CDK-containing macromolecular assemblies

Lead Research Organisation: Newcastle University
Department Name: Northern Institute for Cancer Research

Abstract

The growth and division of cells is strictly controlled at the molecular level by a number of enzymes that include the cyclin dependent protein kinases (CDKs). CDKs 1, 2, 4 and 6 are switched on and off in an orderly sequence, to ensure that cell division starts and stops at the required time. Other members of the CDK family (CDKs 7, 8 and 9) are also important for the control of transcription, the process by which genes are transcribed into messenger RNA that in turn serves as the template for protein synthesis. Regulation of transcription ensures the timely expression of proteins required for cell growth and differentiation. Errors in either the control of cell cycle progression or transcription can lead to uncontrolled cell growth and proliferation. Although CDK family members are closely related in sequence, biological studies have revealed that each has unique properties. Their activities can be regulated both by association with different families of regulatory proteins and by enzymatic modification of the protein sequence by addition of phosphoryl or acetyl residues to specific amino acid side chains. Our work, primarily using the technique of X-ray crystallography, allows us to see the structures of CDKs and the complexes they form at atomic resolution and so to learn how they differ from each other. The aim of this proposal is to build upon our research into the structural and functional properties of CDK/cyclin pairs, to establish how structural and biochemical properties are altered when they are located in multi-protein complexes. To this end we propose to identify and reconstitute selected CDK-containing complexes using heterologous expression systems. We will characterise these complexes by structural, biochemical, and biophysical methods. Aberrant CDK activity has been linked to cancer, neurological diseases, and rheumatoid arthritis In recent years, understanding a particular defect that leads to disease has led to exciting new medicines directed towards a particular target (e.g. the drugs Gleevec, Iressa and Herceptin for cancer treatment). A number of CDK-selective inhibitors are in clinical trials for the treatment of cancer. These agents all act by binding to the CDK active site to block CDK activity. Compounds that block other interactions made by CDK/cyclin complexes represent an alternative target for CDK-directed therapies. The work described in this project will aid the further development of such compounds and may also reveal additional targets for inhibitor development.

Technical Summary

Cyclin-dependent protein kinases (CDKs) play an orchestrating role in cellular processes that include transcriptional regulation and the control of cell-cycle progression. Aberrant CDK activity is implicated in diverse clinical conditions including cancer, stroke, neurodegeneration and HIV infection. Whereas CDKs isolated ex vivo are found in large, multi-protein complexes, the prevailing understanding of the CDK structure-function relationship has largely been deduced from the study of variously phosphorylated monomeric or cyclin-bound CDKs. We aim to extend available understanding of the composition, structure and functional properties of the multiprotein complexes that characterise the different CDKs. In particular we will develop a structural view of the complexes that localise and regulate CDK7 and CDK9 activity in transcriptional control, selected complexes that contain CDK4 and CDK6 and coordinate cell-cycle progression with checkpoint and apoptotic pathways, and complexes of CDKs 1 and 2 that coordinate their activity with the ubiquitin-proteolysis system.

Publications

10 25 50
 
Description CRUK studentship
Amount £130,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 09/2015
 
Description DDERP Discovery Award
Amount £10,000 (GBP)
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2014 
End 12/2015
 
Description JGW Patterson Foundation
Amount £125,828 (GBP)
Organisation Newcastle University 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description MRC Responsive Mode
Amount £2,290,641 (GBP)
Funding ID MR/N009738/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 06/2016 
End 05/2021
 
Description MRC Studentship
Amount £95,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 10/2012 
End 09/2016
 
Title CDK1-cyclin B crystals 
Description CDK1-cyclin B suitable for assay and crystallisation. 
Type Of Material Technology assay or reagent 
Year Produced 2016 
Provided To Others? Yes  
Impact None to date. 
 
