combined with MD simulations (which identified Val84 displacement as a novel conformational descriptor of A2AR activation), a series of known PDE10A inhibitors were shown to dock to the orthosteric site of the A2AR. Subsequent in-vitro analysis confirmed that these compounds bind to the A2AR and exhibit dual-activity at both the A2AR and PDE10A. Furthermore, many of the compounds exhibited promising anti-proliferative effects upon NSCLC cell-lines, which directly correlated with the expression of both PDE10A and the A2AR. Thus, we propose a structure-based methodology, which has been validated in in-vitro binding and functional assays, and demonstrated a promising therapeutic value. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
192. RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis.
- Author
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Mackie, Duncan I., Nielsen, Natalie R., Harris, Matthew, Singh, Smriti, Davis, Reema B., Dy, Danica, Ladds, Graham, and Caron, Kathleen M.
- Subjects
- *
MOLECULAR chaperones , *CHEMOKINE receptors , *DELOCALIZATION energy , *MOLECULAR interactions , *CELL migration - Abstract
Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cellmigration. Specifically, RAMP3 associationwith atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
193. The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activation
- Author
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Ian Winfield, Kerry Barkan, Sarah Routledge, Nathan J. Robertson, Matthew Harris, Ali Jazayeri, John Simms, Christopher A. Reynolds, David R. Poyner, Graham Ladds, Harris, Matthew [0000-0002-7918-5735], Ladds, Graham [0000-0001-7320-9612], and Apollo - University of Cambridge Repository
- Subjects
CLR ,MAP Kinase Signaling System ,Endocrinology, Diabetes and Metabolism ,Amino Acid Motifs ,Calcitonin Receptor-Like Protein ,RAMPs ,Molecular Dynamics Simulation ,RC648-665 ,Receptors, Corticotropin-Releasing Hormone ,Diseases of the endocrine glands. Clinical endocrinology ,GPCRs ,Protein Structure, Tertiary ,Receptor Activity-Modifying Protein 1 ,Receptors, G-Protein-Coupled ,Endocrinology ,HEK293 Cells ,Protein Domains ,signaling bias ,Cyclic AMP ,Receptors, Glucagon ,Humans ,Calcium Signaling ,mutagenesis ,Original Research ,Receptors, Calcitonin Gene-Related Peptide - Abstract
The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit.
- Published
- 2022
194. RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis
- Author
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Matthew Harris, Duncan I. Mackie, Reema B. Davis, Kathleen M. Caron, Graham Ladds, Smriti Singh, Danica Dy, Natalie R Nielsen, Harris, Matthew [0000-0002-7918-5735], Ladds, Graham [0000-0001-7320-9612], and Apollo - University of Cambridge Repository
- Subjects
Bioluminescence Resonance Energy Transfer Techniques ,Receptors, CCR3 ,Allosteric regulation ,Neovascularization, Physiologic ,chemokine receptors ,Context (language use) ,Receptor Activity-Modifying Protein 3 ,Chemokine receptor ,Cell Movement ,Humans ,Receptor ,G protein-coupled receptor ,Receptors, CXCR ,Multidisciplinary ,Receptor activity-modifying protein ,G-protein–coupled receptors ,Chemistry ,guided cell migration ,Chemotaxis ,Biological Sciences ,endosomal sorting ,Cell biology ,HEK293 Cells ,Signal transduction ,receptor-activity–modifying proteins ,Lysosomes ,Signal Transduction - Abstract
Receptor-activity-modifying proteins (RAMPs) are single transmembrane-spanning proteins which serve as molecular chaperones and allosteric modulators of G-protein-coupled receptors (GPCRs) and their signaling pathways. Although RAMPs have been previously studied in the context of their effects on Family B GPCRs, the coevolution of RAMPs with many GPCR families suggests an expanded repertoire of potential interactions. Using bioluminescence resonance energy transfer-based and cell-surface expression approaches, we comprehensively screen for RAMP interactions within the chemokine receptor family and identify robust interactions between RAMPs and nearly all chemokine receptors. Most notably, we identify robust RAMP interaction with atypical chemokine receptors (ACKRs), which function to establish chemotactic gradients for directed cell migration. Specifically, RAMP3 association with atypical chemokine receptor 3 (ACKR3) diminishes adrenomedullin (AM) ligand availability without changing G-protein coupling. Instead, RAMP3 is required for the rapid recycling of ACKR3 to the plasma membrane through Rab4-positive vesicles following either AM or SDF-1/CXCL12 binding, thereby enabling formation of dynamic spatiotemporal chemotactic gradients. Consequently, genetic deletion of either ACKR3 or RAMP3 in mice abolishes directed cell migration of retinal angiogenesis. Thus, RAMP association with chemokine receptor family members represents a molecular interaction to control receptor signaling and trafficking properties.
