Drug discovery processes were initially based on the ideology that genetic mutation or dysfunction is responsible for a disease. Thus, most drug discovery approaches were target-based screening, and molecules were tested on specific molecular targets to find a drug that induces/inhibits it.
Drug discovery methodology has since evolved to include phenotypic screening methods that differ from target-based screening as it studies not the interaction between two molecules but the final cell phenotype. High Content Screening (HCS) is a phenotypic screening tool that identifies molecules or peptides that alter the phenotype of a cell to a preferable form. It enables us to study the whole cell and the resultant effects of the molecule being studied on the entirety of the cell and not just the target molecules.
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Methodology
High-content screening is an extremely powerful tool that combines automated multicolor fluorescence imaging and high-throughput quantitative data analysis. The methodological steps include initially incubating cells with an in-vitro-based assay or molecule from a library. These are labeled with fluorescent labels to study specific live parameters such as viability or proliferation.
Afterward, cells are fixed and stained by immunocytochemistry or fluorescent probes to study the expression or localization of proteins of interest. The data from fluorescent microscopy is acquired from various fluorescent peaks of absorption and emission and their quantitative analysis. The data from the analyzed image is then formatted and organized for ranking, clustering, statistics, and machine learning.
Applications of HCS
HCS has various applications that can be largely separated into three groups: drug discovery, toxicology, and functional genomics.
HCS is pivotal in drug discovery as it can screen primary compounds, secondary confirmations, target identification, and study mechanism of actions. HCS assays can study molecules from a library and observe whether the cell has altered to the desired phenotype. Phenotypic screening, such as HCS, are particularly useful in complex and multifactorial diseases where the mechanism of action is unknown. Phenotypic screening allows for the interrogation of the proteome in its pathophysiologically relevant environment and enables a greater chance of identifying disease mechanisms and potential drug therapies.
Barriers to this include the scalability of these assays and their associated limitations, such as cost, complexity, and multi-step process. It also requires well-trained personnel to ensure high-quality assays and care with accurate dispensing and thorough plate washing.
HCS is also considered a type of toxicological assay used in the drug discovery process as it studies the effect of a molecule or a stimulus on a whole cell and its effect on the environment. This has greatly reduced the reliability of animal studies to study toxicity which can be costly and inconsistent in predicting human toxicity. It offers many advantages over biochemical toxicology assessments, such as single-cell level end-point assessment. This enables focus on particular cell types and a better understanding of cellular toxicity and its mode of action. Assays can also be customized where end-points are selected to identify specific toxicity reactions.
The imaging aspect of HCS means it is a more sensitive measure and assessment tool when compared to biochemical assays. Cytotoxic compounds can be easily identified using imaging that allows cell counting in contrast with alternative methods such as ATP content. Studies have proven that the accuracy with which cytotoxic compounds are identified is twice as high with HCS cell counting compared to ATP content assays. The imaging concept is not novel to HCS, but imaging was a descriptive tool previously as opposed to the automated quantitative analytical tool it is with HCS.
Furthermore, HCS has been used in genomic screening to identify genes responsible for specific biological processes. It has also been utilized in studying genetic interactions and characterizing them. Genomic screening uses the knowledge gained from human genome sequencing to identify the role of specific genes in cellular processes by studying the phenotype. The study of functional genomics is highly reliable and useful in identifying drug targets.
High-content Screening is a powerful and reliable tool that uses automated imaging and high-throughput quantitative data analysis for various applications. HCS has gained increasing traction as automated technology and fluorescent microscopy has advanced. It can be used to identify drug targets by screening an extensive library of compounds for desired phenotypic effects on a cell. HCS can also study mechanisms of action and potential therapies in complex diseases with an unknown pathology. This also uses genomic screening to identify genes with a role in cellular mechanisms.
Additionally, HCS is well-known for its role in toxicological studies and identifying cytotoxic compounds early in the drug development process. HCS has proven to offer a great advantage in furthering our understanding of biological processes, identifying drug targets, and improving toxicological studies. HCS holds great promise in fostering these understandings and contributing significantly.
Sources:
- Heynen-Genel, S., Pache, L., Chanda, S. K., & Rosen, J. (2012). Functional genomic and high-content screening for target discovery and deconvolution. Expert opinion on drug discovery, 7(10), 955–968. https://doi.org/10.1517/17460441.2012.711311
- Li, S., Xia, M. (2019) Review of high-content screening applications in toxicology. Arch Toxicol 93, 3387–3396. https://doi.org/10.1007/s00204-019-02593-5
- de Conto, V. (2022, February 10). What is High Content Screening (HCS)? HCS Pharma. https://hcs-pharma.com/what-is-high-content-screening-hcs/
- Persson, M., Løye, A. F., Jacquet, M., Mow, N. S., Thougaard, A. V., Mow, T., & Hornberg, J. J. (2014). High-content analysis/screening for predictive toxicology: application to hepatotoxicity and genotoxicity. Basic & clinical pharmacology & toxicology, 115(1), 18–23. https://doi.org/10.1111/bcpt.12200
- Li, Z., Cvijic, M. E. & Zhang, L. in Comprehensive Medicinal Chemistry III (Elsevier, 2017) (eds Chackalamannil, S., Rotella, D. & Ward, S. E.) 362–387; https://doi.org/10.1016/B978-0-12-409547-2.12328-5
- Lansdowne, L. E. (2022, June 30). The Role of Phenotypic Screening in Drug Discovery. Drug Discovery from Technology Networks. www.technologynetworks.com/.../the-role-of-phenotypic-screening-in-drug-discovery-293422
Further Reading