Publications by authors named "Augustin Amour"

31 Publications

Exploring PI3Kδ Molecular Pathways in Stable COPD and Following an Acute Exacerbation, Two Randomized Controlled Trials.

Int J Chron Obstruct Pulmon Dis 2021 3;16:1621-1636. Epub 2021 Jun 3.

Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, UK.

Background: Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) exerts corrective effects on the dysregulated migration characteristics of neutrophils isolated from patients with chronic obstructive pulmonary disease (COPD).

Objective: To develop novel, induced sputum endpoints to demonstrate changes in neutrophil phenotype in the lung by administering nemiralisib, a potent and selective inhaled PI3Kδ inhibitor, to patients with stable COPD or patients with acute exacerbation (AE) of COPD.

Methods: In two randomized, double-blind, placebo-controlled clinical trials patients with A) stable COPD (N=28, randomized 3:1) or B) AECOPD (N=44, randomized 1:1) received treatment with inhaled nemiralisib (1mg). Endpoints included induced sputum at various time points before and during treatment for the measurement of transcriptomics (primary endpoint), inflammatory mediators, functional respiratory imaging (FRI), and spirometry.

Results: In stable COPD patients, the use of nemiralisib was associated with alterations in sputum neutrophil transcriptomics suggestive of an improvement in migration phenotype; however, the same nemiralisib-evoked effects were not observed in AECOPD. Inhibition of sputum inflammatory mediators was also observed in stable but not AECOPD patients. In contrast, a placebo-corrected improvement in forced expiratory volume in 1 sec of 136 mL (95% Credible Intervals -46, 315mL) with a probability that the true treatment ratio was >0% (Pr(θ>0)) of 93% was observed in AECOPD. However, FRI endpoints remained unchanged.

Conclusion: We provide evidence for nemiralisib-evoked changes in neutrophil migration phenotype in stable COPD but not AECOPD, despite improving lung function in the latter group. We conclude that induced sputum can be used for measuring evidence of alteration of neutrophil phenotype in stable patients, and our study provides a data set of the sputum transcriptomic changes during recovery from AECOPD.
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http://dx.doi.org/10.2147/COPD.S309303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184158PMC
June 2021

The 5-Phosphatase SHIP2 Promotes Neutrophil Chemotaxis and Recruitment.

Front Immunol 2021 19;12:671756. Epub 2021 Apr 19.

Centre for Inflammation Research, Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh, United Kingdom.

Neutrophils, the most abundant circulating leukocytes in humans have key roles in host defense and in the inflammatory response. Agonist-activated phosphoinositide 3-kinases (PI3Ks) are important regulators of many facets of neutrophil biology. PIP3 is subject to dephosphorylation by several 5' phosphatases, including SHIP family phosphatases, which convert the PI3K product and lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) into PI(3,4)P2, a lipid second messenger in its own right. In addition to the leukocyte restricted SHIP1, neutrophils express the ubiquitous SHIP2. This study analyzed mice and isolated neutrophils carrying a catalytically inactive SHIP2, identifying an important regulatory function in neutrophil chemotaxis and directionality and in neutrophil recruitment to sites of sterile inflammation , in the absence of major defects of any other neutrophil functions analyzed, including, phagocytosis and the formation of reactive oxygen species. Mechanistically, this is explained by a subtle effect on global 3-phosphorylated phosphoinositide species. This work identifies a non-redundant role for the hitherto overlooked SHIP2 in the regulation of neutrophils, and specifically, neutrophil chemotaxis/trafficking. It completes an emerging wider understanding of the complexity of PI3K signaling in the neutrophil, and the roles played by individual kinases and phosphatases within.
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http://dx.doi.org/10.3389/fimmu.2021.671756DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8089392PMC
April 2021

Optimization of Orally Bioavailable PI3Kδ Inhibitors and Identification of Vps34 as a Key Selectivity Target.

J Med Chem 2020 01 8;63(2):638-655. Epub 2020 Jan 8.

Cellzome GmbH , GlaxoSmithKline , Meyerhofstrasse 1 , 69117 Heidelberg , Germany.

Optimization of a lead series of PI3Kδ inhibitors based on a dihydroisobenzofuran core led to the identification of potent, orally bioavailable compound . Selectivity profiling of compound showed similar potency for class III PI3K, Vps34, and PI3Kδ, and compound was not well-tolerated in a 7-day rat toxicity study. Structure-based design led to an improvement in selectivity for PI3Kδ over Vps34 and, a focus on oral phramacokinetics properties resulted in the discovery of compound , which showed improved toxicological outcomes at similar exposure levels to compound .
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http://dx.doi.org/10.1021/acs.jmedchem.9b01585DOI Listing
January 2020

Converging TLR9 and PI3Kgamma signaling induces sterile inflammation and organ damage.

