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 (  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


More non specific inhibitors ….

After last week’s article on colloidal aggregation in cell based assay formats, another paper dealing with non specific inhibitors caught my attention: (, published by workers at Genentech. It covers their experiences with non specific inhibitors in a panel of assays, but particularly focusing on how these relate to the compound supply and the order of reagent addition in an assay.

Advances in compound management and dispensing technologies have lead to ARP screening (assay ready plates) where small volumes of test compounds at high concentration are added to the assay plate in advance of screening.  Reagents then are added to the plates on the day of screening. The main advantage being that ARP’s can be frozen ahead of the assay to allow greater flexibility of workflow, and have become a widely adopted method of screening for industrial groups.

The Authors investigated the use of ARP’s across seven different biochemical screens (six kinase programmes and one protease).  They observed a far higher hit rate with the same set of compounds when they added the enzyme as the first addition to the ARP, compared to addition of substrate as the first reagent, (this does also assume that there was no change in assay sensitivity with change of order reagents- unlikely but stranger things happen with assays).

They ran two different sets of compound libraries, a Kinase focused set and a random HTS subset against the seven targets. With the HTS set they saw a greater rate of hit rate reduction compared to the kinase focused set when the order of reagents was changed, suggesting that this extra hits were due to a non specific mechanism of inhibition on the targets. These assays also included the conventional supplements of  0.01% Triton X and 0.01% BGG (bovine gamma globulin) believed to reduce the effects of compound aggregation, which suggests that both carrier protein and detergents have to be carefully optimised for each target to be effective in this role.  The suspect “hit” compounds were further proved to be non specific inhibitors by use of further analytical techniques such as SPR (Surface Plasmon resonance) and DLS (Dynamic light scattering). The suggested mode of action as non specific inhibitors is that these compounds are aggregating and forming a colloidal with target protein and preventing its action on the substrate. This is enhanced when you have a high concentration of compound pre-incubated with your target protein, as in ARP screening.

Another interesting finding from the authors is that BGG is a more optimal carrier protein to use compared to BSA (Bovine Serum albumin), as the latter seems to show a greater reduction of true inhibitors potency due by its binding of compounds.  This is particularly interesting to me as I’ve seen many assays where BSA is added, and the reasons usually given to its presence, is to help the target protein activity.  It should probably be removed or replaced if the assay does not need it for a specific reason.

Personally I feel the use of ARP’s can be beneficial in assay screening groups, and also I’ve seen that time dependence can be a factor in potency determinations on compounds (e.g. a pre-incubation of compounds with target protein does change the potency determination).  However the results of this group suggest pre-incubating your compounds in this ARP format could increase your chance of enriching your hits with non-specific inhibitors, and cause confusion with incorrect SAR, (and nobody likes to see an upset med chemist!)

Overall nothing beats a good, early screening cascade to identify non specific inhibitors and other false positives early on, but from these recommendations you can make it a little easier on yourself.