Title CDK2/cyclin A crystals 
Description Crystals of CDK2/cyclin A suitable for small molecule inhibitor soaks/co-crystallisation 
Type Of Material Technology assay or reagent 
Year Produced 2006 
Provided To Others? Yes  
Impact Large number of CDK2/cyclin A/ATP-competitive inhibitor structures determined that have had an impact on the development of protein kianse inhibitors 
 
Title CDK9/cyclin T crystals 
Description Crystals of CDK9/cyclin T suitable for use in determining binding modes of ATP-competitive inhibitors. Information is useful for the structure-aided design of selective CDK9 inhibitors in academia / pharmaceutical industry. 
Type Of Material Technology assay or reagent 
Year Produced 2010 
Provided To Others? Yes  
Impact Structure determination of CDK9/cyclin T/inhibitor complex structures. 
 
Description Analysis of CDK9/cyclin T complexes by mass spectrometry 
Organisation ExSAR Corporation
Country United States 
Sector Private 
PI Contribution Provision of CDK9/cyclin T/BRD4 and /HEXIM1 protein complexes of sufficient purity for H/D exchange analysis. Initial complex characterisation by X-ray crystallography and NMR.
Collaborator Contribution Access to equipment, significant intellectual input and experimental expertise.
Impact Determination of molecular model for both the CDK9/cyclin T/BRD4 and /HEXIM1 complexes. Manuscript in preparation. Multidisciplinary: Mass spectrometry, X-ray crystallography, NMR
Start Year 2011
 
Description CDC37/HSP90/CDK complexes 
Organisation University of Sussex
Department Genome Damage and Stability Centre
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK4/6 complexes, biochemical, biophysical and structural characterisation of CDK/CDC37 and CDK/CDC37/HSP90 complexes
Collaborator Contribution Provision of CDC37 and HSP90 proteins, biochemical, biophysical and structural characterisation of CDK/CDC37 and CDK/CDC37/HSP90 complexes
Impact PMID: 29091774
Start Year 2013
 
Description CDK PTMs 
Organisation Leiden University Medical Center
Country Netherlands 
Sector Multiple 
PI Contribution Generation of CDK1- and CDK2-cyclin complexes.
Collaborator Contribution Identification of novel post-translational modifications on CDKs. Characterisation by mass spectrometry
Impact Exchange of reagents.
Start Year 2015
 
Description CDK inhibitors 
Organisation University of South Australia
Country Australia 
Sector Academic/University 
PI Contribution Provision of CDK-cyclin complexes for structure determination bound to ATP-competitive inhibitors
Collaborator Contribution Provision of potent and selective CDK ATP-competitive inhibitors.
Impact Multidisciplinary. Chemical syntheses and assay at University of South Australia, protein structure determination by X-ray crystallography at Newcastle University. Two papers published to date when collaborator was working at the University of Nottingham.
Start Year 2013
 
Description CDK1 and CDK2 complexes with RINGO/Speedy regulators 
Organisation Institute for Research in Biomedicine (IRB)
Country Spain 
Sector Academic/University 
PI Contribution Preparation of CDK1 and CDK2 proteins together with various regulators. Biochemical and structural characterisation of CDK-containing complexes.
Collaborator Contribution Biochemical characterisation of RINGO/Speedy proteins. Provision of unpublished results relating to RINGO/Speedy protein expression and characterisation. Provision of cDNAs, mutants and other RINGO/Speedy reagents. Mouse models to interrogate the functions of RINGO/Speedy in vivo
Impact Collaborator provided data that assisted in the preparation of an application to the MRC for programme grant support.
Start Year 2015
 
Description CDK1/cyclin B/inhibitor complexes 
Organisation Astex Pharmaceuticals
Department Astex Therapeutics Ltd
Country United Kingdom 
Sector Private 
PI Contribution Provision of CDK1/cyclin B protein for structure determination.
Collaborator Contribution Provision of proprietary CDK1/cyclin B-selective inhibitors
Impact None as of 11/2015
Start Year 2013
 
Description CDK2 inhibitor studies 
Organisation Astex Pharmaceuticals
Country United States 
Sector Private 
PI Contribution Provision of CDK/cyclin complexes for structure determination by X-ray crystallography
Collaborator Contribution Provision of proprietary CDK inhibitors and cell-based assay protocols and reagents
Impact PMID 29063678, PMID 29118092, PMID 28005359 Multidisciplinary: small molecule chemical synthesis, biochemical and cellular assays, X-ray crystallography, drug design
Start Year 2013
 