Sci Rep 2019 12 13;9(1):19085. Epub 2019 Dec 13.

Department of Biochemistry and Immunology, Institute of Biological Sciences, Feredal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Toll-like receptor 9 (TLR9) and Phosphatidylinositol-3-kinase gamma (PI3Kγ) are very important effectors of the immune response, however, the importance of such crosstalk for disease development is still a matter of discussion. Here we show that PI3Kγ is required for immune responses in which TLR9 is a relevant trigger. We demonstrate the requirement of PI3Kγ for TLR9-induced inflammation in a model of CpG-induced pleurisy. Such requirement was further observed in inflammatory models where DNA sensing via TLR9 contributes to disease, such as silicosis and drug-induced liver injury. Using adoptive transfer, we demonstrate that PI3Kγ is important not only in leukocytes but also in parenchymal cells for the progression of inflammation. We demonstrate this crosstalk between TLR9 and PI3Kγ in vitro using human PBMCs. The inhibition of PI3Kγ in CpG-stimulated PBMCs resulted in reduction of both cytokine production and phosphorylated Akt. Therefore, drugs that target PI3Kγ have the potential to treat diseases mediated by excessive TLR9 signalling.
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http://dx.doi.org/10.1038/s41598-019-55504-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910931PMC
December 2019

An investigation of the anti-inflammatory effects and a potential biomarker of PI3Kδ inhibition in COPD T cells.

Clin Exp Pharmacol Physiol 2017 Sep;44(9):932-940

The University of Manchester, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University Hospital of South Manchester NHS Foundation Trust, The University of Manchester, Manchester, UK.

Lymphocyte numbers are increased in the lungs of chronic obstructive pulmonary disease (COPD) patients. Phosphatidylinositol-3-kinase delta (PI3Kδ) is involved in lymphocyte activation. We investigated the effect of PI3Kδ inhibition on cytokine release from COPD lymphocytes. We also evaluated phosphorylated ribosomal S6 protein (rS6) as a potential biomarker of PI3Kδ activation. Peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage (BAL) cells isolated from healthy never smokers (HNS), smokers (S) and COPD patients were stimulated to induce a T cell receptor response. The effects of a PI3Kδ specific inhibitor (GSK045) on cytokine release and rS6 phosphorylation were measured by Luminex and flow cytometry respectively. The effects of GSK045 on cytokine production from PHA stimulated chopped lung samples were investigated. GSK045 reduced cytokine release from PBMCs, BAL cells and chopped lung. Inhibition was greatest in the chopped lung model, with approximately 80% inhibition of interferon (IFN) γ, interleukin (IL)-2, IL-17 and IL-10. PI3Kδ inhibition suppressed rS6 phosphorylation in unstimulated airway T-lymphocytes by up to 60%. Inhibition of PI3Kδ suppressed T cell cytokine production in COPD patients. rS6 phosphorylation shows potential as a biomarker to assess PI3Kδ activity.
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http://dx.doi.org/10.1111/1440-1681.12784DOI Listing
September 2017

Evolution of a Novel, Orally Bioavailable Series of PI3Kδ Inhibitors from an Inhaled Lead for the Treatment of Respiratory Disease.

J Med Chem 2016 08 27;59(15):7239-51. Epub 2016 Jul 27.

GlaxoSmithKline R&D , Gunnels Wood Road, Stevenage SG1 2NY, U.K.

A four-step process of high-quality modeling of existing data, deconstruction, identification of replacement cores, and an innovative synthetic regrowth strategy led to the rapid discovery of a novel oral series of PI3Kδ inhibitors with promising selectivity and excellent in vivo characteristics.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00799DOI Listing
August 2016

Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition.

Am J Respir Crit Care Med 2016 10;194(8):961-973

1 Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

Rationale: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied.

Objectives: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition.

Methods: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays.

Measurements And Main Results: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures.

Conclusions: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.
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http://dx.doi.org/10.1164/rccm.201509-1818OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067816PMC
October 2016

Genome-wide transcription profiling in neutrophils in acute respiratory distress syndrome.

Lancet 2015 Feb;385 Suppl 1:S55

University of Cambridge, Cambridge, UK. Electronic address:

Background: Acute respiratory distress syndrome (ARDS) is characterised by diffuse neutrophil-mediated alveolar inflammation. Recently, we demonstrated that blood polymorphonuclear leucocytes (PMNs) in ARDS are basally activated, and exhibit aberrant oxidative burst and survival responses. The molecular mechanisms governing ARDS PMN function and longevity are incompletely understood. We aimed to use genome-wide transcriptional profiling of ARDS blood PMNs to explore underlying disease mechanisms and identify therapeutic targets aimed at manipulating PMN function and longevity.

