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.

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