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Session 3: SER/THR KINASES I

Chair: Prof. Janet Lord, University of Birmingham
 

  Lectures and Oral Communications in Red are from members of our Consortium.
 
   



The PKC Superfamily as Therapeutic Targets
Prof. Peter Parker, Cancer Research, UK
 

    Protein Kinases as Targets for Anti-Leukaemic Therapy
Prof. Janet Lord, University of Birmingham, UK
 
    cAMP-Dependent Protein Kinase
Prof. Stein-Ove Døskeland, University of Bergen, Norway
 
    Structural Aspects of the Cyclin Dependent Kinases
Prof. Dame Louise Johnson, University of Oxford, UK
 
    CDK-Inhibitors: Selectivity and Cellular Mechanisms of Action
Prof. Laurent Meijer, CNRS (Roscoff), France
 
     


The PKC Superfamily as Therapeutic Targets

Prof. Peter Parker, Cancer Research, UK

Manu De Rycker1, Brenda Kostelecky1, Sven Kjaer1, Philip Whitehead1, Jos Joore2, Peter Goekjian3, Neil McDonald1 and Peter J Parker1.
1London Research Institute, CR-UK, London, UK. 2Pepscan Systems, Lelystad, Netherlands. 3UMR 5181, Université Claude Bernard-Lyon 1, Villeurbanne France.

 
   

The protein kinase C (PKC) superfamily are implicated in controlling a variety of cellular processes. Physiologically, non-redundant functions for specific isoforms have been identified in the CNS, immune functions, the vasculature and early development. Pathologically, there is accumulating evidence for dysfunctional roles in a number of cancers. The balance of physiological and pathological data indicates that certain members of the PKC superfamily provide a suitable therapeutic index that lends them to the drug development process. Within the EU framework 6 Protein Kinase Consortium we have been working on structure based routes to the

Peter Parker

Prof. Peter Parker

identification of novel, selective PKC superfamily inhibitors. The context of these objectives and the developments in this area will be presented.

 

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Protein Kinases as Targets for Anti-Leukaemic Therapy

Prof. Janet Lord, University of Birmingham

Janet M Lord, P. Hampson and K Wang
MRC Centre for Immune Regulation, Birmingham University Medical School, Birmingham B15 2TT, UK


Leukaemia is cancer of the blood cells. Like most cancers, leukaemic cells show a variety of alterations in genes controlling cell proliferation (e.g. Flt3, cyclinD) or apoptosis (bcl2, abl). Protein kinases have become prime targets for anti-leukaemic therapy since the success of the tyrosine kinase inhibiting drug Glivec in the treatment of patients with chronic myeloid leukaemia. Our own interest is in Protein Kinase C (PKC) as a therapeutic target for Acute Myeloid Leukaemia (AML). AML is characterised by a block in the differentiation of haematopoietic stem cells to cells of the myeloid lineage, leading to an abnormal accumulation of immature precursors. PKC, a family of 12 signalling isoenzymes that regulate many cell processes including proliferation, differentiation and apoptosis, has already been the target of several novel anti-cancer agents. In particular, two isoenzymes, PKC-
a and PKC-d, appear to play specific roles in tumor promotion and suppression. Studies will be described that have used a plant derived broad specificity activator of PKC, Ingenol-3 angelate (PEP005), as a treatment for AML. To date we have shown that this diterpene ester is able to induce apoptosis in leukaemic cell lines as well as primary AML cells isolated from patients with AML. Of 60 samples screened only 8 were unresponsive to PEP005 and unresponsiveness was mainly among the less differentiated sub-type of AML (M1). Expression of PKC-delta was required for responsiveness to PEP005. In a second study we are using novel synthetic PKC-alpha activating agents produced by Professor Jari Yli-Kauhaluoma. These agents are being used to target Chronic Lymphocytic Leukaemia (CLL), which is associated with altered PKC-alpha expression. Only those leukaemic cell lines expressing PKC alpha can respond by entering apoptosis and primary human CLL blasts are also very sensitive to these agents.
 


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cAMP-Dependent Protein Kinase
Prof. Stein Ove Døskeland
Endre Kjærland, Kristin Viste, Rune Kleppe, Stein Ove Døskeland
Department Biomedicine, Med. Faculty, University of Bergen, Norway
 

Background: The cAMP-dependent protein kinase (cA-PK, PKA) is conserved from yeast to mammals. The R subunit is responsible for subcellular anchoring (to AKAP´s). It has two cAMP binding sites from which have evolved the regulatory cAMP sites in ion channels and Epac. Epac activates the small G-protein Rap.

