Phosphodiesterase-4 isoforms: intracellular targeting, regulation and potential therapeutic targets

Lead Research Organisation: University of Glasgow
Department Name: School of Life Sciences

Abstract

Chronic Obstructive Pulmonary Disease is the fourth leading cause of death in the world. It causes lung damage through chronic inflammation. Raising the levels of a substance called cAMP inside lung cells can stop this. Potential new medicines are drugs that bind tightly to PDE4 enzymes to stop them breaking down cAMP. Unfortunately these drugs cause nausea and vomiting, preventing their use. We’ve found that there are many PDE4 subtypes but that only certain of these need to be inhibited to stop inflammation. We aim to devise novel ways to inhibit these important PDE4 subtypes, rather than all PDE4 enzymes, in order to produce effective medicines with minimal or no side-effects. To do this we will exploit our discovery that individual PDE4 subtypes have unique ‘postcodes’ built into them that allow them to be targeted to exactly the right place in cells for them to do their job properly. Our aim is to find molecules that block this correct targeting so as to stop them working properly, providing new COPD medicines. As our drugs focus on just the PDE4 subtype involved in COPD they should not have the side effects caused by current drugs that inhibit all PDE4 subtypes.

Technical Summary

cAMP is a pivotal second messenger able to exert a panoply of cell type specific effects. A multigene family of phosphodiesterases (PDEs) provide the sole means for degrading cAMP and are thus poised to play a key regulatory role in cells. Highly specialised regulatory properties, coupled with cell specific patterns of expression, provide each cell type with a sophisticated and unique means of regulating intracellular cAMP through PDE action. This diversity offers potential for developing highly specific therapeutic agents. There is great interest in developing PDE4 family selective inhibitors for treating Chronic Obstructive Pulmonary Disease (COPD), asthma and other diseases. However, the therapeutic deployment of PDE4 inhibitors has been limited by side effects, such as nausea. It is now appreciated that PDE4 activity is attributable to a large family of isoforms. This has prompted the notion that selective inhibition of particular PDE4 isoforms may maximise therapeutic benefit whilst minimising side effects. Four PDE4 genes produce around 20 isoforms that each have a unique N terminal region and either both UCR1/2 or just UCR2 regulatory domains. Individual PDE4 isoforms have unique functional roles that are attributable to defined intracellular targeting / association with protein complexes. This provides a major underpinning of compartmentalised cAMP signalling. We aim to determine the functional role and basis by which the N terminal and UCR1/2 tailor intracellular targeting, interaction with signal scaffold proteins and regulation by phosphorylation of specific PDE4 isoforms. Our studies have implications for the identification of novel means of generating inhibitors selective for specific PDE4 isoforms by preventing specific PDE4 isoforms from being targeted to their functionally relevant sites in cells. This provides a route to overcome problems in making effective isoform-selective inhibitors by traditional means, namely directed at the active site, as the PDE4 catalytic unit is identical within PDE4 sub-families & very similar between them. Identification of PDE4 interacting proteins has potential for identifying novel biomarkers for COPD. Analysis of post-translational modification of isoforms has potential for appreciating aberrant control of specific PDE4 isoforms in disease. In particular we will focus on (i) PDE4A4, which is selectively up-regulated in COPD macrophages; (ii) PDE4B2, which has been implicated as of importance in regulating macrophage functioning; (iii) PDE4D5, which is up-regulated in macrophages of smokers, regulates beta2AR functioning through beta-arrestin sequestration and is induced by hypoxia in smooth muscle cells and (iv) PDE4D3, which is the major PDE4 in monocytes.

Publications

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Houslay MD (2009) Disrupting specific PDZ domain-mediated interactions for therapeutic benefit. in British journal of pharmacology

 
Description FP6 (co-PI)
Amount £222,000 (GBP)
Organisation Sixth Framework Programme (FP6) 
Sector Public
Country European Union (EU)
Start 10/2006 
End 09/2009
 
Description Network of Excellence (co-PI)/Center for Translational Molecular Medicine (Netherlands)
Amount £208,000 (GBP)
Organisation Center for Translational Molecular Medicine (CTMM) 
Sector Academic/University
Country Netherlands
Start 10/2009 
End 09/2012
 
Description Project Grant (co-PI)
Amount £322,000 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2010 
End 09/2013
 
Description Transatlantic Network of Excellence (co-PI)
Amount £500,000 (GBP)
Organisation Transatlantic Networks of Excellence in Cardiovascular Research Program 
Sector Academic/University
Country France
Start 10/2006 
End 09/2011
 
Title DNA-PK nuclear-cytoplasmic trafficking 
Description Use of EPAC agonists to promulgate the nuclear exit of DNA-PKc. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
Impact 1. Suggests the potential therapeutic use of EPAC agonists as radio- or chemo-sensitizers by removing DNA-PKc from the nucleus and so hindering repair of double stranded breaks. 2. First identification of a mechanism where cAMP provides dual inhibitory and stimulatory inputs through different effectors. 
 