Methods: GeneChip Affymetrix oligonucleotide arrays were used to assess global transcriptional profiles in highly pure PMNs from ventilated patients fulfilling the Berlin ARDS definition (n=10), in freshly isolated PMNs from age-matched and sex-matched healthy volunteers (n=10), and in healthy volunteer PMNs exposed in vitro to recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) (1 ng/mL for 6 h). Ingenuity Pathway Analysis software was used to map probes identified as important onto specific pathways.

Findings: Transcriptomic analysis showed that 1319 genes were altered in ARDS PMNs relative to healthy volunteer PMNs. Compared with well established reference databases, the gene expression profile in ARDS PMNs showed near-complete correlation to datasets derived from patients with sepsis and burns. Transcripts enriched in ARDS PMNs were differentially expressed in known functional network pathways associated with cancer, cellular compromise, apoptotic mechanisms, and chemotaxis. Of the observed gene changes, only 292 (22%) were seen in healthy volunteer PMNs after exposure to rhGM-CSF, of which 216 showed the same directional change as ARDS PMNs.

Interpretation: Existing genome-wide studies in ARDS use total blood leucocytes; our study is the first, to our knowledge, to use unbiased global genomic profiling of highly pure ARDS blood PMNs in parallel with age-matched and gender-matched healthy volunteer PMNs treated with rhGM-CSF. Collectively our results show that ARDS PMNs display important de-novo transcriptional activity. The global transcriptomic changes were consistent with the observed aberrant ARDS PMN survival and functional phenotype that we have previously reported, and show near-complete correlation to existing sepsis and burns datasets, but only limited transcriptomic overlap with healthy volunteer PMNs treated with rhGM-CSF.

Funding: National Institute for Health Research, GlaxoSmithKline.
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http://dx.doi.org/10.1016/S0140-6736(15)60370-1DOI Listing
February 2015

Optimization of Novel Indazoles as Highly Potent and Selective Inhibitors of Phosphoinositide 3-Kinase δ for the Treatment of Respiratory Disease.

J Med Chem 2015 Sep 3;58(18):7381-99. Epub 2015 Sep 3.

Refractory Respiratory Inflammation DPU, and ‡Allergic Inflammation DPU, Respiratory Therapeutic Area, GlaxoSmithKline R&D , Gunnels Wood Road, Stevenage, SG1 2NY, U.K.

Optimization of lead compound 1, through extensive use of structure-based design and a focus on PI3Kδ potency, isoform selectivity, and inhaled PK properties, led to the discovery of clinical candidates 2 (GSK2269557) and 3 (GSK2292767) for the treatment of respiratory indications via inhalation. Compounds 2 and 3 are both highly selective for PI3Kδ over the closely related isoforms and are active in a disease relevant brown Norway rat acute OVA model of Th2-driven lung inflammation.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00767DOI Listing
September 2015

Targeting phosphoinositide 3-kinase δ for the treatment of respiratory diseases.

Ann N Y Acad Sci 2013 Mar;1280:35-9

Refractory Respiratory Inflammation Discovery Performance Unit, Respiratory Therapy Area, GlaxoSmithKline, Stevenage, Hertfordshire, United Kingdom.

Asthma and chronic obstructive pulmonary disease (COPD) are characterized in their pathogenesis by chronic inflammation in the airways. Phosphoinositide 3-kinase δ (PI3Kδ), a lipid kinase expressed predominantly in leukocytes, is thought to hold much promise as a therapeutic target for such inflammatory conditions. Of particular interest for the treatment of severe respiratory disease is the observation that inhibition of PI3Kδ may restore steroid effectiveness under conditions of oxidative stress. PI3Kδ inhibition may also prevent recruitment of inflammatory cells, including T lymphocytes and neutrophils, as well as the release of proinflammatory mediators, such as cytokines, chemokines, reactive oxygen species, and proteolytic enzymes. In addition, targeting the PI3Kδ pathway could reduce the incidence of pathogen-induced exacerbations by improving macrophage-mediated bacterial clearance. In this review, we discuss the potential and highlight the unknowns of targeting PI3Kδ for the treatment of respiratory disease, focusing on recent developments in the role of the PI3Kδ pathway in inflammatory cell types believed to be critical to the pathogenesis of COPD.
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http://dx.doi.org/10.1111/nyas.12039DOI Listing
March 2013

Targeting phosphoinositide 3-kinase δ for allergic asthma.

