Screening


compound destabilised the protein in a thermal shift assay. Chemical analogues of the favoured compound only revealed ‘flat’ SAR, elevating con- cerns regarding the mode of activity. Finally, crys- tallography identified contaminating zinc in the samples (a by-product of compound synthesis) as leading to a metal-mediated oligomerisation of the protein which manifested itself in the original assay as functional inhibition. Technically correct, but not a promising avenue for further work towards developing a drug-like inhibitor. The second exam- ple is a twist on the problems of aggregators. Seminal work by Shoichet et al14 revealed that some compounds form aggregates in solvents. The classical description of aggregators is as forming micellar-type structures that can effectively absorb the protein target in an assay, which is observed as inhibition of target function. Of course, the specific structures that these aggregates adopt will be dic- tated by the physicochemical properties of the compound, its concentration and the environment of the assay – one reason why detergents are com- monly included in screening assays to disrupt aggregation. What is probably less well appreciat- ed is that some aggregates can adopt interesting structures that allow them to act as specific inhibitors. Blevitt et al15 describe the characterisa- tion of an inhibitor of the interaction between TNF and the TNF receptor. Rather than describing a pharmacologically-useful mechanism of inhibition, they used crystallography to identify an aggregate of five molecules mimicking the struc- ture of the TNF subunit. This would replace one of the genuine subunits in the active trimeric target protein leading to a conformational change and apparent inhibition. Both of these examples illus- trate the requirement for a range of techniques to


be available to the screening group together with the expertise to perform and interpret the results.


Assay optimisation and the use of a robustness set An approach to minimise the impact of these prob- lems is to ensure the screening assay is fit-for-pur- pose by developing knowledge of the target’s sensi- tivity to common mechanisms of interference. To help achieve this we have established a ‘robustness set’ of compounds comprising those known to be ‘bad actors’ in high throughput screens, eg redox cycling compounds, aggregators, chelators, coloured, fluorescent and reactive. Testing these compounds in an assay highlights which classes may cause problems and we typically redesign the assay to eliminate or reduce the apparent sensitivi- ty to these mechanisms. An example of this is from a potential metabolic oncology target, phospho- fructokinase (PFK). Using a well-established method of monitoring ADP production, ADP Hunter by DiscoverX, we developed a basic assay using the same buffer conditions as used by our academic collaborator. While all of the usual assay quality metrics, S/B, %CV, Z’ and reference com- pound potency were exemplary, when the robust- ness set was screened, 90% of the compounds showed greater than 20% inhibition of PFK with no particular preference for their class of interfer- ence (Figure 1). Our experience is that if a target is activated or inhibited by >25% of the robustness set then it is likely to suffer from particularly high hit rates when screened against a ‘normal’ com- pound library and likely suffers from some form of environmental sensitivity. In this case, the basic assay buffer did not contain any reducing agent. As a rule, it is best practice to protect any cytosolic


Figure 2 Thermal shift assay data showing the concentration- dependent stabilisation of target protein by an orthosteric reference inhibitor


Drug Discovery World Fall 2019


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