Description CDK2 inhibitor studies 
Organisation Cancer Research UK
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Provision of CDK/cyclin complexes for structure determination by X-ray crystallography
Collaborator Contribution Provision of proprietary CDK inhibitors and cell-based assay protocols and reagents
Impact PMID 29063678, PMID 29118092, PMID 28005359 Multidisciplinary: small molecule chemical synthesis, biochemical and cellular assays, X-ray crystallography, drug design
Start Year 2013
 
Description CDK2 inhibitor studies 
Organisation Newcastle University
Department School of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK/cyclin complexes for structure determination by X-ray crystallography
Collaborator Contribution Provision of proprietary CDK inhibitors and cell-based assay protocols and reagents
Impact PMID 29063678, PMID 29118092, PMID 28005359 Multidisciplinary: small molecule chemical synthesis, biochemical and cellular assays, X-ray crystallography, drug design
Start Year 2013
 
Description CDK4/6-Androgen Receptor 
Organisation Newcastle University
Department Northern Institute for Cancer Research Newcastle
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK and cyclin constructs and protein for in vitro and cell-based studies to elucidate CDK-androgen receptor interactions
Collaborator Contribution Provision of appropriate cell lines and expertise for cell-based studies of androgen receptor function (transfection, si RNA, ChIP and lenti-viral-mediated knockdown),
Impact No publications as of 11/2015. Results of collaborative experiments were included in a subsequent application to the MRC for programme grant funding.
Start Year 2013
 
Description CDK7 ATP-competitive inhibitors 
Organisation Harvard University
Department Department of Biological Chemistry & Molecular Pharmacology (BCMP)
Country United States 
Sector Academic/University 
PI Contribution Co-crystallisation of CDK7 with ATP-competitive inhibitors for structural studies
Collaborator Contribution In vitro and in vivo functional characterisation of inhibitors as leads for drug design.
Impact As yet no outputs, CDK7 still in crystallisation trials. Multi-disciplinary structure/function study to identify leads for anti-cancer drug design. X-ray crystallography, chemical synthesis, in vitro biochemical and cell-based assays.
Start Year 2012
 
Description Cellular studies of CDK4 
Organisation Newcastle University
Department Northern Institute for Cancer Research Newcastle
Country United Kingdom 
Sector Academic/University 
PI Contribution Generation of constructs for cellular studies and supporting biochemical, biophysical and structural characterisation
Collaborator Contribution Provision of expertise in cellular studies and cell imaging
Impact Generation of stable transfected cell lines for further functional characterisation. Collaboration is multidisciplinary: cell culture, FACS and imaging at NICR, biochemical, biophysical and structural characterisation of CDK4-containing complexes at Newcastle.
Start Year 2010
 
Description Determination of CDK9/cyclin T/ATP-competitive inhibitor structures 
Organisation University of Nottingham
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of CDK9/cyclin T for structure determination of inhibitor complexes by X-ray crystallography
Collaborator Contribution Provision of CDK9-specific inhibitors for co-crystallisation studies for structure lead inhibitor design. Hold a CRUK DDERP award (01/01/14-31/12/15) on which we are a collaborator and receive £5000/annum towards consumables.
Impact Multidisciplinary: inhibitor synthesis and in vitro assays at Nottingham, structural characterisation started at Oxford. Activity now transferred to NICR, Newcastle University. Two papers published: 1. Shao,H., Shi,S., Huang, S., Hole, A.J., Abbas, A.Y., Baumli, S., Liu,X., Lam, F., Foely, D., Fischer, P.M., Noble, M.E.M., Endicott, J.A., Pepper, C. and Wang, S. (2013) Substituted 4-(thiazol-5-yl)-2-(phenylamino)pyrimidines are highly active CDK9 inhibitors: synthesis, x-ray crystal structure, SAR and anti-cancer activities. J.Med. Chem. 56:640-59. 2. Hole, A.J., Baumli, S., Shao, H., Shi, S., Huang, S., Abbas, A.Y., Liu, X., Lam, F., Pepper, C., Fischer, P.F., Wang, S., Endicott, J.A. and Noble, M.E.M. (2013) Comparative binding of 2-amino-4-heteroaryl-pyrimidine inhibitors to CDK2 and CDK9. J.Med. Chem. 56:660-70. 3. Successful application for further funding: CRUK Science Committee - Drug Discovery Project Award (PI P. Fischer, Nottingham University, Award ref: C18648/A17648)
Start Year 2009
 
