Structural studies on P-TEFb and its role in regulation of transcription elongation

Lead Research Organisation: University of Oxford
Department Name: Biochemistry

Abstract

Transcription is the process during 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 transcription regulation can lead to uncontrolled cell growth and proliferation. Positive transcription elongation factor b (P-TEFb) controls the elongation phase of transcription that is carried out by RNA polymerase II. Not only is P-TEFb essential for transcription of the vast majority of cellular genes, it is also a critical host cellular cofactor for the human immunodeficiency virus HIV. HIV uses P-TEFb to enable the transcription of its own genome by the cellular transcription apparatus. Increases in P-TEFb activity are also associated with other diseases, for example cardiac hypertrophy and breast cancer. Our study aims not only to contribute to a better understanding of how P-TEFb recognizes its substrates and how its activity is regulated within the cell, but also to identify the mode of action of specific inhibitors of P-TEFb. One such inhibitor is Flavopiridol, a potential drug against cancer that is already in clinical trials. These studies will form the basis for future structure-aided drug discovery initiatives to develop therapeutics targeting P-TEFb. Within the cell P-TEFb activity is controlled by its association with positive and negative regulatory factors, called BRD4 and HEXIM1/7SK RNA, respectively. Our knowledge of the mechanisms by which these regulatory factors control P-TEFb activity would be greatly assisted by knowing their structures in molecular detail. Protein structures can be determined using the techniques of X-ray crystallography and electron microscopy. The aim of the proposed research is to use both these techniques to characterise the molecular details of the active site as well as the organization of the large inhibitory complex. This information will be useful in furthering our understanding of how transcriptional activity is controlled at the stage of transcription elongation and will also facilitate our understanding of how HIV uses the P-TEFb to promote transcription of its own genes. Ultimately we may be able to exploit this knowledge for the treatment of disease.

Technical Summary

In eukaryotes control of transcription is critical for the timely expression of genes during cell growth and differentiation. The different stages of transcription initiation, elongation, 3?end formation and termination, are tightly regulated by different factors which assemble on the 52-repeat C-terminal domain (CTD) of the largest subunit of RNA polymerase II (pol II). Patterns of CTD phosphorylation and dephosphorylation change during the transcription cycle, providing a dynamic assembly platform for the appropriate proteins. Positive transcription elongation factor b (P-TEFb) is a cyclin dependent kinase, CDK9, which phosphorylates the CTD as well as negative elongation factors and thereby promotes processive transcription elongation. Brd4 and HEXIM1/7SK RNA, are respectively positive and negative regulators that dynamically control P-TEFb activity. While Brd4 recruits P-TEFb to sites of active transcription via its bromo domains, association with HEXIM1/7SK RNA leads to the formation of an inactive RNA/protein complex. Association with Brd4 or HEXIM1/7SK RNA is mutually exclusive and stress treatment causes a conversion of the inactive HEXIM1/7SK RNA to the active Brd4-bound form of P-TEFb. The functional equilibrium between the active and inactive forms is disturbed in several major human diseases, such as cardiac hypertrophy, breast cancer and Human immunodeficiency virus (HIV) infection. HIV type 1 uses P-TEFb as a specific cofactor for efficient transcriptional elongation of its genome. To provide a greater understanding of the molecular interactions that mediate P-TEFb substrate selectivity we have recently solved the structure of the CDK9/cyclinT sub-complex. We plan to use SPR and ITC to analyse the interaction of P-TEFb with its substrates and use the insights from these studies to identify CDK9/cyclinT/substrate complexes for crystallisation and structural analysis. The involvement of P-TEFb in cardiac hypertrophy, breast cancer and HIV make it a possible drug target for the treatment of these diseases. Determination of structures for P-TEFb in complex with the clinically relevant CDK9 inhibitor Flavopiridol, and DRB (5,6-dichloro-1-b-D-ribufuranosylbenzimidazole) will allow us to elaborate the binding modes of potent CDK9 inhibitors within the ATP binding site. The second part of the project will address the mechanism by which the natural CDK9 inhibitor HEXIM1/7SK RNA inhibits P-TEFb. A combination of electron microscopy and X-ray crystallography will be used to tackle this complex structural challenge and we will elucidate the relationships between structure and function within the P-TEFb/HEXIM1/7SK RNA complex by in vitro transcription assays.
 
Description Newcastle-Monash Seedcorn Award
Amount £3,000 (GBP)
Funding ID OSR/0190/MONA/MN01 
Organisation Newcastle University 
Sector Academic/University
Country United Kingdom
Start 04/2015 
 
Description Wellcome Trust Studentship
Amount £130,500 (GBP)
Funding ID 083113/Z/07/A 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2008 
End 09/2011
 
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 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 Determination of structures of CDK9/cyclin T ATP-competitive inhibitors. 
Organisation Palacky University
Country Czech Republic 
Sector Academic/University 
PI Contribution Determination of structures of CDK9/cyclin T/ATP-competitive inhibitor complexes.
Collaborator Contribution Provision of small molecule inhibitors for co-crystallisation studies to 1) improve structure resolution and 2) as part of a structure-aided inhibitor design program. Complementary biochemical and cellular characterisation of inhibitors.
Impact Structure determination of CDK9/cyclin T/inhibitor complex structures. One joint review published: Perspective of cyclin-dependent kinase 9 (CDK9) as a drug target. Krystof V, Baumli S, Fürst R. Curr Pharm Des. 2012;18(20):2883-90. Review.
Start Year 2009
 
Description EM structure determination of pTEFb 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Institute of Mineralogy, physical materials and Cosmochemistry IMPMC, Paris
Country France 
Sector Public 
PI Contribution Provision of samples for EM analysis
Collaborator Contribution Provision of electron microscope for data collection and associated support.
Impact Structure determination of a pTEFb-inhibitory complex in progress.Multidisciplinary, EM structure determination assisted by knowledge of constituent proteins determined by X-ray crystallography.
Start Year 2009
 
Description Structural and functional characterisation of CDK9/cyclin T ATP-competitive inhibitors 
Organisation National Center for Scientific Research (Centre National de la Recherche Scientifique CNRS)
Department Biological Station, Roscoff
Country France 
Sector Academic/University 
PI Contribution Determination of structures of CDK9/cyclin T/ATP-competitive inhibitor complexes.
Collaborator Contribution Provision of small molecule inhibitors for co-crystallisation studies to 1) improve structure resolution and 2) as part of a structure-aided inhibitor design program. Complementary biochemical and cellular characterisation of inhibitors.
Impact Structure determination of CDK9/cyclin T/inhibitor complex structures. Manuscript now published (Genes and Cancer, 2010). Multidisciplinary collaboration involving compound synthesis, cellular assays, and X-ray crystallography.
Start Year 2008
 
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