By Tony Fong
After lying low for the past number of years, Kinexus Bioinformatics is readying to launch two peptide arrays that its chief executive said would together provide a new level of information about which protein kinases should be targeted for therapeutic intervention.
This week, the Vancouver, BC, firm launched a new synthesis peptide facility, and with it new peptide array services for the research community. In about three months, Kinexus' Founder and President Steven Pelech told ProteoMonitor, the company anticipates launching a peptide substrate microarray "optimized to conform to the kinase-substrate specificities of 500 different human protein kinases."
In addition, it anticipates launching a microarray that uses peptides patterned after human phosphorylation sites that Kinexus believes are functionally important.
Kinexus, which has 18 full-time employees, is currently in discussions with an undisclosed firm to produce the microarrays, which initially would be used in its in-house service business. However, they could also be sold as products by the end of the year along with the company's protein kinase microarrays, which were launched as a service last month, Pelech said.
The microarrays would represent Kinexus' first product offerings.
The company also is "looking to launch the mass spec services" for targeted analysis of phosphorylation sites. Pelech did not provide a timeline for such a launch.
Kinexus was launched in 1999 as a proteomics and bioinformatics company, but in recent years it has operated in stealth mode. And until last month, when it announced the launch of its protein kinase microarray service, it had not publicly said anything about what it was doing in more than two years.
Far from hibernating, though, the company has been busy building its infrastructure, which in turn has allowed it to make its recent launches and positioned it to make upcoming ones, Pelech said.
In particular, Kinexus has been building its library of protein kinases and phosphorylation sites. Its business has historically concentrated on cell-signaling proteins, in particular protein kinases. About 400 diseases, including cancer, diabetes, and Alzheimer's disease, have been linked to defects in cell-signaling systems, and one-third of all drug-discovery R&D is directed at protein kinases as drug targets, according to Pelech.
Of the approximately 23,000 protein-encoding genes in the human protein, about 515 encode protein kinases, he added.
Kinexus' efforts have been focused on protein kinase phosphorylation. In humans, there are about 500,000 phosphorylation sites, Pelech said, meaning each protein kinase targets on average more than 1,000 phosphorylation sites. Of those 500,000 sites, 75,000 have been experimentally confirmed and gathered into a database Kinexus created, called PhosphoNet.
The 75,000 human phosphorylation sites are contained in 12,400 proteins. More than half of the phosphorylation sites in PhosphoNet are serine phosphosites, about 18 percent are threonine phosphosites and almost 24 percent are tyrosine phosphosites.
Using its bioinformatics capabilities, the company has further identified "with pretty accurate prediction" the 500,000 human phosphorylation sites, Pelech said, and mapped the specificities of about 500 human protein human kinases, about three times what is available elsewhere.
Of special note is that Kinexus has mapped tyrosine phosphorylation, which accounts for only about 4 percent of all phosphorylations but is of particular importance in cancer research.
Serine phosphorylation accounts for more than three-quarters of all the phosphorylations, while threonine makes up 20 percent.
"Half the known oncogenes encode protein kinases and defects in these enzymes where they have a gain of function contributes to cancer," he said. "And what happens is this tyrosine phosphorylation, although it's very rare compared to the other types, is actually very linked to cell growth, and so a lot of the drugs that are in the market today … target protein tryrosine kinases."
By linking these specificities of the 500 kinases to the 500,000 phosphorylation sites, Kinexus has established the predicted connections. "So we've defined 25 million-plus different type connections between the kinases and their target proteins, and often the target proteins are themselves protein kinases," Pelech said.
Of those 25 million connections, only between 5 and 10 percent are "absolutely critical" to the function of a cell and to understanding human disease process. For the 75,000 phosphorylation sites for humans contained in PhosphoNet, Kinexus has performed an evolutionary analysis linking the sites to 22 other species, and eventually hopes to do the same for all 500,000 sites.
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By doing so, the company aims to define the critical 5-10 percent of the 25 million connections. "Once you define the most critical connections, the objective is to develop tools that are based on those critical connections — to develop microarray technologies that allow you to track [them] with any crude cell lysate … and see how they're affected in disease and in response to treatment," Pelech said.
"This is very vital to the development of diagnostic biomarkers and defining the therapeutic drug targets [that] are likely to be the protein kinases in the first place," he said.
Kinexus launched its first microarray, an antibody-based technology that tracks phosphorylation sites and proteins, in 2005. The company followed up with lysate microarrays that take lysates from cells that are printed as individual spots so "we can track hundreds of proteins in hundreds of different clinical samples."
Most recently, Kinexus introduced its protein kinase microarray service offering with 200 recombinant human protein kinases for screening. In a statement, the company said that the service has wide applications including drug-target counter screening, identification of novel kinase substrates, and discovery and testing of protein kinase-protein and protein kinase-compound interactions.
The two microarrays that the company plans to launch in the coming months are peptide arrays with peptide substrates that will contain 15 amino acid sequences of phosphorylation sites. There are phosphorylation sites in "important" proteins, Pelech said, which essentially turn the proteins "on" or "off."
"What we're doing is, by our evolutionary analysis, picking those phosphorylation sites in all species that are non-bacterial that are the most highly conserved in evolution that are the most important," he added. "When you monitor those sites, it gives you a sense whether those proteins are 'on' or 'off.'"
With one of the microarrays that is poised for launch, Kinexus will be able to take crude cell lysates from healthy tissue and diseased tissue, and then test them separately in an array of 5,000 to 25,000 peptide sequences "corresponding to these important target proteins."
The kinases in the crude lysates will be tested "to phosphorylate the individual peptides separately to each spot of printing in the different peptide," Pelech said. By doing so, Kinexus can detect differential effects, which in turn allows it to ascertain "which protein in that cell is likely to be phosphorylated by the same kinase and that that phosphorylation regulates that protein."
"So we know that protein may be controlled," which can then provide information about which substrates and targets are likely to be regulated by the kinase, Pelech said.
The other peptide microarray being targeted for launch allows Kinexus to assay hundreds of protein kinases simultaneously in cell and tissue lysates, and then to determine which kinases are regulated.
"So when you combine the information from both chips, you now have very powerful tools for defining which kinases are affected … and which are the most likely important targets of those kinases in that system," Pelech said.
He added that Kinexus also will be developing protein kinase microarrays for non-human species that have had their genomes sequenced, such as yeast. Because so much data has been generated for yeast, Kinexus will be using that model system to develop a microarray in order to validate its technology, Pelech said.
While there are numerous firms that are very good at printing microarrays, Pelech said the bottlenecks are at the content level and being able to "deconvolute the data. … There's no point in tracking thousands of genes or thousands of proteins if you don't know what it means."
Kinexus' energy is focused on developing probes to track proteins and especially phosphorylation sites in proteins. Pelech described the work of his company as "reverse-engineering of the human operating system, and we think we've cracked it." The first generation of products from Kinexus is directed at healthcare "because that's where we think this defines important disease diagnostic markers, this being the phosphorylation sites."
Further into the future, though, the technology can be used for other research areas such as agriculture, forestry, and energy, he said.
And while the company has not historically offered mass spec services, Pelech said the offering is within Kinexus' sight line. That service is presently being developed, he said, adding "We may have a partner for that in the very near future that, coupled with what we do, we may be able to offer services where we can track thousands of phosphorylation sites that are novel in crude cell lysates. …Presently there are only maybe a handful of labs that will do this."
He declined to provide any financial information about Kinexus, but said that it is profitable. The company has more than 1,300 laboratories in 33 countries that it counts as clients of its proteomics services. "Most of the world's major pharmaceutical companies" are Kinexus clients, he added.