Biochem Soc Trans 2012 Feb;40(1):240-5

Biological Reagents and Assay Development, Platform Technology and Science, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK.

Chronic inflammation in the lung has long been linked to the pathogenesis of asthma. Central to this airway inflammation is a T-cell response to allergens, with Th2 cytokines driving the differentiation, survival and function of the major inflammatory cells involved in the allergic cascade. PI3Kδ (phosphoinositide 3-kinase δ) is a lipid kinase, expressed predominantly in leucocytes, where it plays a critical role in immune receptor signalling. A selective PI3Kδ inhibitor is predicted to block T-cell activation in the lung, reducing the production of pro-inflammatory Th2 cytokines. PI3Kδ is also involved in B-cell and mast cell activation. Therefore the inhibition of PI3Kδ should dampen down the inflammatory cascade involved in the asthmatic response through a wide breadth of pharmacology. Current anti-inflammatory therapies, which are based on corticosteroids, are effective in controlling inflammation in mild asthmatics, but moderate/severe asthmatic patients remain poorly controlled, experiencing recurrent exacerbations. Corticosteroids have no effect on mast cell degranulation and do not act directly on B-cells, so, overall, a PI3Kδ inhibitor has the potential to deliver improvements in onset of action, efficacy and reduced exacerbations in moderate/severe asthmatics. Additionally, PI3Kδ inhibition is expected to block effects of Th17 cells, which are increasingly implicated in steroid-insensitive asthma.
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http://dx.doi.org/10.1042/BST20110665DOI Listing
February 2012

Kinetic analysis of the inhibition of matrix metalloproteinases: lessons from the study of tissue inhibitors of metalloproteinases.

Methods Mol Biol 2010 ;622:435-50

Tissue inhibitors of metalloproteinases (TIMPs) are a group of highly potent inhibitors of matrix metalloproteinases (MMPs) and disintegrin metalloproteinases (ADAMs). The high affinity and "tight-binding" nature of the inhibition of MMPs or ADAMs by TIMPs presents challenges for the determination of both equilibrium and dissociation rate constants of these inhibitory events. Methodologies that enable some of these challenges to be overcome are described in this chapter and represent valuable lessons for the in vitro assessment of MMP or ADAM inhibitors within a drug discovery context.
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http://dx.doi.org/10.1007/978-1-60327-299-5_25DOI Listing
April 2010

The identification of beta-hydroxy carboxylic acids as selective MMP-12 inhibitors.

Bioorg Med Chem Lett 2009 Oct 6;19(19):5760-3. Epub 2009 Aug 6.

GSK Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, United Kingdom.

A new class of selective MMP-12 inhibitors have been identified via high throughput screening. Crystallization with MMP-12 confirmed the mode of binding and allowed initial optimization to be carried out using classical structure based design.
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http://dx.doi.org/10.1016/j.bmcl.2009.07.155DOI Listing
October 2009

Kinetic assay for characterization of spleen tyrosine kinase activity and inhibition with recombinant kinase and crude cell lysates.

Anal Biochem 2009 Jan 15;384(1):56-67. Epub 2008 Aug 15.

Center for Signal Transduction Research, Cellular Analysis/BioDiscovery Division, Invitrogen, Hopkinton, MA 01748, USA.

Spleen tyrosine kinase (Syk) is involved in the activation of cells implicated in allergic or autoimmune diseases and certain cancers. Therefore, Syk inhibitors may prove to be effective in treating diseases where Syk activity or expression is increased or deregulated. We developed a continuous and direct (noncoupled) fluorescence intensity assay for measuring Syk activity using purified recombinant enzyme or crude lysates generated from anti-immunoglobulin M (IgM) antibody-treated RAMOS cells. The assay is based on the chelation-enhanced fluorophore 8-hydroxy-5-(N,N-dimethylsulfonamido)-2-methylquinoline (referred to as Sox), which has been incorporated into a peptide substrate selected for robust detection of Syk activity. This homogeneous assay is simple to use, provides considerably more information, and has been adapted to a 384-well, low-volume microtiter plate format that can be used for the high-throughput identification and kinetic characterization of Syk inhibitors. The assay can be performed with a wide range of adenosine triphosphate (ATP) concentrations and, therefore, can be used to analyze ATP-competitive and ATP-noncompetitive/allosteric kinase inhibitors. Measurement of Syk activity in RAMOS crude cell lysates or immunoprecipitation (IP) capture formats may serve as a physiologically more relevant enzyme source. These Sox-based continuous and homogeneous assays provide a valuable set of tools for studying Syk signaling and for defining inhibitors that may be more effective in controlling disease.
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http://dx.doi.org/10.1016/j.ab.2008.07.040DOI Listing
January 2009

Antithrombotic potential of GW813893: a novel, orally active, active-site directed factor Xa inhibitor.