PKA and other cAMP receptors as potential drug targets:
The C-subunit of PKA has still not been specifically targeted – the much used H-89 is a strong inhibitor of other protein kinases, like ROCK. This is unfortunate since PKA inhibits RhoA and thereby ROCK action - an improved inhibitor is required.

The R subunit of PKA can be successfully targeted by stimulatory cAMP analogs (like N6-benzoyl-cAMP) and inhibitory analogs (like Rp-8-Br-cAMPS). Only when two cAMP analogs, each preferring one of the two sites of R, are combined will selective activation of isozymeI or II of PKA be achieved. Isozyme selectivity is required to focus an effect on cell types expressing mainly one isozyme (e.g. type II in fat or type I in leukemia cells).

Anchoring of PKA can be abolished by competing peptides, and is an obvious target for novel, small, membrane-permeable molecules.

Epac is selectively activated by 8-CPT-2´-O-Me-cAMP. Specific inhibitors are under development.
PKA may be an unexpected target in cells exposed to proteasome inhibitors. Note that the level of R subunit can influence PKA activity even at saturating cAMP.

Activation of PKA type I and CREB-dependent transcription of Bim induces rat leukemia cell death only when cyclin-dependent protein kinase 5 (cdk5) is active. This model offers a test for modulators of PKA and cdk5.

 

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Structural Aspects of the Cyclin Dependent Kinases
Prof. Dame Louise N. Johnson,
Laboratory of Molecular Biophysics, Biochemistry Department, University of Oxford, Oxford OX1 3QU, UK and Diamond light Source, Chilton, Berks, UK.
 

Protein kinases are key components of cell signalling pathways. Defects in these processes lead to diseases such as cancer, diabetes and arthritis and hence protein kinases have become targets for drug design and therapy. We recently reviewed progress in this field with reference to kinase inhibitors that are in clinical trials or in the clinic and for which structural information is available [1]. In this talk I shall review some of our work with reference to structural studies on cell cycle protein kinases [2] and I shall expand the discussion to consider wider aspects of substrate recognition with reference to CDK2/cyclin A, CDK2/cyclin E [3], CDK7 [4] and polo-like kinase [5].

References:
[1] Noble, M.E., et al. Science, 2004, 303, 1800. [2] Davies, T.G., et al.. Nature Structural Biology, 2002, 9, 745. [3] Honda, R. et al., Embo J, 2005, 24, 452. [4] Lolli, G., et al., Structure (Camb), 2004, 12, 2067. [5] Cheng, K.-Y., et al. EMBO J., 2003, 22, 5757.

 

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CDK-Inhibitors: Selectivity and Cellular Mechanisms of Action
Prof. Laurent Meijer,
Cell Cycle Laboratory, Station Biologique de Roscoff, C.N.R.S., BP 74, 29682 ROSCOFF cedex, Bretagne, France
 

Cyclin-dependent kinases (CDKs) regulate the cell division cycle, apoptosis, transcription, essential neuronal functions, viral replication. CDKs and their physiological regulators show multiple abnormalities in human cancers. CDKs also regulate b-amyloid formation and tau hyperphosphorylation, two hallmarks of Alzheimer’s disease. Pharmacological inhibitors of CDKs have thus a strong potential for the treatment of cancers, neurodegenerative diseases (Alzheimer’s, Parkinson’s, Nieman-Pick type III, stroke, etc…), diabetes, viral infections, unicellular parasites. Some of our early CDK inhibitors have reached the pre-clinical and clinical stages of pharmaceutical evaluation. For instance, roscovitine (CYC202, Seliciclib), is currently undergoing phase 2 clinical trials against leukaemia, lung and breast cancers, and phase 1 trials against various kidney diseases. It is undergoing pre-clinical animal evaluation against Alzheimer’s disease stroke and Niemann-Pick’s disease type C. Other families of kinase inhibitors are currently being developed in the laboratory, such as the bis-indole indirubins.

Over 100 CDK inhibitors have been identified, among which more than forty have been co-crystallized with CDK2 or CDK5. These co-crystal structures are extremely helpful to design further derivatives with increased potency and selectivity. These kinase inhibitors all target the ATP-binding pocket of the catalytic site of their targets. The actual selectivity of most compounds, and thus the underlying mechanism of their cellular effects, is poorly known. We have developed affinity chromatography using immobilized inhibitors as a straightforward approach to identify the actual targets of kinase inhibitors. Results show that although some compounds are quite selective, single target products are very unlikely to be discovered. This may in fact turn out to be an advantage as cells are unlikely to develop resistance to multiple target drugs.

The selectivity and intracellular mechanism of action of roscovitine has been extensively studied and will be presented as a representative example of the multiple effects of CDK inhibitors in cells, tissues and organisms.

 

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