Title Mapping of potential ubiquitination sites 
Description Spot immobilized peptide arrays used for in vitro ubiquitination in order to determine potential sites of modification. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact Has potential for rapid and cheap site identification that otherwise would have to be undertaken by exhaustive and expensive mutagenesis. 
 
Title PDE expression profiling 
Description We have shown that PDE expression profiling can provide a potential diagnostic indicator for different stages of prostate cancer. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This has led to three patents being filed. 
 
Title PDE4 antisera 
Description antisera specific for PDE4 subfamilies and various PDE4 isoforms 
Type Of Material Antibody 
Year Produced 2009 
Provided To Others? Yes  
Impact High quality antisera developed both by ourselves and in collaboration with Millipore to aid our work and that of the academic community. 
 
Title PDE4/beta-arrestin peptide disruptor 
Description A cell permeable peptide that disrupts the association of PDE4D5 with beta-arrestin and so promulgates beta2-adrenoceptor signalling. 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact PDE4 inhibitors have potential utility in asthma and COPD but their deployment has been hindered due to side effects of emesis because active-site directed inhibitors act on all 25+ isoforms. This technology shows that disrupting the targeting of a specific disease-associated isoform can lead to the 'functional' inhibition of a single species. This addresses the PDE4D5 isoform, which regulates beta2-adrenoceptor signalling and causes resistance to the action of beta2 agonists used in asthma treatment as it is up-regulated with their chronic use. Our data suggests that the development of peptidomimetics or small molecules that disrupt the association of PDE4D5 with beta-arrestin will promulgate beta2-adrenoceptor signalling and so could be used in cases of beta2-agonist (eg salbutamol) resistant patients. 
 
Description Cardiac IKs K+ channel 
Organisation University of Washington
Department Department of Pharmacology
Country United States 
Sector Academic/University 
PI Contribution Project development Reagent provision. Mapping protein-protein interfaces with peptide arrays. Development of mutant PDE4 species.
Collaborator Contribution Identification of a novel system where PDE4 is involved
Impact Publications
Start Year 2008
 
Description DISC1 partnerships 
Organisation Johns Hopkins University
Department Department of Psychiatry and Behavioral Sciences
Country United States 
Sector Academic/University 
PI Contribution Project development. Protein-protein mapping reagent generation DISC1 expression
Collaborator Contribution novel in vivo system access novel system for analysing the PDE4 partner, DISC1
Impact Publications
Start Year 2007
 
Description Identification of PDE4A8, a novel cAMP phosphodiesterase 
Organisation F. Hoffmann-La Roche AG
Country Global 
Sector Private 
PI Contribution Allowed us access to a novel PDE4A clone without having to generate it ourselves. Created useful contacts.
Collaborator Contribution Allowed us access to a novel PDE4A clone without having to generate it ourselves. Created useful contacts.
Impact Contributed experimental research work for a joint publication involved in the identification and characterisation of a novel cAMP phosphodiesterase.; Allowed us access to a novel PDE4A clone without having to generate it ourself. Given access to a company that may lead to future collaborations. Created useful contacts.
Start Year 2007
 
Description Novel allosteric regulators of PDE4 
Organisation deCODE genetics
Country Iceland 
Sector Private 
PI Contribution Analyzed the effect of a novel PDE4 selective inhibitor in one of our assays systems to determine if there was potential for collaboration. deCode has since gone into chapter 11 bankruptcy so the project could not continue.
Collaborator Contribution Allowed us access to novel PDE4 inhibitors, to novel structural data and to purified PDE4 proteins with possibility for joint research/publication and future funding.
Impact Allowed us access to novel PDE4 inhibitors, to novel structural data and to purified PDE4 proteins with possibility for joint research/publication and future funding.
Start Year 2008
 
Description PDE and immunosuppression 
Organisation Yeshiva University
Department Department of Molecular Pharmacology
Country United States 
Sector Academic/University 
PI Contribution Joint project management PDE4 reagents PDE4 analsyes Ubiquitin ananlsyes Peptide mapping
Collaborator Contribution Identification of a novel system where PDE4 is involved
Impact Publications Novel reagents
Start Year 2007
 
