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1.
PLoS One ; 19(5): e0298864, 2024.
Article in English | MEDLINE | ID: mdl-38753630

ABSTRACT

Fibrotic remodeling is the primary driver of functional loss in chronic kidney disease, with no specific anti-fibrotic agent available for clinical use. Transglutaminase 2 (TG2), a wound response enzyme that irreversibly crosslinks extracellular matrix proteins causing dysregulation of extracellular matrix turnover, is a well-characterized anti-fibrotic target in the kidney. We describe the humanization and characterization of two anti-TG2 monoclonal antibodies (zampilimab [hDC1/UCB7858] and BB7) that inhibit crosslinking by TG2 in human in vitro and rabbit/cynomolgus monkey in vivo models of chronic kidney disease. Determination of zampilimab half-maximal inhibitory concentration (IC50) against recombinant human TG2 was undertaken using the KxD assay and determination of dissociation constant (Kd) by surface plasmon resonance. Efficacy in vitro was established using a primary human renal epithelial cell model of tubulointerstitial fibrosis, to assess mature deposited extracellular matrix proteins. Proof of concept in vivo used a cynomolgus monkey unilateral ureteral obstruction model of chronic kidney disease. Zampilimab inhibited TG2 crosslinking transamidation activity with an IC50 of 0.25 nM and Kd of <50 pM. In cell culture, zampilimab inhibited extracellular TG2 activity (IC50 119 nM) and dramatically reduced transforming growth factor-ß1-driven accumulation of multiple extracellular matrix proteins including collagens I, III, IV, V, and fibronectin. Intravenous administration of BB7 in rabbits resulted in a 68% reduction in fibrotic index at Day 25 post-unilateral ureteral obstruction. Weekly intravenous administration of zampilimab in cynomolgus monkeys with unilateral ureteral obstruction reduced fibrosis at 4 weeks by >50%, with no safety signals. Our data support the clinical investigation of zampilimab for the treatment of kidney fibrosis.


Subject(s)
Fibrosis , GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Renal Insufficiency, Chronic , Animals , Humans , Male , Rabbits , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized/pharmacology , Disease Models, Animal , Fibrosis/drug therapy , GTP-Binding Proteins/antagonists & inhibitors , GTP-Binding Proteins/metabolism , GTP-Binding Proteins/immunology , Kidney/pathology , Kidney/drug effects , Kidney/metabolism , Macaca fascicularis , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/pathology , Transglutaminases/antagonists & inhibitors , Transglutaminases/metabolism
2.
Br J Pharmacol ; 179(11): 2697-2712, 2022 06.
Article in English | MEDLINE | ID: mdl-34879432

ABSTRACT

BACKGROUND AND PURPOSE: Transglutaminase type 2 (TG2) catalyses formation of ε-(γ-glutamyl)-lysine bonds between proteins, including those of the extracellular matrix (ECM). Elevated extracellular TG2 leads to accelerated ECM deposition and reduced clearance that underlies tissue scarring and fibrosis. Many transglutaminase inhibitors exist and allowed for proof-of-concept studies in disease models, but their lack of specificity for the TG2 isoform, and/or poor pharmacokinetic/pharmacodynamic properties have limited their clinical application. We sought to develop a high affinity TG2-specific antibody against extracellular TG2 activity, with characteristics suitable for therapeutic development. EXPERIMENTAL APPROACH: Individual human TG2 domains were used to immunize mice and generate hybridomas. Supernatants were screened for inhibition of recombinant human TG2 activity, with TG2 specificity determined by ELISA. KEY RESULTS: Thirteen TG2-specific, hybridoma supernatants inhibited human transamidation activity. Each hybridoma was cloned and the antibody mapped to an epitope in the TG2 core domain, using phage display panning of a TG2 fragment library. Four distinct inhibitory epitopes were determined. The most effective antibodies (AB1, DC1, and BB7) bound to amino acids 313-327 (catalytic core), with an IC50 of approximately 6-7 nM. The antibodies inhibit TG2 in human cells and block ECM accumulation in a primary human proximal tubular epithelial cell model of fibrosis. Only 7 antibodies inhibited rat TG2, all with higher IC50 values. CONCLUSIONS AND IMPLICATIONS: We identified a preferred inhibitory epitope in human TG2, developed antibodies with required characteristics for clinical development, and established that targeted inhibition of extracellular TG2 transamidation activity is sufficient to modify fibrotic remodelling.