J Cardiovasc Pharmacol 2008 Jul;52(1):66-71

GlaxoSmithkline, Cardiovascular & Urogenital Diseases Center of Excellence, King of Prussia, Pennsylvania, USA.

Background: Factor Xa (FXa) has been a target of considerable interest for drug development efforts aimed at suppressing thrombosis. In this report, a new orally active, small molecule, active-site directed FXa inhibitor, GW813893, has been profiled in a succession of in vitro and in vivo assays involved in its preclinical characterization as a potential antithrombotic therapeutic.

Methods: In vitro profiling of GW813893 consisted of assessing its inhibitory potential against FXa and a broad panel of related and unrelated enzymes and receptors. Additionally, the FXa inhibition potential of GW813893 was assessed in prothrombinase and plasma-based clotting assays. In vivo characterization of GW813893 consisted of thrombosis studies in a rat inferior vena cava model, a rat carotid artery thrombosis model, and a rabbit jugular thrombosis model. Bleeding studies were conducted in a rat tail transection model. Ex vivo determinations of compound effects on FX and clotting activity were also undertaken.

Results: GW813893 was more than 90-fold selective over all enzymes tested, and it inhibited FXa and prothrombinase activity with a Ki of 4.0 nM and 9.7 nM, respectively. In vivo, GW813893 concentration-dependently suppressed thrombotic activity in all models tested. The antithrombotic activity correlated with the suppression of plasma-based clotting activity and the inhibition of plasma FX activity (P < 0.02). Over the antithrombotic dose-range, an increased bleeding diathesis was not observed.

Conclusion: These experiments demonstrate that GW813893 is a potent, selective, orally active inhibitor of FXa. The data suggest that GW813893 has robust antithrombotic potential at doses that have no detectable hemostasis liability. Collectively, the profile suggests that GW813893 has the preclinical pharmacology underpinnings of an oral antithrombotic therapeutic.
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http://dx.doi.org/10.1097/FJC.0b013e31817e9b9eDOI Listing
July 2008

TIMP-3 inhibition of ADAMTS-4 (Aggrecanase-1) is modulated by interactions between aggrecan and the C-terminal domain of ADAMTS-4.

J Biol Chem 2007 Jul 30;282(29):20991-8. Epub 2007 Apr 30.

GlaxoSmithKline, New Frontiers Research Park, Harlow, Essex CM19 5AW, United Kingdom.

ADAMTS-4 (aggrecanase-1) is a glutamyl endopeptidase capable of generating catabolic fragments of aggrecan analogous to those released from articular cartilage during degenerative joint diseases such as osteoarthritis. Efficient aggrecanase activity requires the presence of sulfated glycosaminoglycans attached to the aggrecan core protein, implying the contribution of substrate recognition/binding site(s) to ADAMTS-4 activity. In this study, we developed a sensitive fluorescence resonance energy transfer peptide assay with a K(m) in the 10 microm range and utilized this assay to demonstrate that inhibition of full-length ADAMTS-4 by full-length TIMP-3 (a physiological inhibitor of metalloproteinases) is enhanced in the presence of aggrecan. Our data indicate that this interaction is mediated largely through the binding of glycosaminoglycans (specifically chondroitin 6-sulfate) of aggrecan to binding sites in the thrombospondin type 1 motif and spacer domains of ADAMTS-4 to form a complex with an improved binding affinity for TIMP-3 over free ADAMTS-4. The results of this study therefore indicate that the cartilage environment can modulate the function of enzyme-inhibitor systems and could have relevance for therapeutic approaches to aggrecanase modulation.
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http://dx.doi.org/10.1074/jbc.M610721200DOI Listing
July 2007

A broad-spectrum fluorescence-based peptide library for the rapid identification of protease substrates.

Proteomics 2006 Apr;6(7):2112-20

Proteolysis Research Group, School of Biochemistry and Microbiology, Leeds Institute of Genetics, Health and Therapeutics, University of Leeds, Leeds, UK.