Description PDE in Psychiatric disease 
Organisation Pfizer Ltd
Department Psychiatry Pfizer
Country United Kingdom 
Sector Private 
PI Contribution Expertsie on PDEs Reagents Anayses of PDE actiity and expression Peptide mapping
Collaborator Contribution reagents access to novel in vivo systems
Impact Publications Imminent signing of a contract for PDE4:DISC1 research
Start Year 2008
 
Description PDE4 and vascular permeability 
Organisation Queen's University
Department Department of Pathology & Molecular Medicine
Country Canada 
Sector Academic/University 
PI Contribution Project management PDE4 activity and expression analyses
Collaborator Contribution Joint project management. Access to unique in vivo models
Impact Publications
Start Year 2007
 
Description PDE4 antisera and cell lines 
Organisation Merck
Department MilliporeSigma
Country United States 
Sector Private 
PI Contribution Jointly generating antisera to PDE4 isoforms and sub-families. We are supplying know-how and testing; they are generating the antisera and cell lines. Our lab has received 16000 GBP to offset costs plus a share in the reagents generated and Univ Glasgow will obtain a share in royalties for any sales
Collaborator Contribution Jointly generating antisera to PDE4 isoforms and sub-families. We are supplying know-how and testing; they are generating the antisera and cell lines. Our lab has received 16000 GBP to offset costs plus a share in the reagents generated and Univ Glasgow will obtain a share in royalties for any sales
Impact Jointly generating antisera to PDE4 isoforms and sub-families. We are supplying know-how and testing; they are generating the antisera and cell lines. Our lab has received 16000 GBP to offset costs plus a share in the reagents generated and Univ Glasgow will obtain a share in royalties for any sales
Start Year 2008
 
Description PDE4 dimerisation 
Organisation University of Alabama at Birmingham
Department Department of Radiation Oncology
Country United States 
Sector Academic/University 
PI Contribution Led study.
Collaborator Contribution Yeast 2-hybrid expertise; generated mutants
Impact Publications being written up. Impact on understanding the dynamics of PDE4 partnerships in cells. Impact on understanding selective inhibition
Start Year 2007
 
Description PDE4 selective Inhibitors 
Organisation Basilea Pharmaceutica
Country Switzerland 
Sector Private 
PI Contribution We have received funds to analyse novel PDE4 inhibitors with a view to potential further interactions with the company.
Collaborator Contribution Supply of novel inhibitors
Impact We have received funds to analyse novel PDE4 inhibitors with a view to furthering interactions should the company continue work in this new area.
Start Year 2007
 
Description PDE4A and sleep 
Organisation University of Pennsylvania
Department Department of Neuroscience
Country United States 
Sector Academic/University 
PI Contribution Project management Analsysis of PDE4 activity and expression Provision of reagents
Collaborator Contribution Publications. Access to in vivo analyses
Impact Publications
Start Year 2008
 
Description PDE4D5 and FAK:RACK1 complexes in direction sensing complexes 
Organisation Cancer Research UK
Department Edinburgh Cancer Research UK Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution we describe a novel "direction-sensing" pathway that links the integrin effector focal adhesion kinase (FAK), the molecular scaffold protein RACK1, and activity of one of its client proteins, PDE4D5, a cAMP-degrading phosphodiesterase. we identified the key complexes, mapped the interaction surfaces and developed disruptors
Collaborator Contribution Provison of a novel system to explore the functional role of our RACK1:PDE4D5 complex
Impact Publication. We are developing small molecules / peptide disruptors as potential therapeutics
Start Year 2007
 
Description RACK1 signalling functions 
Organisation University College Cork
Department BioSciences Institute
Country Ireland 
Sector Academic/University 
PI Contribution Defining protein protein interactions with peptide arays; generating mutants;
Collaborator Contribution Expertise in FAK; novel cell / in vivo systems.
Impact Publications Peptide disruptor development.
Start Year 2007
 
Description Reverse transcriptase inhibitor actions on PDE4 
Organisation Purdue University
Department Department of Medicinal Chemistry and Molecular Pharmacology
Country United States 
Sector Academic/University 
PI Contribution Analsysi of small molecules in various PDE4 assay systems
Collaborator Contribution Provison of small molecules for analyses in our Labs
Impact Publications
Start Year 2007
 
Description Small molecule PDE4 subfamily inhibitors 
Organisation University of Texas at San Antonio
Department Department of Biology
Country United States 
Sector Academic/University 
PI Contribution provision of pde4 sub-family specific assay systems provision of cell-based cell systems for anti-inflammatory analyses project development
Collaborator Contribution provision of small molecule PDE inhibitors from a novel yeast-based screen
Impact developing molecules for publication / IP
Start Year 2009
 
