High-throughput screening is well known as an efficient method for drug discovery and disease mechanism researches. But how to carry out drug screening experiments? How to guarantee the correctness of the lead compounds that screened out? Let's take a look at how high-throughput screening is carried out by using two papers as references.

Screening experiments can typically be divided based on the experimental level into cell-based and biochemical assays:

Biochemical assays are often applied in target-based studies to investigate the inhibitory effects or binding activities of test compounds on target proteins. There are various detection methods available for biochemical assays, which can be chosen based on specific usage purposes and research subjects.

When the target is unknown, the target protein is difficult to isolate or purify, or the testing phenotypes only exist at the cellular level, it is necessary to conduct cell-based assays. Cell-based assays can assess the overall effect of compounds on cells, including regulation, toxicity, or metabolic conditions within the cells.

Compound libraries are the experimental subject for conducting high-throughput screening and serves as the main element of the screening experiments. Selection of a compound library that matches the experimental goal is the key for high-throughput screening.

MCE offers a diverse range of compound libraries to meet various study needs.

Now let's take a look at how the researchers conducted the screening experiments in the published literature by using the MCE compound library!

The cytokine burst induced by SARS-CoV-2 is closely related to the severity and mortality rate of COVID-19. Therefore, the authors of this paper aimed to screen and identify anti-inflammatory drugs to treat COVID-19. The authors constructed a CAR-T cell model targeting SARS-CoV-2-S protein (SARS-CoV-2-S CAR-T) and stimulated with the spike protein to simulate the T cell response in COVID-19 patients, that is, the release of a large amount of cytokines, as well as the unique memory, exhausted and regulatory T cell phenotypes.

Combining this cell model and the infection phenotype, the authors aimed to identify small molecule compounds that effectively inhibit cytokine release through high-throughput screening. Initially, they screened based on the inhibition rates of IL8 and IFNγ release by ELISA. Subsequently, they further evaluated the toxicity of the compounds on T cells. Finally, they identified four compounds that met the screening criteria from 1049 FDA-approved drugs (Figure 1). In subsequent experiments, the authors also demonstrated the potential of these four small molecules in early clinical treatment through cell and in vivo assays^[1].

The missense mutations in TP53 can impair its own tumor suppressor function and confer oncogenic activity, thereby promoting carcinogenesis. Therefore, conducting researches on TP53 missense mutations is of great significance. The authors discovered that mutations in the TAD or DBD of p53 can lead to different cellular localization patterns and protein partners. Thus, the authors intended to use high-throughput screening to test whether the unique protein interactions between p53 TAD mutants and DBD mutants would affect different signaling pathways, thereby generating different pro-tumor or anti-tumor effects.

Therefore, the author conducted a screening using a customized inhibitors library (303 compounds) targeting signal transduction and metabolic pathways to identify and verify the specific signaling pathways that p53 TAD and DBD mutant cells rely on for growth or survival. The results of the cell viability assay revealed that selective inhibition of the PI3K/AKT/mTOR signaling pathway and biosynthesis would significantly reduce the survival rates of TAD mutant cells and DBD mutant cells (Figure 2). Western Blot analysis showed that the levels of phosphorylated AKT, S6K, ERK, and mTOR were elevated in TAD mutant cells. These data indicate that the survival or growth of p53 TAD and DBD mutant cells depends on different mechanisms and exhibits different activation patterns within the intracellular signaling pathways^[2].

The above two articles provide some ideas for the selection and screening experiments. No matter which type of experiment is chosen, it is essential to ensure that the experimental method can prove the experimental purpose and has the characteristics of reproducibility of results, economic efficiency, and high-throughput feasibility.