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Small-Molecule Probes – Future Helpmates in Diagnostics and Monitoring

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These probes typically consist of one or several fragments (usually a nucleotide analogue and a peptide) that are responsible for the selective recognition of the target, and a fluorescent dye that serves as a reporter, showing where the probe and target are located according to Darja Lavõgina, senior researcher at the Institute of Chemistry, University of Tartu. “Basically, we set out to develop small ‘bugs’ that light up when they see their partner engaged in suspicious activity,” she said.

What is more, while the activity of the target can be monitored, it is also possible to intercept it. “Therefore, we expect our probes are useful for both fundamental research (from the aspect of dissection of crucial intracellular pathways) as well as for the pharmaceutical industry,” Lavõgina added.

Very Informative Protein Kinases

Kaido Viht, researcher at the Institute of Chemistry, explained that more than 100 small compounds targeting protein kinases are under scrutiny in clinical trials, mostly as anticancer drugs, and around 30 have already reached the market. “However, as the process of drug development is extremely time-consuming and costly, it would be extremely beneficial to subject the drug candidates to an elaborate screening before they enter clinical trials. On the other hand, much attention has recently been focused on the role of protein kinases as cancer biomarkers,” he said.

Schematic structure of fluorescent probes targeting the protein kinase Aurora A. If exposed to low-wavelength UV-irradiation, probes generate active radical species that result in the formation of a cova-lent complex between the probe and kinase. Published in Lavogina et al., Bioorg Med Chem Lett. 2015.
Schematic structure of fluorescent probes targeting the protein kinase Aurora A. If exposed to low-wavelength UV-irradiation, probes generate active radical species that result in the formation of a cova-lent complex between the probe and kinase. Published in Lavogina et al., Bioorg Med Chem Lett. 2015.

Viht also explained that a biomarker is a naturally occurring molecule owing to the excessive presence of which a disease can be diagnosed or monitored. So, although protein kinases are intracellular proteins, higher levels of them have been detected in bodily fluids in case of several diseases. “Hence, by identifying protein kinases in patient samples, it could be possible to diagnose certain diseases or assess the success of therapies,” Viht concluded.

A fluorescent probe marks the characteristic location of Aurora A in the mitotic cell. The probe was ap-plied onto live HeLa cells 1 h prior to fixation and staining with an Aurora-A selective antibody.
A fluorescent probe marks the characteristic location of Aurora A in the mitotic cell. The probe was ap-plied onto live HeLa cells 1 h prior to fixation and staining with an Aurora-A selective antibody.

The project “Small-molecule probes for studies of protein kinase signalling (Aurora A/PIM/NuMa pathway) in live mitotic cells” started in January 2013. For now, the group has managed to design selective fluorescent probes for 3 targets (Aurora A, Haspin and CK2), and developing probes targeting another 4 protein kinases (Aurora B and Pim1-3) is still in progress. “Our probes have shown extremely high efficiency in biochemical experiments and measurements in cell lysates,“ Lavõgina said.

Viht added that they have also worked intensely on creating strategies enabling the delivery of the probes into live cells, as the cells tend to protect themselves from ‘invaders’. “We could also successfully affect the intracellular pathways of the protein kinase CK2, an important target of research with therapeutic potential,” he said.

Visualization of the selectivity profile of a probe targeting the protein kinase CK2. Out of 140 protein ki-nases, the compound affected its target CK2 the most. Data from Viht et al., Bioconjug Chem. 2015.
Visualization of the selectivity profile of a probe targeting the protein kinase CK2. Out of 140 protein ki-nases, the compound affected its target CK2 the most. Data from Viht et al., Bioconjug Chem. 2015.

Encouraging Results

The application of probes in cell cultures of cancerous and healthy cell lines also yielded some encouraging results. For instance, the location of Aurora A could be monitored in cells prior to cell division, and the cells could be prevented from completing division according to Lavõgina. Still, she added that there is still room for improvement. “In general, we realised the extreme delicacy of the intracellular balances of targets, possible off-targets and probes that affect the adequacy of monitoring signalling pathways.”

PhD student Katrin Kestav said that another target for which they have been trying to develop the probes is the Greatwall kinase. “Greatwall was discovered only recently and plays an important role in cell division. On the other hand, it is also a ‘tricky’ kinase, as not much information is available about Greatwall to date—this definitely slows down creating effective probes for it,” she said.

The project lasts until the end of the year. Lavõgina has a clear vision for the future: “Now that we have effective, selective and biologically stable probes targeting protein kinases, we would like to focus more closely on the role of protein kinases as biomarkers of diseases. After we have conclusively formulated the list of targets that we can take a closer look at, we will specify the set of protein kinase-related diseases in relation to which our probes will be tested for the diagnosis and monitoring of patients.”

Kestav added that in developing diagnostic methods, the ease of use and cost-effectiveness of a method are also important aspects. “We believe that our compounds will be of great value for such novel diagnostics platforms,” she said.

This article was supported by the European Regional Development Fund through Estonian Research Council.

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