- Published
- 2019
195. Suppression of Proliferation of Human Glioblastoma Cells by Combined Phosphodiesterase and Multidrug Resistance-Associated Protein 1 Inhibition
- Author
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Dewi Safitri, David S. Bailey, Graham Ladds, Maksym V. Kopanitsa, Liliya Kopanitsa, Kopanitsa, Liliya [0000-0002-3752-2887], Ladds, Graham [0000-0001-7320-9612], Bailey, David S [0000-0002-9343-3408], Apollo - University of Cambridge Repository, and Bailey, David S. [0000-0002-9343-3408]
- Subjects
Combination therapy ,QH301-705.5 ,Cell Survival ,Phosphodiesterase Inhibitors ,proliferation ,Phosphodiesterase 3 ,multidrug resistance-associated protein 1 ,drug combination ,Antineoplastic Agents ,Catalysis ,Article ,Inorganic Chemistry ,chemistry.chemical_compound ,Cell Line, Tumor ,Cyclic AMP ,Humans ,Biology (General) ,Physical and Theoretical Chemistry ,Phosphodiesterase inhibitor ,QD1-999 ,Molecular Biology ,Cyclic GMP ,Spectroscopy ,Cell Proliferation ,Phosphoric Diester Hydrolases ,Organic Chemistry ,Trequinsin ,glioblastoma ,Phosphodiesterase ,Drug Synergism ,General Medicine ,Computer Science Applications ,Chemistry ,chemistry ,Toxicity ,Cancer research ,Quinolines ,Pyrazoles ,Multidrug Resistance-Associated Protein 1 ,PDE10A ,Multidrug Resistance-Associated Proteins ,phosphodiesterase inhibitor - Abstract
The paucity of currently available therapies for glioblastoma multiforme requires novel approaches to the treatment of this brain tumour. Disrupting cyclic nucleotide-signalling through phosphodiesterase (PDE) inhibition may be a promising way of suppressing glioblastoma growth. Here, we examined the effects of 28 PDE inhibitors, covering all the major PDE classes, on the proliferation of the human U87MG, A172 and T98G glioblastoma cells. The PDE10A inhibitors PF-2545920, PQ10 and papaverine, the PDE3/4 inhibitor trequinsin and the putative PDE5 inhibitor MY-5445 potently decreased glioblastoma cell proliferation. The synergistic suppression of glioblastoma cell proliferation was achieved by combining PF-2545920 and MY-5445. Furthermore, a co-incubation with drugs that block the activity of the multidrug resistance-associated protein 1 (MRP1) augmented these effects. In particular, a combination comprising the MRP1 inhibitor reversan, PF-2545920 and MY-5445, all at low micromolar concentrations, afforded nearly complete inhibition of glioblastoma cell growth. Thus, the potent suppression of glioblastoma cell viability may be achieved by combining MRP1 inhibitors with PDE inhibitors at a lower toxicity than that of the standard chemotherapeutic agents, thereby providing a new combination therapy for this challenging malignancy.
