The demand for new drugs is pushing companies to develop cell-based assays that focus on a specific cellular event, potentially leading to tailored and more potent therapies. These assays also hold promise to predict toxicity, improve extrapolation to humans, and identify optimal leads earlier in the drug-discovery process. The companies highlighted in this article will share recent developments in this field at two upcoming conferences: Informa Life Sciences “Cell-Based Assay” conference to be held in London and CHI’s “Cell and Tissue-based Assays for HTS” meeting to be held in Philadelphia.
There are many factors to consider before and during cell-based assays, says Lisa Minor, Ph.D., principal scientist, drug discovery, Johnson & Johnson. Some of these include whether the assay has the right signal, the correct biological event is measured, and the right cells are used.
Another challenge is cell lines. “What you’re really concerned with is that the cell doesn’t have endogenous expression of your target and that the only event you’re seeing is what you’ve added to the cell.” A recent concern focuses on cell lines that are contaminated with other cell lines, or may not be the right species. Dr. Minor says several companies are developing tools to qualify for species and tissue type.
The biggest trend in cell-based assays, says Dr. Minor, is label-free technology. “You can pick up minor cell changes you can’t see with a microscope, and without having to do multiple assays on the same cells.” She says everyone would like to use primary cells, but they are limited in number and their characteristics are difficult to maintain. Electrophysiology with high throughput is also an emerging technology. Although Dr. Minor says better electrophysiology systems are needed, she is not sure about improvements to other technologies yet. “You don’t realize you need something until somebody shows up with an idea.”
DiscoverRx recently updated a platform developed years ago by its CEO for cell-based assays. “We’ve put some new improvements and twists on it and those are what have made it amenable to measuring protein-protein interaction events in live cells,” explains Keith Olson, Ph.D., vp of R&D.
The PathHunter™ assays incorporate a target protein fused to a b-galactosidase (b-gal) peptide expressed in the cytoplasm. Another portion of b-gal (enzyme activator) is expressed only in the nucleus. When these two interact, active enzyme is formed, generating signal. This can detect protein translocation to the nucleus or nuclear envelope degradation during mitosis.
All the assays run on any chemiluminescence-capable plate reader with a single reagent addition step, do not require special instrumentation or antibody labeling. An advantage of using an enzyme approach is that it amplifies the signal—up to a twenty or thirty signal to background ratio. “One of the challenges of FRET and HRET, the two most common ways to address protein-protein interactions, is that they don’t generate a lot of signal. After you finish building an assay, you may only have a two- to threefold signal to background ratio,” Dr. Olson states.
In addition, the system is tunable. “We’ve been able to modulate the b-gal reporter system so we have essentially no background association of the two components. The only way to generate signal is to have productive protein-protein interactions.”
The company also offers the PathHunter b-Arrestin Assays for over 100 G-protein coupled receptors (GPCRs). These are high-throughput screening assays for monitoring GPCR activation following ligand stimulation, without imaging instrumentation, fluorescent protein tags, or radioactivity. It provides direct measure of b-Arrestin binding to the GPCR of interest during activation, making it useful for de-orphanizing novel GPCRs. Dr. Olson says the company has recently started a program to look at transcription factors and cytoplasmic kinases involved in general cell signaling events.