Over the past decade, the genomics revolution has encouraged scientists to perform target-based drug discovery.
However, among new first-in-class drugs that have emerged in recent years, over 60% have come from phenotypic screening using cell-based assays.
This was a critical and important point made by Steve Ludbrook, Ph.D., cellular section head at GlaxoSmithKline, at the recent SMi “Cell-based Assays” conference.
“As a result, it makes sense to look at some of the more complex assays for our screening projects,” he said.
Peter Simpson, Ph.D., associate director, assay sciences group at AstraZeneca, added, “Many of the current cell assays we’re using are not mimicking the diseases we’re targeting closely enough. For example, if you’re using monolayer CHO cells grown in fetal calf serum, then they’ll behave differently to human cells growing in a tumor.”
Simon Barry, Ph.D., associate director of the oncology iMED at AstraZeneca, agreed.
“In oncology, 3-D cell culture or co-culture assays are very useful in helping us to understand the biology of targets or effects of drugs in more detail. However, we can’t use this information alone. To increase success, we need to think more about the patient as a whole,” he noted.
“In addition to tumor biology, learning more about the effects of a drug on the patient using accessible clinical biomarkers, as for example with serum chemokine/cytokine profiles, would help us use our drugs more effectively.”
According to Stefan Przyborski, Ph.D., founder and CSO of Reinnervate, a spinout company from U.K.-based Durham University, complex cell cultures offer more physiologically relevant models than conventional 2-D cell culture for some applications.
“2-D cell culture limits cell-cell interactions, as up to 50 percent of the cell surface is against plastic and most of the other 50 percent is against the media, so cell signaling mechanisms between adjacent cells are constrained,” he explained.
“Also, if you grow cells in a monolayer, the cytoskeleton remodels. This can have significant effects on the nucleus and, in turn, gene transcription and protein translation, so cells behave in a significantly different manner. By growing cells in 3-D scaffolds, you can get complex interactions between cells and it reduces the stress on cells of being cultured in a monolayer.”
He cited the example of skin models and showed that it is difficult to achieve a stratum corneum layer with existing epidermal models of skin.
“Most skin models are simple and only consist of the epidermis with limited formation of the stratum corneum layer, which is an important component of the skin if you want to test barrier function and drug penetration,” said Dr. Przyborski.
Primary Cells—A Good Starting Point?
Complex cell assays show promise in a number of areas. “Using primary cells such as whole blood cells can reduce the cycle time for lead optimization,” noted Nathan Bays, Ph.D., research fellow at Merck. “They can be used to redeploy existing leads, as the assays are more predictive for disease modification and can provide earlier pharmacological validation for targets identified using genetic/genomic data.”
As an example, Dr. Bays described a screening campaign to determine which compounds used previously to treat cancer could be redeployed to treat inflammatory diseases such as asthma and rheumatoid arthritis. Screening was performed directly in human whole blood, and assays measured changes in protein phosphorylation as well as markers of immune cell activation.
Dr. Bays reported that flow cytometers are in constant use at Merck’s Boston-based research facility, with applications ranging from biomarker discovery to weekly compound titration assays and library screening.
“Using flow cytometry we can measure multiple antigens simultaneously, and a key advantage is we can identify and purify specific cell types,” explained Dr. Bays.
“We now have in place robust automation for safe handling of whole blood, which could be translated to other assays. We are seeing a significant return on our investment because we’re able not only to provide key SAR data using primary immune cells, but we have the ability to perform very focused pharmacological target validation and revise our inhibitor profiles using solid data from disease-relevant cells.”
GSK’s Dr. Ludbrook also spoke about an interesting application of primary cells for drug screening. His group is using frozen primary PBMCs (peripheral blood mononuclear cells) taken from multiple donors to look for IFN-γ inhibition in modified ELISA screens, the Meso Scale Discovery platform, and the proximity ligation assay (PLA).
“We like PLA because it uses the power of qPCR to improve the sensitivity of an ELISA assay. The workflow is not too challenging and we can screen in 384-well plates using automation,” he said.
“The added sensitivity of PLA may enable the screening of rare cells and analytes using this method. To date we have screened one million compounds using the Meso Scale Discovery assay and have found selective leads that inhibit IFN-γ and are potentially useful for disease areas such as colitis.”