Subject(s)
GTP-Binding Proteins , Protein Glutamine gamma Glutamyltransferase 2 , Animals , Epitopes , Fibrosis , GTP-Binding Proteins/metabolism , Immunologic Factors , Mice , Rats , Transglutaminases/chemistry , Transglutaminases/metabolism
3.
Nephron ; 142(4): 328-350, 2019.
Article in English | MEDLINE | ID: mdl-31048591

ABSTRACT

BACKGROUND: Tubulointerstitial fibrosis is a key feature of chronic kidney diseases leading to renal failure. It is characterised by the infiltration of fibroblasts and aberrant accumulation of extracellular matrix (ECM) proteins, which are associated with progressive loss of renal function. Integrins play a major role in fibrosis, but the mechanisms through which they do this are not fully understood. OBJECTIVE: Using a complex cell system, we test the hypothesis that integrins are pro-fibrotic via regulation of functional interactions between tubular epithelial cells and renal fibroblasts. METHOD: Contact co-culture of human primary renal proximal tubular epithelial cells and renal fibroblasts promoted the spontaneous accumulation of a mature ECM rich in interstitial collagens, which was considerably in excess of that seen in the individual mono-cultures. Both cell types persisted throughout the culture and were capable of expressing multiple ECM components. RESULTS: While ECM accumulation was inhibited by the clinically proven anti-fibrotic, nintedanib, and was partially abrogated by transforming growth factor ß neutralisation, its levels did not return to basal, indicating additional pathways were implicated in the pro-ECM response. Application of anti-integrin blocking antibodies and small molecules demonstrated a major role of the αV integrins in the ECM accumulation during fibroblast: epithelial cell interactions. CONCLUSION: Integrin-mediated pathways can facilitate the spontaneous accumulation of ECM during fibroblast: epithelial cell interactions, and this direct renal co-culture assay system could provide a translational in vitro assay for investigating novel pathways involved in the pro-ECM response and the screening of renal anti-fibrotic agents.


Subject(s)
Extracellular Matrix/metabolism , Fibrosis/metabolism , Integrins/metabolism , Kidney Diseases/metabolism , Cells, Cultured , Humans , In Vitro Techniques
4.
Sci Rep ; 7(1): 18111, 2017 12 21.
Article in English | MEDLINE | ID: mdl-29269854

ABSTRACT

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

5.
Biol Open ; 6(10): 1423-1433, 2017 Oct 15.
Article in English | MEDLINE | ID: mdl-29032370

ABSTRACT

Activated fibroblasts are considered major drivers of fibrotic disease progression through the production of excessive extracellular matrix (ECM) in response to signals from damaged epithelial and inflammatory cells. Nevertheless, epithelial cells are capable of expressing components of the ECM, cross-linking enzymes that increase its stability and are sensitive to factors involved in the early stages of fibrosis. We therefore wanted to test the hypothesis that epithelial cells can deposit ECM in response to stimulation in a comparable manner to fibroblasts. We performed immunofluorescence analysis of components of stable, mature extracellular matrix produced by primary human renal proximal tubular epithelial cells and renal fibroblasts in response to cytokine stimulation. Whilst fibroblasts produced a higher basal level of extracellular matrix components, epithelial cells were able to deposit significant levels of fibronectin, collagen I, III and IV in response to cytokine stimulation. In response to hypoxia, epithelial cells showed an increase in collagen IV deposition but not in response to the acute stress stimuli aristolochic acid or hydrogen peroxide. When epithelial cells were in co-culture with fibroblasts we observed significant increases in the level of matrix deposition which could be reduced by transforming growth factor beta (TGF-ß) blockade. Our results highlight the role of epithelial cells acting as efficient producers of stable extracellular matrix which could contribute to renal tubule thickening in fibrosis.