- Published
- 2021
- Full Text
- View/download PDF
196. Transcriptomics-Based Phenotypic Screening Supports Drug Discovery in Human Glioblastoma Cells
- Author
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Vladimir Shapovalov, Liliya Kopanitsa, David S. Bailey, Lavinia-Lorena Pruteanu, Graham Ladds, Kopanitsa, Liliya [0000-0002-3752-2887], Ladds, Graham [0000-0001-7320-9612], Bailey, David S [0000-0002-9343-3408], Apollo - University of Cambridge Repository, and Bailey, David S. [0000-0002-9343-3408]
- Subjects
0301 basic medicine ,Drug ,Cancer Research ,Phenotypic screening ,media_common.quotation_subject ,medicine.medical_treatment ,Mardepodect ,Druggability ,Computational biology ,Drug-inducible Gene Expression ,Tumor antigens ,Article ,Transcriptome ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Regorafenib ,drug targets ,medicine ,RC254-282 ,media_common ,Drug discovery ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Immunotherapy ,Cell cycle ,3. Good health ,030104 developmental biology ,Oncology ,chemistry ,030220 oncology & carcinogenesis ,Glioblastoma - Abstract
Simple Summary Glioblastoma (GBM) remains a particularly challenging cancer, with an aggressive phenotype and few promising treatment options. Future therapy will rely heavily on diagnosing and targeting aggressive GBM cellular phenotypes, both before and after drug treatment, as part of personalized therapy programs. Here, we use a genome-wide drug-induced gene expression (DIGEX) approach to define the cellular drug response phenotypes associated with two clinical drug candidates, the phosphodiesterase 10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib. We identify genes encoding specific drug targets, some of which we validate as effective antiproliferative agents and combination therapies in human GBM cell models, including HMGCoA reductase (HMGCR), salt-inducible kinase 1 (SIK1), bradykinin receptor subtype B2 (BDKRB2), and Janus kinase isoform 2 (JAK2). Individual, personalized treatments will be essential if we are to address and overcome the pharmacological plasticity that GBM exhibits, and DIGEX will play a central role in validating future drugs, diagnostics, and possibly vaccine candidates for this challenging cancer. Abstract We have used three established human glioblastoma (GBM) cell lines—U87MG, A172, and T98G—as cellular systems to examine the plasticity of the drug-induced GBM cell phenotype, focusing on two clinical drugs, the phosphodiesterase PDE10A inhibitor Mardepodect and the multi-kinase inhibitor Regorafenib, using genome-wide drug-induced gene expression (DIGEX) to examine the drug response. Both drugs upregulate genes encoding specific growth factors, transcription factors, cellular signaling molecules, and cell surface proteins, while downregulating a broad range of targetable cell cycle and apoptosis-associated genes. A few upregulated genes encode therapeutic targets already addressed by FDA approved drugs, but the majority encode targets for which there are no approved drugs. Amongst the latter, we identify many novel druggable targets that could qualify for chemistry-led drug discovery campaigns. We also observe several highly upregulated transmembrane proteins suitable for combined drug, immunotherapy, and RNA vaccine approaches. DIGEX is a powerful way of visualizing the complex drug response networks emerging during GBM drug treatment, defining a phenotypic landscape which offers many new diagnostic and therapeutic opportunities. Nevertheless, the extreme heterogeneity we observe within drug-treated cells using this technique suggests that effective pan-GBM drug treatment will remain a significant challenge for many years to come.
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- 2021
197. CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells
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Matthew Harris, Davide Gianni, David R. Poyner, Tessa de Vries, Niamh Mullooly, Antoinette MaassenVanDenBrink, Dewi Safitri, Ashley J. Clark, Graham Ladds, Mark Wigglesworth, Wigglesworth, Mark [0000-0002-6421-3414], Ladds, Graham [0000-0001-7320-9612], Apollo - University of Cambridge Repository, and Internal Medicine
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0301 basic medicine ,Agonist ,Calcitonin ,medicine.drug_class ,QH301-705.5 ,45/41 ,Calcitonin Gene-Related Peptide ,Peptide Hormones ,Medicine (miscellaneous) ,Endogeny ,13/106 ,Calcitonin gene-related peptide ,General Biochemistry, Genetics and Molecular Biology ,96/95 ,Receptors, G-Protein-Coupled ,03 medical and health sciences ,Adrenomedullin ,0302 clinical medicine ,Receptor pharmacology ,631/45/776/814 ,38/23 ,medicine ,Cyclic AMP ,Humans ,Biology (General) ,Receptors, Adrenomedullin ,Receptor ,Extracellular Signal-Regulated MAP Kinases ,Cells, Cultured ,G protein-coupled receptor ,Chemistry ,631/92/436/2387 ,Calcitonin Receptor-Like Protein ,article ,Endothelial Cells ,Cell biology ,030104 developmental biology ,13/95 ,General Agricultural and Biological Sciences ,030217 neurology & neurosurgery ,Endogenous agonist ,Receptors, Calcitonin Gene-Related Peptide - Abstract
Agonist bias occurs when different ligands produce distinct signalling outputs when acting at the same receptor. However, its physiological relevance is not always clear. Using primary human cells and gene editing techniques, we demonstrate endogenous agonist bias with physiological consequences for the calcitonin receptor-like receptor, CLR. By switching the receptor-activity modifying protein (RAMP) associated with CLR we can “re-route” the physiological pathways activated by endogenous agonists calcitonin gene-related peptide (CGRP), adrenomedullin (AM) and adrenomedullin 2 (AM2). AM2 promotes calcium-mediated nitric oxide signalling whereas CGRP and AM show pro-proliferative effects in cardiovascular cells, thus providing a rationale for the expression of the three peptides. CLR-based agonist bias occurs naturally in human cells and has a fundamental purpose for its existence. We anticipate this will be a starting point for more studies into RAMP function in native environments and their importance in endogenous GPCR signalling., Clark et al. explore the ability of ligands to activate the calcitonin-like receptor (CLR) in primary endothelial cells, and the influence of co-expressed receptor-activity modifying proteins (RAMPs). Their study reveals that GPCR agonist bias occurs naturally in human cells and plays a fundamental role in providing unique functions to endogenous agonists.