Description EM structure determination of P-TEFb complexes 
Organisation Monash University
Country Australia 
Sector Academic/University 
PI Contribution Preparation of samples for EM analysis.
Collaborator Contribution Expertise in EM data collection and analysis of protein-RNA complexes. Access to a Titan Krios microscope.
Impact With advice from partners, successful generation of samples suitable for data collection. Structure determination in progress.
Start Year 2015
 
Description Structure/function studies of CDK and CDK regulatory protein complexes 
Organisation Ludwig Institute for Cancer Research
Department Oxford Branch
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Generation of constructs for cellular studies, proteins and supporting biochemical, biophysical and structural characterization
Collaborator Contribution Provision of expertise in cellular studies and cell imaging for functional studies.
Impact Two manuscripts published: (1) Lu, M., Breyssens, H., Salter, V., Zhong, S., Hu, Y., Baer, C., Sullivan, A., Brown, N.R., Endicott, J.A., Knapp, S., Kessler, B., Middleton, M.R., Siebold, C., Jones, Y., Sviderskaya, E.V., Cebon, J., John, T., Goding, C.T. and Lu, X. (2013) Restoring p53 function in human melanoma cells by inhibiting mdm2 and cyclinB1/cdk1 phosphorylated nuclear iASPP. Cancer Cell, 23:618-33. (2) Lu, M., Zak, J., Chen, S., Sanchez-Pulido, L., Severson, D.T., Endicott, J., Ponting, C.P., Schofield, C.J., and Lu, X. (2014) A code for RanGDP binding in ankyrin repeats defines a nuclear import pathway. Cell 157:1130-450. Collaboration is multidisciplinary: cell culture, FACS and imaging at Oxford, biochemical, biophysical and structural characterisation of CDK-containing complexes at Newcastle.
Start Year 2009
 
Description CRUK Race for Life (Newcastle) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Public engagement in cancer research. Short talk and thank you to women running a "Race for Life".

Highlights the work of the Institute - interested groups ask for building tours and to meet scientists.
Year(s) Of Engagement Activity 2012,2014,2016,2017
 
Description Department Open Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Open Day posters describing the group's research. Tour of the facilities. Open Day well attended by sixth form students looking to study at Oxford.

An opportunity to inform sixth form science teachers that we welcome students for work experience placements.
Year(s) Of Engagement Activity 2013
 
Description School Workshop (Newcastle) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact 6 x 2hour sessions each attended by between 12-24 year12-13 students which got them to think about serendipity in drug design and asked them to use molecular graphics to design a drug to overcome a drug-resistant target (British Science week Newcastle, 2013). Engagement in British Science week event (Durham, 2016).

Part of an ongoing program of outreach coordinated by members of staff at the Northern Institute for Cancer Research to the local community. Impact monitored by locally based CRUK Outreach Officer.
Year(s) Of Engagement Activity 2013,2015
 
Description School visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Engaging biology and chemistry students at a local school to think about where cancer medicines have come from and will come from in the future. Students had lots of questions and particularly enjoyed the interactive graphics.

Visits such as these have encouraged pupils in year 12 to approach us for a work experience/short summer placement in the lab
Year(s) Of Engagement Activity 2014
 
Description School visit (Newcastle) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Visit to a class of 20 year 1 children to introduce them to doing experiments and analysing the results- chromatography of inks and discussion of life being a scientist as examples.
Year(s) Of Engagement Activity 2014,2015,2016,2017