6.
Sci Rep ; 7(1): 9963, 2017 08 30.
Article in English | MEDLINE | ID: mdl-28855577

ABSTRACT

Fibrosis is a common driver of end-stage organ failure in most organs. It is characterised by excessive accumulation of extracellular matrix (ECM) proteins. Therapeutic options are limited and novel treatments are urgently required, however current cell-based high-throughput screening (HTS) models to identify molecules affecting ECM accumulation are limited in their relevance or throughput. We report a novel sensitive approach which combines in situ fluorescent staining of accumulated decellularised ECM proteins with automated high-content microscopy. Using this method to measure ECM accumulation in a kidney cell model, we demonstrated good agreement with established radiolabelled amino acid incorporation assays: TGFß1 delivered a potent pro-fibrotic stimulus, which was reduced by TGFß antibody or the anti-fibrotic nintedanib. Importantly, our method also provides information about matrix organisation: the extent of ECM accumulation was unaffected by the BMP antagonist Gremlin-1 but a pronounced effect on matrix fibrillar organisation was revealed. This rapid, straightforward endpoint provides quantitative data on ECM accumulation and offers a convenient cross-species readout that does not require antibodies. Our method facilitates discovery of novel pro- and anti-fibrotic agents in 384-well plate format and may be widely applied to in vitro cell-based models in which matrix protein deposition reflects the underlying biology or pathology.


Subject(s)
Extracellular Matrix/chemistry , Fibrosis/pathology , Kidney Diseases/pathology , Microscopy, Fluorescence/methods , Proteins/analysis , Automation, Laboratory/methods , Cells, Cultured , Epithelial Cells/pathology , Humans , Models, Biological , Staining and Labeling/methods
7.
Mol Cancer Res ; 13(4): 620-635, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25548099

ABSTRACT

UNLABELLED: Salt-inducible kinase 2 (SIK2) is a multifunctional kinase of the AMPK family that plays a role in CREB1-mediated gene transcription and was recently reported to have therapeutic potential in ovarian cancer. The expression of this kinase was investigated in prostate cancer clinical specimens. Interestingly, auto-antibodies against SIK2 were increased in the plasma of patients with aggressive disease. Examination of SIK2 in prostate cancer cells found that it functions both as a positive regulator of cell-cycle progression and a negative regulator of CREB1 activity. Knockdown of SIK2 inhibited cell growth, delayed cell-cycle progression, induced cell death, and enhanced CREB1 activity. Expression of a kinase-dead mutant of SIK2 also inhibited cell growth, induced cell death, and enhanced CREB1 activity. Treatment with a small-molecule SIK2 inhibitor (ARN-3236), currently in preclinical development, also led to enhanced CREB1 activity in a dose- and time-dependent manner. Because CREB1 is a transcription factor and proto-oncogene, it was posited that the effects of SIK2 on cell proliferation and viability might be mediated by changes in gene expression. To test this, gene expression array profiling was performed and while SIK2 knockdown or overexpression of the kinase-dead mutant affected established CREB1 target genes; the overlap with transcripts regulated by forskolin (FSK), the adenylate cyclase/CREB1 pathway activator, was incomplete. IMPLICATIONS: This study demonstrates that targeting SIK2 genetically or therapeutically will have pleiotropic effects on cell-cycle progression and transcription factor activation, which should be accounted for when characterizing SIK2 inhibitors.


