Drug companies are no strangers to protein biomarkers, with pharma firms exploring their use for tasks like measuring drug efficacy and selecting patient cohorts for clinical trials.
In recent years, the industry has also begun using targeted mass spec to measure biomarkers for drug absorption, distribution, metabolism, elimination, and toxicity – or ADMET – research, with quantitative techniques like selected-reaction monitoring MS offering potentially improved ways of modeling drug behavior.
"Where we're really using [targeted mass spec] right now, is to help with our in vitro-in vivo correlations," Pfizer senior scientist Larissa Balogh told ProteoMonitor, noting that her group focuses primarily on drug transporter proteins, an understanding of which can be key to predicting a compound's activity.
Discrepancies between transporter protein expression levels in pre-clinical in vitro and animal models and the human systems the drugs are ultimately intended for can limit the usefulness of such modeling, Balogh said. By using SRM-MS to better measure these differences in protein expression, her group hopes to improve the accuracy of its models.
"Variations in protein transporter expression are a major limiting factor for us," she said. "In that respect, despite all the other things that we know, the cell is really kind of a black box in terms of a quantitative understanding of their expression, and that can really limit a model's power in terms of predicting [things like] human PK or drug-drug interactions."
Approaches like measuring mRNA levels or quantifying transporters via immunoassays can also be used for this work, Balogh said, but, she added, both of these techniques have significant drawbacks.
While "a lot of people use [an antibody approach], and it can be helpful," she said, "you have to have an antibody, which in a lot of cases we don't, and that can take a long time to generate."
"And antibodies can have a lot of cross-reactivity," she added. "That's one of the great things about using this targeted quantification by mass spec method. When applied right you really are targeting only your specific protein of interest, so you really don't have that cross-reactivity issue."
With mRNA-based assays, Balogh said "we've seen that there can be a big disconnect between your mRNA transcript levels and what your actual functional protein expression is due to post-translational mechanisms."
David Lustig, vice president of business development at Optivia Biotechnology, a biotech firm specializing in transporter protein work for drug development, seconded Balogh's comments, telling ProteoMonitor that "the best answer is to actually quantify the transporter protein itself in the membrane."
"That's where mass spec comes in," he said. "If you can isolate the membrane, flush away the contents inside the cell, look at the membrane only, and then use mass spectrometry to quantify how much of the protein is there, that's the Holy Grail, that's the answer that you want."
Traditionally complicating such assays, Lustig noted, has been the challenge of extracting the plasma membranes – where transporter proteins are located – for mass spec analysis. In an interview with ProteoMonitor last week, however, Tetsuya Terasaki, a professor at the Tohoku University Graduate School of Pharmaceutical Sciences in Japan, noted that advances in mass spec technology have made it possible to measure transporter expression levels in tissue homogenate samples.
In particular, his lab has been using AB Sciex's TripleTOF 5600 instrument, which, he said, has allowed the researchers to work with tissue homogenate due to its high sensitivity and low signal-to-noise ratio.
In March 2010, Terasaki launched a company called Proteomedix Frontiers that aims to apply targeted mass spec to drug discovery and development, including ADMET work (PM 08/05/2011). According to Terasaki, his lab has developed SRM-MS assays for more than 400 proteins that could be important in drug development, including more than 200 membrane transport proteins. His company plans to launch by the end of the year a kit with SRM-MS assays for seven human transport proteins identified by the US Food & Drug Administration as being especially important for monitoring drug-drug interactions.
Balogh said that Proteomedix Frontiers was the first company she was aware of that is focusing on mass-spec based transporter quantification for drug research, but, she noted, many pharma companies have begun doing such work internally.
"It's definitely becoming popular," she said. "At a recent conference we saw people from Eli Lilly, from Merck – definitely at all the major drug companies there are people who are starting to work on this. It's definitely not just Pfizer."
"I've seen a big increase in it over the last two years," Balogh said, adding that while there was still in some cases methods development work to be done, pharma companies were working to implement mass spec-based quantitation of receptor proteins into their regular drug discovery workflows, "because obviously if you can make these models more predictive it will speed up the drug-discovery process."
Balogh's group primarily uses an AB Sciex API 4000 machine for their work, she said, although "when we're first selecting the peptide [for SRM-MS] sometimes we incorporate a higher resolution instrument to help since at that point we don't have any standards and we actually need high mass accuracy."
In particular, Balogh said, her team has been using a TripleTOF 5600 demo instrument to help with peptide selection.
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