Comprehensive target screening by label-free cell microarray profiling to reduce animal efficacy and toxicology studies in drug discovery

Current screening strategies used during lead selection only assess whether a candidate drug binds to commonly hit targets, resulting in many off-target interactions only becoming evident during later stages of drug development, or once a drug has reached the market. Being able to identify all protein interactions of lead chemicals during discovery would improve understanding of therapeutic and safety properties of drugs, enabling companies to prioritise development of drugs with favourable efficacy and safety profiles.

The partnership between Phenotox Ltd, Retrogenix Ltd and Sheffield Hallam University has created a novel mass spectrometry-based profiling method that can detect the binding of label-free (unmodified) chemicals to potentially thousands of targets expressed in cell microarrays. This approach could be applied to drug discovery to select lead chemicals with optimal therapeutic off-target effects, or to identify off-targets associated with toxicity, allowing more optimal chemistry to be developed. It could also be applied to phenotypic screen-based drug discovery for target deconvolution of small molecules. This method could, in the short term, avoid unnecessary animal studies for compounds that fail due to lack of efficacy or because of toxicity. In the long term, the technology could further reduce animal use by accelerating the adoption of adverse outcome pathways and systems pharmacology modelling approaches by identifying molecular initiating events.

With the support of CRACK IT Solutions funding, the consortium is now working with Biogen, an US-based company, to further develop technical aspects of the platform such that it is ready for larger scale studies against 500-1000 targets and adoption by end users.

Full details about this CRACK IT Solution can be found on the CRACK IT website.

This study aimed to develop a mass spectrometry (MS)-based system to profile the interaction of unmodified (label-free) chemicals to protein targets of interest. This tool could be used early in drug discovery to identify off-targets associated with toxicity and for identifying targets of small molecules in phenotypic screens, avoiding unnecessary animal studies for compounds that fail due to lack of efficacy or because of toxicity.

In the CRACK IT Challenge: Targeting off-targets, Phenotox Ltd, Retrogenix Ltd and Sheffield Hallam University (SHU) demonstrated that the Advion Liquid Extraction Surface Analysis (LESA) high throughput automated solvent extraction/MS injection system can identify the binding of small molecules to their targets. Through CRACK IT Solutions a new partnership was established with Biogen to develop the LESA tandem MS platform (LESA-MS/MS) towards large scale studies and commercialisation.

The LESA-MS/MS platform detected test compounds and MS standards, spotted on glass slides, down to the level of one femtomole (1 x 10-15 moles). This was within the theoretic range (10 attomoles (10 x 10-18 moles) to 10 femtomoles) of the amount of compound that would be expected to bind to an over-expressed receptor in a transfected cell in the microarray spot. However, significant cross contamination of the test compounds was found in the vehicle control spots, which was caused by the LESA-MS/MS ionisation system. This fault with the LESA-MS/MS meant that test compounds were evaluated using an alternative MS-imaging platform (a matrix-assisted laser desorption ionization (MALDI) MS) for the remainder of the study. Slides consisting of 48 different target sites (GPCRS, ion channels, enzymes and transporters) were prepared and exposed to six test compounds at low, medium and high concentrations. The MALDI-MS was able to detect these compounds, however no specific binding to target expressing cells was obtained, which suggests that the MALDI-MS does not have sufficient sensitivity to detect this (limit of MALDI-MS sensitivity is 5-50 femtomoles). These results indicate that the application of MS, for the detection of small molecule binding to targets expressed in mammalian cells, will require the development of a more reliable high throughput MS analysis platform with sub-femtomolar detection sensitivity.

Discussions with pharmaceutical and agrochemical industries enabled the consortium (Phenotox Ltd, Retrogenix Ltd and SHU) to confirm there is an unmet need and commercial opportunity for label-free small molecule target profiling for both drug discovery and chemical safety assessment. Specific interest in the capability to profile against particular protein target classes (e.g. transporters, ion channels and serine hydrolases enzymes) and support in selection of test compounds was also recognised.  

Further work is required to optimise the LESA-MS/MS platform, making it suitable for use in drug discovery and chemical safety assessment, as this approach could be used as an early toxicity and efficacy screen to reduce the number of toxic or poorly efficacious compounds progressing to animal studies.   

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CRACK IT Solution

Award date:

Jun 2016

Contract amount


Primary 'R'


Scientific Discipline



In vitro techniques


Drug safety
Mass spectrometry