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Global Translational Medicine Role of HTS in cancer therapeutics

3. Phenotype-based screening in 2015, Vincent et al. coined the phenotypic screening
“rule of three” that emphasized on three specific criteria
Despite the continued discovery of therapeutic compounds to design the most predictive phenotypic assay. The first
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by means of target-based drug discovery technologies, the rule is the relevance of the assay system to the disease that
recent drug approval list features drugs discovered using highlights the importance of physiological relevance in
cell-based assays, which constitute approximately half of the assay systems. Advancements in in vitro preclinical
all the high-throughput screens performed. 31,61 Phenotype- studies can be effectively translated to clinical outcomes
based screening offers many advantages over target-based by extensively using primary cells and induced pluripotent
methods due to discovery of multiple protein-level targets stem cells (iPSC)‑derived cells, which are considered to be
or hits and leads in complex biological systems without more representative of human physiology. In the first HTS
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prior knowledge of a direct molecular target. 31 study carried out by McNeish et al., murine embryonic
In recent years, cell-based assays focused on the stem cell-derived neurons were used to screen around two
modulation of a cellular phenotype in a way that million compounds in a library for α-amino-3-hydroxyl-5-
has biological relevance to the disease. These assays methyl-4-isoxazolepropionate (AMPA) subtype glutamate
identify modulators of a pathway of interest in a more receptors. The study demonstrated human translatability
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physiological environment that mimics some aspects of by revealing a similar rank order in hit potency. Hence, the
disease. This approach increases our understanding of phenotypic assays predict more translatability to clinical
the MOA for the disease, providing an additional layer outcome and help in predicting the clinical therapeutic
of information compared to the single-target-based response to a drug.
biochemical approach. Multiple endpoints at various The second criterion is the relevance of the stimulus
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levels are determined by primary endpoints and secondary in achieving the production of the desired phenotype.
endpoints. Primary endpoints such as cell viability and The purpose of stimuli is to target specific signaling
proliferation are followed by secondary endpoints. Once pathways related to the disease caused by the imbalance
primary endpoints are successfully determined, functional of the crosstalk between signaling pathway. This can
assays such as second messenger mobilization (intracellular be achieved by focusing on the highly disease-relevant
calcium fluxes or cAMP) after GPCR activation, 63,64 biological systems with the same genetic background as
reporter gene assays, cell migration, and cytokinesis are depicted by iPSCs. 71-73 In 2012, Lee et al. tested a library
performed. 65-67 of 6,912 small-molecule compounds covering a wide range
A big question that arises is how to determine if a of biologically active and structurally diverse compounds
phenotypic drug screen is effective and what defines a from various commercial sources, including FDA-
good phenotypic drug screen (Figure 2). To answer this, approved drugs. These compounds were screened for
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Figure 2. Challenges to drug development and troubleshooting checklist for identifying and validating targets before clinical trials.

Volume 3 Issue 1 (2024) 7 https://doi.org/10.36922/gtm.2448