Description Stem Cells 
Organisation Scottish Enterprise
Country United Kingdom 
Sector Public 
PI Contribution PDE know how
Collaborator Contribution provided stem cell know how and various state of the art pieces of equipment
Impact PDE2 inhibitor, Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) found to block differentiation and maintains the expression of pluripotency markers in human embryonic stem cells. this has led to a patent
Start Year 2006
 
Description Sub-plasmalemma cAMP compartments 
Organisation University of Washington
Department Department of Pharmacology
Country United States 
Sector Academic/University 
PI Contribution Joint project development. Novel reagents: cDNA constructs, antisera Dominant neative PDE4 approach
Collaborator Contribution Novel system for understanding PDE4 function
Impact Publications
Start Year 2006
 
Description T-cell receptor activation in human primary T cells 
Organisation University of Oslo
Department Biotechnology Centre of Oslo
Country Norway 
Sector Academic/University 
PI Contribution Identification of a novel complex of beta-arretsin/PDE4/Akt regukated by CD28 that controls T-cell receptor activation. Mapping sites, defining function.
Collaborator Contribution Provided access to a novel system for analyses
Impact Publications
Start Year 2007
 
Description cAMP signalling in prostate cancer 
Organisation Koninklijke Philips Electronics N.V.
Department Philips
Country Global 
Sector Private 
PI Contribution I supervise a research student who works full-time in my laboratory. My team provide underpinning expertise of cAMP phosphodiesterases.
Collaborator Contribution Studentship support. Has provided new insights and indicated new directions plus access to novel technologies.
Impact Studentship support. Has provided new insights and indicated new directions plus access to novel technologies. Has led to three patent applications
Start Year 2007
 
Title PDE expression profiling 1 
Description PDE4D7 expression profiling as a diagnostic indicator for stages of prostate cancer 
IP Reference WO2010131195 
Protection Patent application published
Year Protection Granted 2010
Licensed No
Impact Expected to lead to major publications
 
Title PDE expression profliing 2 
Description PDE9 expression profiling as a diagnostic indicator for stages of prostate cancer 
IP Reference WO2010131193 
Protection Patent application published
Year Protection Granted 2010
Licensed No
Impact Exepcted to lead to major publciations
 
Title PDE expression profliing 3 
Description PDE PCR panel as a diagnsotic tool for prostate cancer 
IP Reference WO2010131194 
Protection Patent application published
Year Protection Granted 2009
Licensed No
Impact Expected to lead to major publications
 
Title STEM CELL CULTURE METHODS 
Description We have discovered a novel way of blocking the spontaneous differentiation of hESCs in the absence of exogenous cytokines by supplementing feeder-free conditions with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an established inhibitor of cyclic nucleotide phosphodiesterase- 2 (PDE2). 
IP Reference WO2010084300 
Protection Patent application published
Year Protection Granted 2010
Licensed Yes
Impact generated a publication. enhanced career prospects of involved scientists
 
Title PDE4 antibody panel 
Description A panel of antisera specifc for PDE4 sub-fmailies and specific PDE4 isoforms. Now fully developed and marketing being assessed by Milipore. 
Type Support Tool - For Fundamental Research
Current Stage Of Development Market authorisation
Year Development Stage Completed 2010
Development Status Under active development/distribution
Impact Will provide our group with access to first rate diagnostic reagents essential for our research. Will provide the research community with the same high quality reagents. 
URL http://www.antibodypedia.com/gene/4321/PDE4A/antibody/555536/ABS23
 
Title PDE4 stable cell lines 
Description Marketing being developed by Millipore 
Type Support Tool - For Fundamental Research
Current Stage Of Development Market authorisation
Year Development Stage Completed 2010
Development Status Under active development/distribution
Impact Will provide a novel platform for development of PDE4 selective therapeutics to treat inflammatory disease. 
 
Description Alumni letter 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact described life as a biomedical scientist

hopefully interested people as received feedback
Year(s) Of Engagement Activity 2008
 
Description School - Renfrewshire 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Talk on research in signaling systems and biomedical impact.

Hopefully increased applications to study biomedical sciences
Year(s) Of Engagement Activity 2008
 
Description Science Festival Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Manning of a stand at a science festival event for schools. Continual flow of pupils though.

Enquiries form students about courses and careers
Year(s) Of Engagement Activity 2010
 
Description Sleep Radio Broadcast 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Radio broadcast interview

Email contact from members of public. Contact from clinicians involved in sleep research
Year(s) Of Engagement Activity 2009
 
Description Sleep press release 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact press release on use of PDE4 inhibitors for correcting cognitive defects upon sleep deprivation.

request for information from various journalists and members of the public.
Year(s) Of Engagement Activity 2009