Phenotypic assays observe overall changes in cells or organisms when exposed to a specific treatment or condition. This approach focuses on the “big picture” of how biological systems behave, rather than analyzing individual molecular components, helping researchers understand the observable characteristics that result from biological processes.
Understanding Cellular Responses
Phenotypic assays observe the entire biological system (cells, tissues, or whole organisms) for observable changes. Instead of targeting a single molecule, these assays monitor a range of observable characteristics, or ‘phenotypes,’ in response to a compound or condition. These characteristics can include changes in cellular behavior such as cell growth, cell death, migration patterns, or alterations in cell morphology.
The observations in phenotypic assays can be both qualitative and quantitative. For example, researchers might visually assess changes in cell shape or count the number of cells that have died. This broad observation allows for screening compounds to identify those that induce a desired phenotypic change in a disease-relevant cellular model.
Driving Drug Discovery and Disease Research
Phenotypic assays are impactful in finding new drugs and understanding diseases because they can identify compounds that correct disease-related phenotypes, even when the exact molecular target is unknown. This approach helps in discovering novel mechanisms of action for potential drug candidates. For instance, in cancer research, phenotypic screening can identify compounds that cause tumor cells to die while leaving healthy cells unharmed.
These assays have led to the discovery of important medicines for various diseases. For example, some anti-infective and anti-cancer therapies, such as azacitidine and daptomycin, were discovered using cell-based phenotypic assays where cell death was the observed phenotypic marker. Another example is the discovery of compounds that can re-establish the thin layer of liquid atop lung cells in cystic fibrosis patients, using cell types sourced directly from those patients.
Phenotypic vs. Target-Focused Approaches
Phenotypic assays differ from ‘target-based’ assays, which focus on a specific molecule like a protein or enzyme known to be involved in a disease. Phenotypic assays, in contrast, observe the overall effect a compound has on a biological system without necessarily knowing the initial target. This allows for an unbiased discovery of novel mechanisms, as compounds are selected based on their therapeutic effect rather than their interaction with a predefined target.
A strength of phenotypic assays is their ability to identify unexpected drug targets and their relevance to complex biological systems. They can lead to the discovery of first-in-class drugs, especially in areas where the molecular causes of diseases are not fully understood. While target-based screening has yielded many “best-in-class” drugs, phenotypic screening often identifies “first-in-class” drugs.
Diverse Applications of Phenotypic Assays
Beyond drug discovery, phenotypic assays are versatile and applied in various scientific fields. In toxicology screening, they help identify harmful effects of compounds by observing changes in cell morphology or viability. For instance, a panel of fluorescent imaging probes can simultaneously measure oxidative stress and mitochondrial function in human liver cells to predict drug-induced liver injury.
These assays also contribute to basic biological research, providing insights into fundamental cellular processes. They are used in personalized medicine, where patient-derived cells can be tested to determine individual drug sensitivities. Different models are employed, including:
2D cell cultures.
Physiologically relevant 3D organoids and spheroids.
Whole animal models like zebrafish.
Caenorhabditis elegans (for observing systemic effects and toxicity).