Research helps understand role of protein kinases involved in cancer

In a study conducted under the aegis of the Structural Genomics Consortium, Brazilian scientists have developed synthetic molecules that inhibit the activity of vaccinia-related protein kinases VRK1 and VRK2.

By Karina Toledo  |  Agência FAPESP – Vaccinia-related protein kinases VRK1 and VRK2, which are involved in cell proliferation, are considered potential targets for treatment of prostate, ovarian, colorectal and other kinds of cancer.

To obtain a deeper understanding of the role of these enzymes in human cells, in the contexts of both wellness and disease, Brazilian researchers are working on the development of small synthetic molecules capable of modulating their activity in study models. Recent results of their research have been published in Scientific Reports, an online journal owned by Springer Nature.

“Data in the scientific literature show that VRK1 and VRK2 are found in higher amounts in malignant than normal cells, but we don’t know exactly what they do,” said Rafael Couñago, a researcher at the University of Campinas (UNICAMP) in São Paulo State, Brazil, and first author of the article.

The research is the result of a partnership between the university’s Protein Kinase Chemical Biology Center (SGC-UNICAMP) and Brazilian pharmaceutical company Aché Laboratórios Farmacêuticos. Both are members of the Structural Genomics Consortium (SGC), a public-private partnership that supports drug discovery through open-access research by more than 400 scientists at universities, pharmaceutical companies and nonprofits in several countries. 

Supported by FAPESP and led by researchers Jonathan Elkins, Katlin Massirer, Opher Gileadi and Paulo Arruda, SGC-UNICAMP investigates the functions of protein kinases, which regulate important processes such as cell division, proliferation and differentiation. Although protein kinases are considered priority targets for drug development, it is estimated that only 80 of over 500 protein kinases identified in the human genome have been studied in depth.

“VRKs are called ‘vaccinia-related’ because the first member of the group was identified in the genome of the vaccinia virus, but there are similar molecules in the human genome,” Couñago explained.

Work by other groups, he added, has shown that when VRK levels are artificially reduced in the laboratory (using RNA interference, for example), the cell cycle becomes anomalous. 

“No one knows exactly what role VRKs play in the organism, but it appears to be important because their overexpression is linked to different kinds of tumors,” he said. “We therefore decided to characterize VRK1 and VRK2 in order to be able to develop chemical probes designed specifically to inhibit the action of these kinases.” 

Once chemical probes with the desired characteristics are obtained, the researchers will be able to use them as tools to inhibit the action of VRKs in cancer models. “The goals will be to confirm that inhibiting these enzymes affects tumor cells and to validate them as therapeutic targets,” Couñago said.

Starting point

The research described in the Scientific Reports article began with the screening of two libraries of commercially available kinase inhibitors. The first, known as PKIS (short for Published Kinase Inhibitor Set), was developed by the SGC group at the University of North Carolina at Chapel Hill in the US.

The other library contains kinase inhibitors that have been tested in clinical trials and have known biological activity. Each one comprises some 400 kinase inhibitor molecules.

The researchers used high-throughput screening, a process that uses automation to assay the biological or biochemical activity of hundreds of compounds simultaneously. 

“We scanned the two libraries in search of a starting point for the development of our own chemical probe. Interestingly, we found few inhibitors capable of binding to VRK1 or VRK2,” Couñago said.

The group selected from each library the molecule with the best capacity to bind to the target kinases, and they then crystallized the complexes formed by the inhibitors and kinases so that their three-dimensional structures could be mapped using X-ray crystallography.

“This technique enabled us to plot the spatial positions of all the atoms and showed how the synthetic molecule was linked to the site where it was bound to the ATP [adenosine triphosphate] in the substrate of the kinase. With this information, we were able to suggest modifications to the inhibitor’s structure designed to make its action even more powerful and also more specific, so that it wouldn’t interact with other kinases in the organism,” Couñago said.

The scientists at Aché, led by Cristiano Guimarães, are responsible for structural analysis, molecular modeling, and the design of new compounds. Guimarães heads the company’s Radical Innovation team, made up of medicinal chemists, molecular biologists and pharmacists who specialize in drug discovery and new molecule design.

“So far, we’ve completed the design and synthesis of 97 molecules, which have been tested in vitro against VRK1. This part of the research will be described in an article to be published in early 2018,” said Hatylas Azevedo, Aché’s RD&I manager.

For a molecule to be considered a chemical probe, he added, it must show activity at 50 nanomolar (nM) or less, meaning it can inhibit at least 50% of the kinase’s response or binding at that concentration. The lower the half-maximal inhibitory concentration (IC50), the more powerful the compound.

“BI-D1870, the best molecule found in the screening stage, had an IC50 of 600 nM, but our team has synthesized compounds with an IC50 of less than 100 nM. For some molecules we design, we expect to achieve an IC50 of between 20 and 30 nM,” Azevedo said.

After obtaining a sufficiently powerful molecule, the group will have to evaluate its selectivity by testing the candidate probe against some 320 other kinases to find out whether it interacts with any of them as well as it does with VRK1.

“Next, we’ll see whether our molecule also inhibits the action of VRK1 in a cellular context, which is more complex than in vitro interaction. In this case, the IC50 must be less than 1 µM. After that, we can start testing the chemical probe in experimental models of cancer,” Azevedo said.

The article “Structural characterization of human Vaccinia-Related Kinases (VRK) bound to small-molecule inhibitors identifies different P-loop conformations” (doi:10.1038/s41598-017-07755-y) by Rafael M. Couñago, Charles K. Allerston, Pavel Savitsky, Hatylas Azevedo, Paulo H. Godoi, Carrow I. Wells, Alessandra Mascarello, Fernando H. de Souza Gama, Katlin B. Massirer, William J. Zuercher, Cristiano R. W. Guimarães and Opher Gileadi can be read at: nature.com/articles/s41598-017-07755-y.