Identification of peptide substrates for proteases can be a major undertaking. To overcome issues such as feasibility and deconvolution, associated with large peptide libraries, a 'small but smart' generic fluorescence resonance energy transfer rapid endopeptidase profiling library (REPLi) was synthesised as a tool for rapidly identifying protease substrates. Within a tripeptide core, flanked by Gly residues, similar amino acids were paired giving rise to a relatively small library of 3375 peptides divided into 512 distinct pools each containing only 8 peptides. The REPLi was validated with trypsin, pepsin, the matrix metalloprotease (MMP)-12 and MMP-13 and calpains-1 and -2. In the case of calpain-2, a single iteration step involving LC-MS, provided the definitive residue specificity from which a highly sensitive fluorogenic substrate, (FAM)-Gly-Gly-Gly-Gln-Leu-Tyr-Gly-Gly-DPA-Arg-Arg-Lys-(TAMRA), was then designed. The thorough validation of this 'small but smart' peptide library with representatives from each of the four mechanistic protease classes indicates that the REPLi will be useful for the rapid identification of substrates for multiple proteases.
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http://dx.doi.org/10.1002/pmic.200500153DOI Listing
April 2006

Continuous real-time measurement of tumor necrosis factor-alpha converting enzyme activity on live cells.

Lab Invest 2005 Nov;85(11):1440-8

Department of Anaesthetics and Intensive Care, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK.

Tumor necrosis factor-alpha (TNF) converting enzyme (TACE) is responsible for shedding of various membrane proteins including proinflammatory cytokine TNF. In vivo regulation of TACE is poorly understood mainly due to lack of reliable methodology to measure TACE activity in cell-based assays. Here we report a novel enzyme assay that enables continuous real-time measurement of TACE activity on the surface of live cells. Cells were incubated with a new fluorescent resonance energy transfer peptide consisting of a TACE-sensitive TNF sequence and fluorescein-tetramethylrhodamine (FAM-TAMRA), and enzyme activity was monitored by the rate of increase in fluorescent signal due to peptide cleavage. Validation studies using resting as well as stimulated monocytic cells indicated that the assay was sensitive, reproducible and quantitative. Pharmacological studies with various inhibitors indicated that the observed enzyme activity could largely be ascribed to TACE. Thus, the FAM-TAMRA peptide provides a powerful tool for measurement of constitutive and inducible cellular TACE activity. The principles developed may be applied to analyses of enzyme activity of various sheddases on live cells.
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http://dx.doi.org/10.1038/labinvest.3700340DOI Listing
November 2005

ADAM28 is overexpressed in human non-small cell lung carcinomas and correlates with cell proliferation and lymph node metastasis.

Int J Cancer 2006 Jan;118(2):263-73

Department of Pathology, School of Medicine, Keio University, Tokyo, Japan.

ADAM (a disintegrin and metalloproteinases) are a recently discovered gene family of proteins with sequence similarity to the reprolysin family of snake venom metalloproteinases, and about one-third of the family members have the catalytic site consensus sequence in their metalloproteinase domains. We screened the mRNA expression of 11 different ADAM species with putative metalloproteinase activity in human non-small cell lung carcinomas by RT-PCR, and found that prototype membrane-anchored ADAM28 (ADAM28m) and secreted ADAM28 (ADAM28s) are predominantly expressed in the carcinoma tissues. Real-time quantitative PCR demonstrated that the expression levels of ADAM28m and ADAM28s are significantly 16.8-fold and 9.0-fold higher in the carcinomas than in the non-carcinoma tissues, respectively. In addition, the expression levels of ADAM28m and ADAM28s were significantly higher in the carcinomas with >30 mm in diameter than in those < or =30 mm. The expression levels were also significantly higher in the carcinomas with lymph node metastasis than in those without metastasis. MIB1-positive cell index of the carcinomas had a direct correlation with the expression levels of ADAM28m and ADAM28s (r = 0.667, p < 0.001 and r = 0.535, p < 0.01, respectively). In situ hybridization and immunohistochemistry demonstrated that ADAM28 is expressed predominantly in the carcinoma cells. Immunoblot analysis showed the activated form of ADAM28 in the carcinoma tissues. These data demonstrate for the first time that ADAM28 is overexpressed and activated in human non-small cell lung carcinomas, and suggest the possibility that ADAM28 plays a role in cell proliferation and progression of the human lung carcinomas.
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http://dx.doi.org/10.1002/ijc.21324DOI Listing
January 2006

Role of TIMPs (tissue inhibitors of metalloproteinases) in pericellular proteolysis: the specificity is in the detail.

Biochem Soc Symp 2003 (70):65-80

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.