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- 2021
198. Accelerating cryoprotectant diffusion kinetics improves cryopreservation of pancreatic islets
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Fiona M. Gribble, Krishnaa T. Mahbubani, Michael P. Murphy, Graham Ladds, Kerry Barkan, Jakub Surmacki, John Gamble, Jackie A. Higgins, Frank Reimann, Kourosh Saeb-Parsy, John Casey, Till Moreth, Nikola Dolezalova, Simon Andrews, Anja V. Gruszczyk, Nigel K.H. Slater, Kevin O'Holleran, Francesco Pampaloni, Sam G Galvin, Apollo - University of Cambridge Repository, Galvin, Sam [0000-0001-5910-9471], O'Holleran, Kevin [0000-0003-1039-2127], Mahbubani, Krishnaa [0000-0002-1327-2334], Gribble, Fiona [0000-0002-4232-2898], Reimann, Frank [0000-0001-9399-6377], Andrews, Simon Richard [0000-0002-5006-3507], Murphy, Mike [0000-0003-1115-9618], Ladds, Graham [0000-0001-7320-9612], Slater, Nigel [0000-0002-0207-9440], and Saeb-Parsy, Kourosh [0000-0002-0633-3696]
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0301 basic medicine ,Male ,Islets of Langerhans Transplantation ,02 engineering and technology ,Cryopreservation ,chemistry.chemical_compound ,Mice ,Cryoprotective Agents ,692/699/2743/137 ,ComputingMilieux_COMPUTERSANDEDUCATION ,692/308/575 ,ComputingMilieux_MISCELLANEOUS ,Cells, Cultured ,Multidisciplinary ,geography.geographical_feature_category ,Chemistry ,Diabetes ,article ,Peptide secretion ,021001 nanoscience & nanotechnology ,Islet ,Cell biology ,medicine.anatomical_structure ,Models, Animal ,Medicine ,Permeation and transport ,0210 nano-technology ,Cryoprotectant ,Cell Survival ,Science ,Primary Cell Culture ,GeneralLiterature_MISCELLANEOUS ,Streptozocin ,Diabetes Mellitus, Experimental ,03 medical and health sciences ,Islets of Langerhans ,medicine ,Animals ,Humans ,geography ,Dimethyl sulfoxide ,Pancreatic islets ,Translational research ,Trehalose ,Transplantation ,692/163/2743/137 ,030104 developmental biology ,Diabetes Mellitus, Type 1 ,631/57/2283 - Abstract
Funder: W. D. Armstrong Fund (School of Technology, University of Cambridge), Cryopreservation offers the potential to increase the availability of pancreatic islets for treatment of diabetic patients. However, current protocols, which use dimethyl sulfoxide (DMSO), lead to poor cryosurvival of islets. We demonstrate that equilibration of mouse islets with small molecules in aqueous solutions can be accelerated from > 24 to 6 h by increasing incubation temperature to 37 °C. We utilize this finding to demonstrate that current viability staining protocols are inaccurate and to develop a novel cryopreservation method combining DMSO with trehalose pre-incubation to achieve improved cryosurvival. This protocol resulted in improved ATP/ADP ratios and peptide secretion from β-cells, preserved cAMP response, and a gene expression profile consistent with improved cryoprotection. Our findings have potential to increase the availability of islets for transplantation and to inform the design of cryopreservation protocols for other multicellular aggregates, including organoids and bioengineered tissues.
