Second year of the project

Several hundred natural extracts have been prepared from cyanobacterial and fungal strains and also from plants, pure compounds isolated and preliminary activity screening has been done. In addition several hundred synthetic compounds targeted to various protein kinases have been synthesised.

Groups oriented to synthetic organic chemistry (WP3) use the modern methods of organic synthesis to prepare compounds that are based on natural product screening (WP1) or virtual screening of known chemicals (WP2). These synthesis products can be tested in various protein kinase binding and activity assays to find the hit molecules. This provides essential feed-back information for the molecular modellers and synthetic chemists to refine the chemical structure of the found hits within the iterative process of drug discovery and development.

Protein Kinase Research Consortium (PKRC) chemical library and databases of synthesised and extracted compounds have been uploaded and updated to the intranet. Computational tools for generation of 3D models of protein kinases and for virtual screening of protein kinase-targeted compounds have been developed within Consortium. Tens of thousands of compounds have been screened virtually and the promising hits have been submitted for experimental validation to further testing within Consortium.

A number of the novel compounds show biological activity in various assay systems utilising purified kinase enzyme preparations. Several recombinant protein kinase domains have been expressed and used in the screening for ligand binding. Indeed, there are more than 150 different kinase activity assays available within the Consortium, thus the binding affinity and biological activity of potential protein kinase inhibitors/activators can be screened with the help of extensive collaboration.

Rational drug design relies on three dimensional models of proteins and more specifically of the active site – the ligand binding site. In order to determine the crystal structure of the critical binding sites of different kinases, large quantities of various protein kinases have been produced in cell cultures. Structures of different kinase/inhibitor complexes and a novel protein kinase have been solved. (WP4)

Protein kinases represent a significant therapeutic target in many pathologies as they represent the effectors of cell signaling pathways that control a variety of cell functions including cell proliferation, differentiation and cell death. In WP5 various model systems for a range of diseases have been utilised to test therapeutic potential of novel compounds of both natural and synthetic origin. The diseases include cancer (with leukaemia as a specific disease targeted as well as breast, glia and gastric tumours), epilepsy, heart disease and post-stroke neurodegeneration, leishmaniasis and inflammatory disease. The protein kinases that have been the focus of the project include tyrosine kinases (receptor and cytoplasmic), Protein kinase C, MAP kinases and Cyclin Dependent Kinases. In the second year of the project most partners in WP5 have progressed to second generation screening of agents identified as of interest in year 1, while other have carried out further investigations of compounds that were already identified in year 1 as potential lead compounds.

Several protein kinase inhibitors showed inhibitory activity in vascular smooth muscle proliferation assay and therefore it is possible that in the future protein kinase inhibitors may be used in the treatment of re-stenosis after balloon surgery (angioplasty) of coronary arteries. More than 100 compounds originating from consortium laboratories have been tested in anti-parasite assays against Leishmania species. Some compounds show promising activity and this observation can open new avenues in the treatment strategies of parasite diseases in the future. Leishmanial protein kinases have also been cloned and expressed.

Natural and synthetic compounds have been tested in models of leukaemia and already 3 plant and 3 synthetic PKC-modulating compounds show high potency against leukaemic cells. The effects of selected inhibitors on neuronal excitotoxicity (models of neuronal death identified during stroke) were tested and identified as neuroprotective agents. One protein kinase inhibitor was shown to display neuroprotective effect in an animal model of stroke. The lead compound and its potential successors will be further studied for a possible novel stroke therapy. Some of these inhibitors may also modulate neuronal excitability and may represent a new approach for epileptic treatment.

See also our scientific publications.