Pericellular proteolysis represents one of the key modes by which the cell can modulate its environment, involving not only turnover of the extracellular matrix but also the regulation of cell membrane proteins, such as growth factors and their receptors. The metzincins are active players in such proteolytic events, and their mode of regulation is therefore of particular interest and importance. The TIMPs (tissue inhibitors of metalloproteinases) are established endogenous inhibitors of the matrix metalloproteinases (MMPs), and some have intriguing abilities to associate with the pericellular environment. It has been shown that TIMP-2 can bind to cell surface MT1-MMP (membrane-type 1 MMP) to act as a 'receptor' for proMMP-2 (progelatinase A), such that the latter can be activated efficiently in a localized fashion. We have examined the key structural features of TIMP-2 that determine this unique function, showing that Tyr36 and Glu192-Asp193 are vital for specific interactions with MT1-MMP and proMMP-2 respectively, and hence activation of proMMP-2. TIMP-3 is sequestered at the cell surface by association with the glycosaminoglycan chains of proteoglycans, especially heparan sulphate, and we have shown that it may play a role in the regulation of some ADAMs (a disintegrin and metalloproteinases), including tumour necrosis factor alpha-converting enzyme (TACE; ADAM17). We have established that key residues in TIMP-3 determine its interaction with TACE. Further studies of the features of TIMP-3 that determine specific binding to both ADAM and glycosaminoglycan are required in order to understand these unique properties.
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http://dx.doi.org/10.1042/bss0700065DOI Listing
February 2004

Heparan sulfate regulates amyloid precursor protein processing by BACE1, the Alzheimer's beta-secretase.

J Cell Biol 2003 Oct 6;163(1):97-107. Epub 2003 Oct 6.

School of Biosciences, University of Birmingham, Birmingham, UK.

Cleavage of amyloid precursor protein (APP) by the Alzheimer's beta-secretase (BACE1) is a key step in generating amyloid beta-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with beta-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by alpha-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.
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http://dx.doi.org/10.1083/jcb.200303059DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173449PMC
October 2003

Synthesis and evaluation of delta-lactams (piperazones) as elastase inhibitors.

Bioorg Med Chem Lett 2003 Feb;13(3):387-9

The Oxford Centre for Molecular Sciences and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, UK.

A series of monocyclic delta-lactams (piperazones) was prepared and analysed as inhibitors of porcine pancreatic elastase and human neutrophil elastase.
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http://dx.doi.org/10.1016/s0960-894x(02)00995-2DOI Listing
February 2003

Investigation of bradykinin metabolism in human and rat plasma in the presence of the dual ACE/NEP inhibitors GW660511X and omapatrilat.

J Pept Sci 2002 Nov;8(11):591-600

GlaxoSmithkline, Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK.

Several studies have suggested that the accumulation of bradykinin, or that of one its metabolites BK1-8, is involved in the occurrence of side effects such as AE associated with the use of various ACEi. In this work a novel approach combining HPLC-UV on-line with oaTOF-MS and ICPMS was applied to investigate in human and rat plasma the metabolism of labelled BK (79/81 Br-Phe5) BrBK in the presence of two new dual ACE/NEP inhibitors (GW660511X and omapatrilat) currently under clinical trial. In human plasma the BrBK half-life values in the absence or in the presence of GW660511X (3.8 microM) or omapatrilat (32 nM) were 38.7 +/- 2.4, 51.2 +/- 4.7 and 114.7 +/- 9.3 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe. In the presence of inhibitors, however, the levels of these resultant metabolites were different. Unlike GW660511X, omapatrilat abolished the production of BrBK1-5 and BrBK1-7, suggesting a better ACE inhibition effect over GW660511X as no NEP activity was found. In addition the production of BrBK1-8 was enhanced in the presence of these inhibitors with a greater accumulation being observed with omapatrilat. The production of Br-Phe5 was reduced with GW660511X while no significant change was observed with omapatrilat after 4 h of incubation. In rat plasma the BrBK half-life values in the absence or in the presence of GW660511X (530 nM) or omapatrilat (50 nM) were 9.31 +/- 1.7, 22.06 +/- 3.1 and 25.3 +/- 1.7 min, respectively and BrBK was degraded into BrBK1-8, BrBK1-7, BrBK1-5 and Br-Phe5 plus BrBK2-9, BrBK4-8 and BrBK2-8 metabolites not found in human plasma. GW660511X and omapatrilat reduced the production of BrBK1-5 and BrBK1-7 with more effect being observed with omapatrilat. GW660511X and omapatrilat increased the production of both BrBK1-8 and Br-Phe5 but not that of BrBK4-8 and BrBK2-8. This study shows that the potency of GW660511X in comparison with omapatrilat is more than 100-fold lower in human, but less than 10-fold lower in rat plasma, suggesting that rat may not be a suitable in vivo model for the evaluation of ACE/NEP inhibition in relation to effects in humans.
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http://dx.doi.org/10.1002/psc.419DOI Listing
November 2002

Enzyme accessibility and solid supports: which molecular weight enzymes can be used on solid supports? An investigation using confocal Raman microscopy.

Chemistry 2002 Aug;8(16):3769-72

University of Southampton, Department of Chemistry, UK.

