Aggregation false positives in cell based assays?


An article dealing with the common problem of compounds that are false positives in screening assays was published recently (http://www.ncbi.nlm.nih.gov/pubmed/23437772).  One cause of compounds acting as false positives in screening assays is that they can self-aggregate, forming colloidal particles. This aggregation effectively sequesters the protein from its target and prevents activity. This has been a common problem in drug screening assays, particularly with soluble protein methods. In this publication the Shoichet lab at University of California, and others, have been investigating a further scope of the problem by examining  GPCR assays using a cell based format.

They took four compounds that were known to form aggregates and measured the activity against a variety of receptors using the Beta- Arrestin assay. The results show that these compounds were acting as antagonists against the receptors when they were stimulated with their agonist ligand, and this activity could be reversed with the addition of detergent or the use of centrifugation.

They also observed inverse agonism when the compounds were tested against the receptor in the absence of the activating ligand of the receptor, maybe via membrane perturbation.

It all highlights a type of assay artefact, which was thought to be more prevalent in soluble protein assays, can also have a bearing in cell based formats.The steps show by the authors (centrifugation and detergent usage) should be included to reduce the chance of false positives even if you are using a cell based method.

gw1Figure extracted from: Sassano, M. F., Doak, A. K., Roth, B. L., & Shoichet, B. K. (2013). Colloidal aggregation causes inhibition of g protein-coupled receptors. Journal of medicinal chemistry, 56(6), 2406–14. doi:10.1021/jm301749y

 

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