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Bioactive compound libraries are composed of small molecules and can be used for high-throughput screening in biomedical research, medicinal chemistry and chemical biology. These screening libraries provide researchers with bioactive compounds spanning a wide range of targets or research areas, or may be focussed on a particular target type, research area or application.
Compound libraries can be used in a multitude of different assays and experimental paradigms, with differing biological outcomes and endpoints. This blog post provides examples of how compound libraries can be used for target validation, model/assay development, and drug repurposing.
When using a compound library for target validation, researchers aim to demonstrate that their hypothesis about a target and its involvement in a cellular or disease process is correct, by using compounds with a known target protein.
For example, Markussen et al used the Tocriscreen Kinase Inhibitor Library to identify glycogen synthase kinase 3 (GSK3) as a negative regulator of fibroblast growth factor 21 (FGF21) in brown adipose tissue (BAT). BAT activity has beneficial metabolic functions that may have therapeutic potential in disorders such diabetes and obesity, as it is able to improve systemic insulin sensitivity and glucose homeostasis, and increase tissue oxygen consumption. This paper investigated the signaling pathways that regulate expression of Fgf21 in BAT, and identified two GSK3 inhibitors that increased Fgf21 expression and oxygen consumption in brown adipocytes.
Figure 1: A kinase inhibitor screen identified novel regulators of Fgf21 expression in response to β-adrenergic stimulation. Fgf21 expression was analysed in cultured brown adipocytes, pre-treated with 10 µM kinase inhibitor for 1 hour prior to β-adrenergic stimulation. Adapted from Markussen et al, 2018.
Drug repurposing, also known as drug repositioning, involves investigating new therapeutic uses for drugs that already have clinical approval. By leveraging validated safety and efficacy data, this process aims to bring a drug to market faster, with reduced cost. Compound libraries consisting of FDA-approved drugs are an essential starting point for drug repurposing studies.
First identified in the early 2000s, Middle East Respiratory Syndrome is a viral respiratory disorder with the potential to progress into acute respiratory distress, which is caused by the MERS coronavirus (MERS-CoV). Shin et al used the Tocriscreen FDA-approved Drugs Library to identify compounds that have antiviral action against MERS-CoV in a cell-based antiviral screening assay. Huh-7 cells were infected with MERS-CoV and treated with compounds, which identified Saracatinib, a potent and selective Src tyrosine kinase family inhibitor, as having antiviral activity against MERS-CoV.
Figure 2: Saracatinib inhibits the MERS-CoV life cycle in vitro. Huh-7 cells were infected with MERS-CoV and treated with saracatinib for 24h, after which culture supernatant and cell lysates were collect. A) Viral particle release determined by plaque assay. B) MERS-CoV nucleocapsid (N) protein levels in infected cells. C & D) Quantification of MERS-CoV RNAs ORF1a and upE by RT-qPCR, normalized to GAPDH. Adapted from Shin et al (2019).
When using a compound library during model or assay development, researchers aim to validate the utility of their model/assay using compounds with known action.
The blood-brain barrier (BBB) prevents conventional chemotherapeutic agents from reaching brain tumors such as gliomas, making them very hard to treat with drugs. Traditional methods for testing BBB permeability of compounds in high throughput screening is limited to assessing radio- or fluorescently-labeled compounds passing through a monolayer cell culture. However, in most cases, this does not accurate recapitulate the physical environment of a glioma. Sherman & Rossi developed a 3D ‘BBB plus tumor’ model to allow label-free investigation of brain permeability alongside tumor toxicity. To validate this model they screened compounds from the Tocriscreen Kinase Inhibitor Library for their cytotoxicity against LN-229 glioma spheroids alone and with the BBB model. They identified 27 compounds that showed tumor cytotoxicity in LN-229 glioma spheroids, however addition of the BBB in the 3D ‘BBB plus tumor’ model narrowed this number to 9, showing 18 compounds would not penetrate the BBB and so are suitable for treatment of gliomas.
Figure 3: 3D ‘BBB plus tumor’ model for determining tumor cytotoxicity. LN-229 glioma spheroid viability represented by relative luminescence (RLU) without (A) and with (B) a BBB model. Pink line is average buffer response and green line represents 3 σ below buffer response. Data points below green line were considered hits. Adapted from Sherman & Rossi (2019).
Markussen et al (2018) GSK3 is a negative regulator of the thermogenic program in brown adipocytes. Sci Rep. 22, 3469. PMID 29472592.
Sherman & Rossi (2019) A novel three-dimensional glioma blood-brain barrier model for high-throughput testing of tumoricidal capability. Front Oncol. 9, eCollection 2019. PMID 31131260
Shin et al (2018) Saracatinib inhibits Middle East Respiratory Syndrome-coronavirus replication in vitro. Viruses. 10, 283 PMID 29795047