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- 2021
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199. Structure‐based identification of dual ligands at the A 2A R and PDE10A with anti‐proliferative effects in lung cancer cell‐lines
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Leen Kalash, Marcel Bermudez, Graham Ladds, Ian Winfield, Dewi Safitri, Sabrina Carvalho, Robert C. Glen, Andreas Bender, Kalash, Leen [0000-0002-1751-6658], Safitri, Dewi [0000-0003-2260-3892], Bermudez, Marcel [0000-0002-7421-3282], Glen, Robert [0000-0003-1759-2914], Ladds, Graham [0000-0001-7320-9612], and Apollo - University of Cambridge Repository
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0301 basic medicine ,Technology ,Chemistry, Multidisciplinary ,NSCLC ,law.invention ,Docking ,lcsh:Chemistry ,0302 clinical medicine ,law ,Receptor ,MD simulations ,Computer Science, Information Systems ,lcsh:T58.5-58.64 ,In Silico Structure Generation: Recent Developments, Applications, and Challenges ,Anti-proliferative ,Chemistry ,lcsh:Information technology ,A(2A)R ,Phosphodiesterase ,0303 Macromolecular and Materials Chemistry ,Computer Graphics and Computer-Aided Design ,Computer Science Applications ,030220 oncology & carcinogenesis ,Physical Sciences ,Computer Science, Interdisciplinary Applications ,Lung cancer ,500 Naturwissenschaften und Mathematik::540 Chemie::540 Chemie und zugeordnete Wissenschaften ,Research Article ,Virtual screening ,Anti‐proliferative ,Structure-based design ,Library and Information Sciences ,03 medical and health sciences ,Dual target ,medicine ,Physical and Theoretical Chemistry ,Science & Technology ,PDE10A ,Cancer ,medicine.disease ,Structure‐based design ,030104 developmental biology ,lcsh:QD1-999 ,Docking (molecular) ,Computer Science ,Cancer research ,Triazoloquinazolines ,Suppressor ,A2AR - Abstract
Enhanced/prolonged cAMP signalling has been suggested as a suppressor of cancer proliferation. Interestingly, two key modulators that elevate cAMP, the A2A receptor (A2AR) and phosphodiesterase 10A (PDE10A), are differentially co-expressed in various types of non-small lung cancer (NSCLC) cell-lines. Thus, finding dual-target compounds, which are simultaneously agonists at the A2AR whilst also inhibiting PDE10A, could be a novel anti-proliferative approach. Using ligand- and structure-based modelling combined with MD simulations (which identified Val84 displacement as a novel conformational descriptor of A2AR activation), a series of known PDE10A inhibitors were shown to dock to the orthosteric site of the A2AR. Subsequent in-vitro analysis confirmed that these compounds bind to the A2AR and exhibit dual-activity at both the A2AR and PDE10A. Furthermore, many of the compounds exhibited promising anti-proliferative effects upon NSCLC cell-lines, which directly correlated with the expression of both PDE10A and the A2AR. Thus, we propose a structure-based methodology, which has been validated in in-vitro binding and functional assays, and demonstrated a promising therapeutic value.
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- 2021
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200. Ras activation revisited: role of GEF and GAP systems.
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Hennig, Anne, Markwart, Robby, Esparza-Franco, Manuel A., Ladds, Graham, and Rubio, Ignacio
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G proteins , *GUANINE nucleotide exchange factors , *GTPASE-activating protein , *CELL growth , *CELL differentiation , *GUANOSINE diphosphate , *CHEMICAL equilibrium - Abstract
Ras is a prototypical small G-protein and a central regulator of growth, proliferation and differentiation processes in virtually every nucleated cell. As such, Ras becomes engaged and activated by multiple growth factors, mitogens, cytokines or adhesion receptors. Ras activation comes about by changes in the steady-state equilibrium between the inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states of Ras, resulting in the mostly transient accumulation of Ras-GTP. Three decades of intense Ras research have disclosed various families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) as the two principal regulatory elements of the Ras-GDP/GTP loading status. However, with the possible exception of the GEF Sos, we still have only a rudimentary knowledge of the precise role played by many GEF and GAP members in the signalling network upstream of Ras. As for GAPs, we even lack the fundamental understanding of whether they function as genuine signal transducers in the context of growth factor-elicited Ras activation or rather act as passive modulators of the Ras-GDP/GTP cycle. Here we sift through the large body of Ras literature and review the relevant data for understanding the participation and precise role played by GEFs and GAPs in the process of Ras activation. [ABSTRACT FROM AUTHOR]
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- 2015
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