The tediously long and risk-laden process of translating a discovery in biology to a marketable medical breakthrough starts with identifying a repertoire of molecules that will bring about the desired effect.
What is it, really?
Let´s start with the basics – the definition of phenotype itself. A phenotype is the physical expression of DNA. Phenotypes are caused by the interactions of the many different proteins created by DNA. A “phenotype” is any type of observable or measurable biochemical or physical characteristic of an individual, organism or complex system e.g. heart rate, blood pressure, the length of the nerve fibers that grow from a neuron cell etc. A more noticeable examples of a phenotype is melanin: the pigment responsible for skin, eye and hair color in most mammals.
It is the act of measuring the effect of a substance on the measurable characteristic or the phenotype of a complex system such as a whole cell, tissues or organisms. In context of drug discovery, it is a strategy for the identification of molecules with specific effects on the phenotype of an organism.
Phenotypic screening differs from a more modern concept called Target based screening in, that the latter refers to screening substances that interact with a specific protein target. Target based screening is preferred in situations when the disease biology is well understood and the value of the protein (or RNA) is validated with high confidence.
How is Phenotypic Screening done?
Compounds are tested in cellular assays and/or animals and screened for the desired effect i.e. the desired change in phenotype. Once a compound has been screened, efforts are made to understand its biological target(s) (target deconvolution) and mechanism of action (MoA). The overall strategy is referred to, as classical or forward pharmacology or phenotypic drug discovery (PDD).
At the simplest level, there are two types of phenotypic screening strategies:
In vitro screening
These screens employ cell lines and monitor a single parameter e.g. cellular death, production of a specific protein of interest etc. This method´s biggest advantage: they can be easily adapted to an automated high-throughput format. In situations when the knowledge of the disease pathology is limited, in vitro phenotypic assays may provide more insight than target-based screening alone.
In vivo screening
This kind of screening centers around testing a substance for a specific effect (a medical benefit) in fully assembled biological systems. The strategy involves testing the substance across many different types of animal models representing different disease states e.g. fruit fly (Drosophila melanogaster), zebrafish (Danio rerio) and mice (Mus musculus).
This method offers the advantage of context i.e. the test agent is being interrogated in a fully integrated, assembled biological system or an organism.
How successful is Phenotypic Screening?
Phenotypic Screening has been the basis for discovery of new drugs throughout the history of medicine. Between 1999 and 2008, 37% of the FDA approved drugs, were discovered using phenotypic screening.
Here are some examples of the drugs that went from bench to market using phenotypic screening within the first decade of the 21st century:
- Ziconotide’s analgesic and ranolazine´s anti-anginal effects were measured in animal models.
- The endpoints for ezetimibe, nateglinide and pemirolast were blood cholesterol, blood glucose and cutaneous anaphylaxis, respectively.
- Nitisinone originally developed as an herbicide, was repurposed to treat tyrosineamia its effect on tyrosine metabolism was discovered serendipitously.
Anti-cancer medicines such as azacitidine, daptomycin, linezolid, nelarabine, retapamulin and sirolimus, were discovered using cell-based assays in which cell death was used as a phenotypic marker.
And there are several more examples of successfully approved drugs, discovered by screening against phenotypic markers in cell-based assays or in animals, prior to human testing.
How relevant is Phenotypic Screening today?
Target-based screening has been the method of choice in drug discovery for the past two decades. However, the interest in phenotypic screening is going through a renaissance, creating new drug discovery opportunities. Regardless of the approach, a predictive model of the disease and a high confidence biomarker is crucial to guide drug discovery. Clinical success of a compound is directly proportional to the relevance of the disease model and its associated biomarker(s).
Unfortunately, predictive phenotypic assays and relevant biomarkers are not available for most human diseases. With genome sequencing, we hope to identify specific genotypes, genes and targets that could be used to guide drug discovery. This approach is typically known as reverse pharmacology or target based drug discovery (TDD).
TDD involves making a hypothesis about the relevance of a specific protein target e.g. an enzyme, a cell-signaling receptor, a structural protein etc. in the pathology of a disease, and subsequently screening a library of compounds to measure each compound´s effect on that target.
Target-based approaches are often simpler to execute than phenotypic assays as they involve a specific genetic target. However, this simplicity can also be its Achilles’ heel: a specific gene/protein may play “a” role in the disease pathology but not “the” role i.e. a disease is typically a progressive multi-factorial outcome driven by multiple genetic and environmental factors. This means that there is a high likelihood of surprises as the target-based drug progresses in animal and human testing.
Phenotypic assays seem to better capture the uncertainty of biological systems, letting biology decide the correct answer without the preconceptions and biases of the scientists. Historically, phenotypic screening has a slight advantage when it comes to identifying first-in-class drugs, while target-based screening has yielded more best-in-class drugs.
How will Phenotypic Screening look in 21st Century?
The NEED and the DIFFICULTY, of identifying predictive markers of disease that reflect the organ function accurately, is very high. Target- and phenotype-based strategies are simply two parallel pathways to the same goal – discovering effective and safe pharmaceuticals.
Given that any disease is a progressive multi-factorial phenomenon, a reductionist approach of pursuing a single target, rather than biological networks won´t succeed (as in the development of drug resistance). This necessitates the use of network and systems biology approaches for drug discovery.
Target agnostic approaches using phenotypic assays may offer significant benefit. Researchers are using large-scale behavior-based chemical screens in zebrafish (a popular model organism) to discover compounds with new structures, targets, and functions. Chemical and genetic tools are being built to model specific disease states. Such models create an unbiased phenotypic screen and at the same time elucidate the most disease relevant cellular artifacts thereby creating a target map for researchers. Stem cells grown in artificial microenvironments serve a similar purpose- better understand the disease-causing molecular interaction network. This understanding enables targeted drug discovery and the cell serves as the phenotypic screen to test the effect of the drug on the target as well as the whole-cell-system.
The convergence of advances in genomics, proteomics, RNA biology, imaging technologies, high throughput screening methods portends highly productive times for Pharmaceutical Drug Discovery. Phenotypic screening will remain a vital tool for scientists in their quest to discover high value medicines that will reduce the burden of sickness, globally.
