Once upon a time, especially with the sequencing of the human genome, pharma touted target-based screening as the tool of choice for efficient drug development. In some ways, though, target-based drug discovery hasn’t measured up to its initial billing, due in part to the use of recombinant systems, or to a drug having multiple targets and effects. As a result, researchers and pharmaceutical companies are showing renewed interest in phenotypic-based screening to identify potential drug candidates cheaply and more quickly.
In phenotypic screening, researchers test molecules (i.e., potential drug candidates) in a model system (such as cultured cells) and look for a specific effect (such as increased expression of a particular gene), even though the drug’s mechanism of action may be unknown. Phenotypic screening has an edge over target-based in that it is better at finding drugs that have effects in cells. But the downsides to phenotypic screening include deconvolution of screening results, and usually a lower throughput. Target-based assays are generally faster and easier to interpret.
But both types of screening are likely to be important for future drug development. Today researchers are applying a myriad of diverse technologies to the central idea of phenotypic screening. Here is a sampling of the creative panoply of avenues through the phenotypic screening process available today.
Screening based on genetics & flow cytometry
Horizon Discovery combines CRISPR/Cas9 technology and next-generation sequencing with a pooled-based approach to increase the efficiency of phenotypic screening. Instead of using separate treatments in each well of a plate, for example, they transduce a large batch of cells with a mixture of CRISPR/Cas9 genetic perturbations, each of which are uniquely labeled with genetic barcodes for subsequent identification. After growing out the transduced cells, Horizon conducts phenotypic screening by flow cytometry. “Then we can physically sort populations of cells based on their biomarker response, and deep sequence the cells that show a high response rate,” says Benedict Cross, R&D manager at Horizon Discovery. “That allows us to effectively assign genotype change, or gene perturbations, to a specific biomarker or phenotypic response.” Horizon’s screening system works in model cell lines as well as in more physiological tissues such as primary cells, although the latter can be more challenging.
Intellicyt’s iQue® Screener PLUS platform performs high-throughput flow cytometry of cells in suspension, using a patented system to sample rapidly from multiwell plates, combined with analysis software to transform complex data sets into actionable results. The platform enables large-scale multiplexing experiments, for instance by using beads to capture cytokines or other secreted proteins. “For example, researchers can treat T cells with different compounds, identify different T-cell subpopulations, and examine the viability and proliferation of those subsets, while simultaneously testing which cytokines are being secreted into the media,” says Thomas Duensing, chief technology officer at Intellicyt. The platform is amenable to physiological applications, such as screening the effects of drugs on T cell subsets in whole blood; or analyzing CAR-T constructs used to make patient-derived cancer-killing cells in chimeric antigen receptor T cell therapy. “Researchers use the iQue to look at which constructs have the best functional activity in killing cancer cells, and also to assess other cell functions like which cytokines are secreted,” Duensing adds.
Screening 3D cell cultures
Recent advances in 3D cell culture have more researchers growing cells in 3D conditions to more closely mimic the physiological milieu. At the Scripps Research Institute, Timothy Spicer, senior scientific director in the department of molecular medicine, is teaming up with other researchers and physicians to explore the use of 3D cultures in phenotypic screening. In a recent publication, Spicer’s group developed a method of growing primary pancreatic tumor cells into organoids using magnetically guided nanoshuttle labels developed by n3D Biosciences. With subsequent high-throughput phenotypic drug screening of the organoids, the group found distinct differences compared to conventional cell cultures.
“Ultimately, we think we are finding better and more relevant information, and we are putting those hits into animals for testing, too,” says Spicer. “Besides pancreatic tumors, we are also looking at glioblastomas.” His group is also developing co-cultures of cancer cells and their surrounding stromal cells, such as cancer-associated fibroblasts, because “from a phenotypic standpoint, these co-cultured cells adapt the drug outcome of the tumor cells themselves,” according to Spicer.
High-throughput, imaging-based phenotypic screening
Revvity’s high-content screening (HCS) platforms (the Opera Phenix™ and the lower throughput Operetta CLS™) accomplish phenotypic screening based on imaging. They detect signals by fluorescence microscopy, and incorporate automated multiparametric image and data analysis for high-throughput HCS. Many types of cellular model systems with greater physiological relevance are compatible.
“In addition to 2D cell cultures, primary cells, induced pluripotent stem cell (iPSC)-derived models, cells grown in co-cultures, and 3D culture systems such as spheroids, cysts, organoids, and microtissues can be efficiently used with our HCS platforms,” says Jacob Tesdorpf, portfolio director of imaging and detection instruments at Revvity. Recent phenotypic screening with the Opera Phenix found 20 drugs with activity against the Zika virus that are contenders for drug repurposing.
The key factors for successful phenotypic screens is the quality of the disease model in question and the ability to correctly differentiate between the healthy and diseased phenotypes.
“The key factors for successful phenotypic screens is the quality of the disease model in question and the ability to correctly differentiate between the healthy and diseased phenotypes,” adds Tesdorpf. “Leveraging the power of systems-based assays through a detailed multiparametric observation and description of phenotypes can lead to a more productive discovery pipeline.”
Screening whole animals
Melior Discovery uses in vivo rodent models to focus on drug repositioning, or finding new uses for mid-stage clinical trial drugs that have already been proven safe, but have been discontinued for other reasons. Such candidates represent a huge investment of time and money. “So we begin with a compound that has a whole clinical dossier describing its attributes,” says Andrew Reaume, president and CEO of Melior Discovery. “If the drug truly has an alternate use and we miss it, then that’s a huge opportunity cost — that’s the reason we decided to use an in vivo screening approach.” Melior’s theraTRACE is a collection of translatable animals models of disease that screen phenotypes more efficiently using principles of multiplexing and lean methodology. Their 8-week, 40-model theraTRACE panel is designed to combine tests whenever possible to save time, money, and use of animals.
In addition to drug repositioning, theraTRACE can screen drug candidates that are late-stage but preclinical, as a clinical trial augmentation tool. “The idea is to use theraTRACE as a means of generating hypotheses for alternative indications before entering the clinic and then incorporating additional endpoints or markers (early signals of efficacy) into the early clinical studies to probe those hypotheses in a clinical setting very cost effectively,” explains Reaume. “Identifying alternative indications and further building the product profile early on can potentially save a tremendous amount of time and money going forward.”
Ultimately the most efficient drug development is likely to include both phenotypic- and target-based screening, using the former to find active drugs, then drilling down with target-based screening to gain information about that drug’s mechanism action and effects.