Subject(s)
Autoantibodies/blood , Mitosis , Prostatic Neoplasms/metabolism , Protein Serine-Threonine Kinases/blood , Transcription, Genetic , Apoptosis , Cell Cycle/drug effects , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/genetics , Cyclic AMP Response Element-Binding Protein/metabolism , Gene Expression Regulation, Neoplastic , Humans , Male , Mitosis/drug effects , Prognosis , Prostatic Neoplasms/genetics , Prostatic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Mas , Transcription, Genetic/drug effects
8.
Nucleic Acids Res ; 42(10): 6256-69, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24753418

ABSTRACT

In prostate cancer (PC), the androgen receptor (AR) is a key transcription factor at all disease stages, including the advanced stage of castrate-resistant prostate cancer (CRPC). In the present study, we show that GABPα, an ETS factor that is up-regulated in PC, is an AR-interacting transcription factor. Expression of GABPα enables PC cell lines to acquire some of the molecular and cellular characteristics of CRPC tissues as well as more aggressive growth phenotypes. GABPα has a transcriptional role that dissects the overlapping cistromes of the two most common ETS gene fusions in PC: overlapping significantly with ETV1 but not with ERG target genes. GABPα bound predominantly to gene promoters, regulated the expression of one-third of AR target genes and modulated sensitivity to AR antagonists in hormone responsive and castrate resistant PC models. This study supports a critical role for GABPα in CRPC and reveals potential targets for therapeutic intervention.


Subject(s)
GA-Binding Protein Transcription Factor/metabolism , Prostatic Neoplasms/genetics , Receptors, Androgen/metabolism , Androgen Receptor Antagonists/pharmacology , Animals , Cell Line, Tumor , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic , Gene Regulatory Networks , Humans , Male , Mice , Mice, Inbred NOD , Mice, SCID , Phenotype , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Signal Transduction , Transcription, Genetic
9.
EMBO Mol Med ; 6(5): 651-61, 2014 May.
Article in English | MEDLINE | ID: mdl-24737870

ABSTRACT

Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , E2F1 Transcription Factor/metabolism , Gene Expression Regulation , Prostatic Neoplasms/physiopathology , Receptors, Androgen/metabolism , Repressor Proteins/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Cycle Proteins/metabolism , Disease Models, Animal , E2F1 Transcription Factor/genetics , Gene Expression Profiling , Humans , Male , Mice , Molecular Sequence Data , Prostatic Neoplasms/pathology , Repressor Proteins/genetics , Sequence Analysis, DNA
10.
EMBO J ; 30(13): 2719-33, 2011 May 20.
Article in English | MEDLINE | ID: mdl-21602788

ABSTRACT

The androgen receptor (AR) is a key regulator of prostate growth and the principal drug target for the treatment of prostate cancer. Previous studies have mapped AR targets and identified some candidates which may contribute to cancer progression, but did not characterize AR biology in an integrated manner. In this study, we took an interdisciplinary approach, integrating detailed genomic studies with metabolomic profiling and identify an anabolic transcriptional network involving AR as the core regulator. Restricting flux through anabolic pathways is an attractive approach to deprive tumours of the building blocks needed to sustain tumour growth. Therefore, we searched for targets of the AR that may contribute to these anabolic processes and could be amenable to therapeutic intervention by virtue of differential expression in prostate tumours. This highlighted calcium/calmodulin-dependent protein kinase kinase 2, which we show is overexpressed in prostate cancer and regulates cancer cell growth via its unexpected role as a hormone-dependent modulator of anabolic metabolism. In conclusion, it is possible to progress from transcriptional studies to a promising therapeutic target by taking an unbiased interdisciplinary approach.


Subject(s)
Carcinoma/genetics , Carcinoma/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Receptors, Androgen/physiology , Animals , Base Sequence , Binding Sites/genetics , Biosynthetic Pathways/genetics , Carcinoma/pathology , Cell Line, Tumor , Cell Proliferation , Cluster Analysis , Gene Expression Regulation, Neoplastic , Humans , Male , Metabolism/genetics , Metabolism/physiology , Mice , Models, Biological , Prostatic Neoplasms/pathology , Receptors, Androgen/genetics , Receptors, Androgen/metabolism , Response Elements/genetics , Transplantation, Heterologous
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