by Caitlin Smith
The effect that a drug has on a cell needs to be studied, ultimately, in a cell. Despite the wealth of information that we can glean from molecular and biochemical studies, we really need to observe a drug’s mechanism of action in living cells or in vivo. “Getting closer to physiology is the one biggest concern today,” says François Degorce, director of marketing and communications at Cisbio Bioassays. “Researchers currently address this through approaches like high-content screening, for which they can use unmodified cells or endogenous expressions. Addressing difficult targets such as ion channels and other membrane components through new detection modes with higher throughput is also an important goal.” Here is a look at two new types of cell-based assays that are helping to move researchers closer to physiology.
High throughput cell kinase assays
Cisbio Bioassays is responding to a demand for more high-throughput, nonradioactive, physiological assays using its proprietary HTRF (homogeneous time resolved fluorescence) technology in cell-based formats. Cisbio now offers HTRF-based cellular kinase assays for non-recombinant targets. “These assays are sensitive enough to detect the phosphorylation, in a cell-based format, on endogenous expressing cells—not recombinant,” says Degorce.
Cisbio recently released a cell-based technology platform called Tag-lite™, which lets researchers study many aspects of membrane receptors using the same cellular material. These aspects may include ligand binding, dimerization, second messengers, and other signaling pathway effectors. “Cisbio has since been regularly expanding the Tag-lite product family with new ligands that allow non-radioactive ligand binding assays to be set up,” says Degorce. “Tag-lite also offers critical advantages and assay flexibility in the field of biotherapeutic screening: design of many different assay configurations using the same technology, identification of different types of binders on a given receptor using the same material (pre-labeled cells), and HTRF toolbox and custom labeling.”
Degorce notes that cell-based assays can be used with a number of target types to increase the relevance of assays that used to be only biochemical (e.g. kinases). “A number of important targets, such as epigenetic ones, could potentially be addressed this way,” he says. “The fact that the Tag-lite platform allows the investigation of a receptor under multiple angles using the same technology (binding and receptor function, for instance) is a real breakthrough, as it eliminates the need to develop different basic biological reagents (such as cells) in order to run these assays. Future applications will probably show how these technologies can be optimized for more relevant biological models such as differentiated stem cells.”
Going label-free with drug studies
The ability to screen drug candidates without using labels has advantages for applications in which the presence of labeling molecules might interfere with the screening process itself. Caliper Discovery Alliances & Services (CDAS) recently began using SRU Biosystems’ label-free BIND technology for functional assays. “We use it as part of our GPCR functional platform of assays using recombinant or primary cells for screening, compound profiling, and for specific pharmacology studies, including identification of off-target effects, understanding precisely a drug's mode(s) of action that second messenger assays may miss (such as inverse agonist or partial agonist modes, or multiple signaling pathways), or for uncovering new possible targets for agonist and antagonist compounds,” says Philippe Mourere, senior director of sales and marketing for CDAS. “Label-free data information can be complex to analyze and the CDAS platform of over one thousand optimized in vitro assays can be used to ‘deconvolute’ that holistic information quickly and confirm drug targets, mechanism of actions, or specificity for a given target.”
Mourere adds, “the growing need for better disease markers and companion diagnostic tests will, in my view, drive more interest for tissue- and cell-based immunohistochemistry and high-content screening experiments. The ability to titrate markers in multiplex and in higher throughputs makes those technologies very attractive in both preclinical R&D and diagnostic set up.” However, such developments are not without their challenges. For example, using more physiological, cell-based models in the drug discovery process for screening, lead optimization, and testing safety and toxicity, would be a great advantage. But Mourere says this is still difficult for several reasons, including “limited assay sensitivity when working with endogenous target expression systems or when measuring intracellular markers, limited amounts of primary cells, [and] variability of primary cells batches.”
Mourere has hope that future cell-based assays will include improvements in convenience and optimization, as well as give researchers more options for complex applications. “The developments I envision for the future are cell-based assays supplied as ready-to-use kits with optimized protocols for various cell lines, and primary cells and reagents for more complex biology applications (such as metabolism, migration, and signaling pathways) than existing GPCR or signaling studies,” he says. Doubtless the technology for cell-based assays will progress to the point where, 20 years from now, we will see today’s equipment as archaic—and hopefully, that new technology will get us closer to the physiology.