Automated LC/MS-coupled protein kinase C assay for discovering active compounds from natural sources
Dr. Jouni Jokela, University of Helsinki, Finland
 

New PKC translocation inhibitors
M. Sc. Virpi Talman, University of Helsinki, Finland
 

Effective bisubstrate analogue-type inhibitors of protein kinases for biological applications
Dr. Asko Uri, University of Tartu, Estonia

 

Automated LC/MS-coupled protein kinase C assay for discovering active compounds from natural sources
Dr. Jouni Jokela, University of Helsinki, Finland
 

The aim of this study was to develop a non-radioactive HTS-method capable of detecting protein kinase active compounds from nature, especially from microbial sources. The effect of cyanobacterial, fungal or other natural product extracts on protein kinase C activity was studied on 96/384 well plates using a liquid handling workstation Biomek® FX. After enzymatic reaction well plates were transferred to LCMS and partially LC-separated phosphopeptide was quantififed with MS using 13C-15N-labeled phosphopeptide as internal standard.

Four hundred samples (one 384 well plate + controls) could be analyzed ~overnight (20 h). Quality parameters signal-tobaseline (S/B) ratio from 29 to 32, signal-to-noise (S/N) ratio from 10 to 38 and Z' value from 0.7 to 0.9 showed that the method works reliably. The IC50 values of the PKC inhibitors tested were in good accordance with values from the literature. The method was able to sensitively detect added staurosporin (PKC inhibitor) from LC-fractionated cyanobacterial extracts.

 

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New PKC translocation inhibitors
M. Sc. Virpi Talman, University of Helsinki, Finland
 

Protein kinase C (PKC) is a family of serine/threonine kinases that regulate various cellular processes including cell proliferation and malignant transformation. We have used the X-ray structure of PKC- delta C1b domain with bound phorbol ester as a template for molecular modelling to design ligands which compete with phorbol esters for binding and thus modulate PKC activity. The effect of the most promising ligands on PKC translocation was tested in living cells. HeLa cells transfected with PKC—green fluorescent protein (GFP) constructs were pretreated with the ligands and stimulated with phorbol ester. The translocation of PKC-GFP constructs was visualized with confocal microscopy and quantified from confocal microscopic images captured during the experiments. Three out of eight hydrophobic compounds tested inhibited phorbol ester induced PKC translocation in micromolar concentrations. These novel PKC translocation inhibitors could be used as lead molecules in drug development.

 

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Effective bisubstrate analogue-type inhibitors of protein kinases for biological applications
Dr. Asko Uri, University of Tartu, Estonia
 

Despite serious selectivity problems (all 500 protein kinases and more than 1500 other proteins are able to bind purine nucleotides) the main efforts of drug companies have been concentrated on the development of ATP competitive inhibitors. Development of bisubstrate analogue (biligand) inhibitors that simultaneously bind toATP and protein binding sites could logically give more selective and potent inhibitors of kinases. Bifunctional inhibitors of basophilic PK designed by us comprise moieties targeted at the ATP binding site, adenosine-5?-carboxylic acid, and the protein/peptide substrate binding site-directed arginine-rich peptides. The optimization of the structures of these motifs and the linker has lead to inhibitors with Ki in low nanomolar range. These inhibitors were used as affinity adsorbents with tailored efficacy for chromatography and pull-down assays of kinases. Fluorescently labeled biligand inhibitors were applied for the determination of kinase binding characteristics of nanomolar and micromolar inhibitors with confocal single-molecule and classical optical methods.

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