The accessibility of various solid supports (TentaGel, PEGA 1900, and beaded controlled pore glasses (CPGs)) to a range of enzymes was investigated. The different beaded materials were loaded with the peptide 4-cyanobenzamide-Gly-Pro-Leu-Gly-Leu-Phe-Ala-Arg-OH and incubated with the enzymes MMP-12 (22 kDa), thermolysin (35 kDa), MMP-13 (42.5 kDa), clostridium collagenase (68 kDa), and NEP (90 kDa). The absence/presence of the cyano stretching frequency was measured by means of confocal Raman microscopy. It was found that none of the investigated enzymes could enter the polymer matrices of TentaGel. PEGA 1900 was compatible only with the two smallest enzymes, while beaded CPG was successful even with NEP (90 kDa), proving its superiority over other materials in terms of bio-compatibility.
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http://dx.doi.org/10.1002/1521-3765(20020816)8:16<3769::AID-CHEM3769>3.0.CO;2-VDOI Listing
August 2002

Discovery of further pyrrolidine trans-lactams as inhibitors of human neutrophil elastase (HNE) with potential as development candidates and the crystal structure of HNE complexed with an inhibitor (GW475151).

J Med Chem 2002 Aug;45(18):3878-90

Medicinal Chemistry 1, In Vitro Pharmacology, Systems Research, Respiratory Systems, Stevenage CEDD DMPK, United Kingdom.

Described herein is a modern approach to the rapid preparation and evaluation of compounds as potential back-up drug candidates. GW311616A, 1, a derivative of pyrrolidine trans-lactams, has previously been described as a potent, orally active inhibitor of human neutrophil elastase (HNE) for the treatment of respiratory disease. These properties made it a suitable candidate for development. Described here is the discovery of three further derivatives of pyrrolidine trans-lactams, which fulfill the criteria required for back-up candidates 28, 29, and 32. These include increased activity in inhibiting HNE in human whole blood (HWB) and comparable pharmacokinetic properties, in particular clearance, in two species. To provide a rapid assessment of clearance, cassette dosing in dog was used. Modern array techniques, including the synthesis of mixtures, were used to synthesize compounds rapidly. Having selected three potential compounds as back-up candidates, they were prepared as single enantiomers and profiled in in vitro and in vivo assays and evaluated pharmacokinetically in rat and dog. These compounds are highly potent and selective HNE inhibitors, with a prolonged pharmacodynamic action. Pharmacokinetically, these compounds are comparable with 1 while they are more potent in HWB. Compound 28, however, has a higher clearance. One of these compounds, 32, was cocrystallized with HNE, and features of this structure are described and compared with the cocrystal structure of 1 in porcine pancreatic elastase.
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http://dx.doi.org/10.1021/jm020881fDOI Listing
August 2002

The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs.

FEBS Lett 2002 Jul;524(1-3):154-8

School of Biological Sciences, University of East Anglia, Norwich, UK.

The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane-bound precursors and involved in modulating cell-cell and cell-matrix interactions. ADAM8 (MS2, CD156) has been identified in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane-bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin-gamma, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membrane-associated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane-type metalloproteinases) which have been implicated in protein processing at the cell surface.
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http://dx.doi.org/10.1016/s0014-5793(02)03047-8DOI Listing
July 2002

The C-terminal domains of TACE weaken the inhibitory action of N-TIMP-3.

FEBS Lett 2002 Jun;520(1-3):102-6

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.

Tumor necrosis factor-alpha converting enzyme (TACE) is an ADAM (a disintegrin and metalloproteinases) that comprises an active catalytic domain and several C-terminal domains. We compare the binding affinity and association rate constants of the N-terminal domain form of wild-type tissue inhibitor of metalloproteinase (TIMP-3; N-TIMP-3) and its mutants against full-length recombinant TACE and the truncated form of its catalytic domain. We show that the C-terminal domains of TACE substantially weaken the inhibitory action of N-TIMP-3. Further probing with hydroxamate inhibitors indicates that both forms of TACE have similar active site configurations. Our findings highlight the potential role of the C-terminal domains of ADAM proteinases in influencing TIMP interactions.
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http://dx.doi.org/10.1016/s0014-5793(02)02776-xDOI Listing
June 2002

Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-alpha-converting enzyme.

Biochem J 2002 May;364(Pt 1):227-34

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, U.K.

We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-alpha (TNF-alpha)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (K(app)(i)) and association rates (k(on)) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60 pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-alpha demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-alpha. Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-alpha shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1222565PMC
http://dx.doi.org/10.1042/bj3640227DOI Listing
May 2002
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