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.

Colloidal Aggregation in Cell Culture

We’re told phenotypic screening is the answer to the lack of drug discovery productivity, ‘Return to the old ways and old-style productivity will return’. Well, a paper has just been published in ACS Chemical Biology which may throw some interesting light on this approach.

Brian Shoichet et al have had a long standing interest in the physico-chemical properties of screening molecules. Their first paper in this series, published in Journal of Medicinal Chemistry in 2002, arrived just in time to salvage some of my tarnished reputation with the screening group at a previous company. We were working on inhibitors of a novel antibacterial target and for some time had been following a set of compounds with flat mM SAR. All active molecules were highly lipophilic. No matter which solubilising groups you added to improve physico-chemical properties, activity always disappeared. Only as we added successive fluorines and iodines did activity increase. The observation that lipophilic molecules (such as the ones we were making) tended to form aggregates in solution and sequester protein was a ‘eureka moment’ for us. It explained our SAR (or lack of it) and allowed both chemists and biochemists to climb down from the increasingly confrontation positions we’d been adopting at project meetings! Our follow-up antibacterial projects had a strong focus on physico-chemical properties and bacterial membrane penetration.

At the same company we also had a ‘skunkworks’ project, run in our spare time, in which we used an oncology cell line, developed by a friend during his PhD, to screen some focused sets of compounds. We were looking for phenotypic responses to compound. Once again, screening initially at 20mM single concentration, we discovered some mM hit compounds but none of the nM activity we’d been hoping for.

Perhaps throwing some light on our results, Shoichet et al have used transmission electron microscopy (TEM) to show that some oncology drugs have a propensity to form colloidal aggregates inside cells (not just in biochemical assays). The aggregates act as reservoirs of compound within cells effectively reducing the cellular concentration of the compound. Thus, in contrast to the effect observed for biochemical assays, this can be expected to lead to a raised false negative hit rate. Active molecules will look less active because their effective cellular concentration is lower. The obvious answer is to add detergent to the system but of course most detergents are toxic to cells. However, they discovered that 0.025% Tween-80 was non-toxic in their cell lines. In the presence of compound the detergent was sufficient to break up the colloidal aggregates almost completely. They comment that ‘the monomeric drug forms were substantially more toxic than the colloidal forms, which consistently showed no significant anti-proliferative effects.’ So, returning to our ‘skunkworks’ project, perhaps some of our mM hits would have been considerably more potent had we developed the assay with a small amount to detergent to break up aggregates…we’ll never know!

The authors then go on discuss the observation that Evan’s Blue, a dye frequently used to measure vasculature leakage in tumours, also readily forms aggregates. In the past the concentration of Evans Blue dye within tumours (“enhanced permeability and retention (EPR) effect”) had been ascribed to the binding of Evans Blue to albumin and then transport of albumin across leaky membranes. The authors suggest this is not the case but that Evans Blue colloidal aggregates may cross cellular membranes anyway, bound or unbound to albumin. They conclude with the intriguing suggestion that an ability to form aggregates may be required to enable the concentration of certain anti-cancer drugs in tumours.

‘Evans Blue colloids are 120 nm in radius and will likely, themselves, permeate tumor tissue via the EPR effect, absent any protein. Indeed, such behaviour might, under the right circumstances, be true of other molecules, including colloid forming drugs. This would have a profound effect on their distribution and efficacy in vivo and may merit further study.