Screening


Figure 3 Representative thermal shift


data showing the ‘second peak’ profile of most of the screening hits


References 1 Simpson, PB, Reichman, M. Nature Reviews Drug Discovery, 2014, 13(1), 3-4. 2 Urquart, L. Nature Reviews Drug Discovery, 2019, 18(4), 245. 3 Drewry, DH, Wells, CI, Zuercher, WJ, Willson, TM. SLAS Discovery, 2019, 1-10. DOI: 10.1177/2472555 219838210. 4Waldmann, H, Valeur, E, Guéret, SM, Adihou, H, Gopalakrishnan, R, Lemurell, M, Grossmann, TN, Plowright, AT. Angew. Chem. IEE, 2017, 56(35)10249-10323. 5 Scannell, J. Forbes, 2015, onforb.es/1ZdvQbc. 6 Editorial, Nature Biotechnology, 2014, 32(2), 109. 7 Association of British Pharmaceutical Industries report http://www.abpi.org. uk/media/1372/the-changing- uk-drug-discovery- landscape.pdf.


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enzyme with a strong reducing environment as structurally and functionally important cysteine residues can easily be oxidised when exposed to a variety of chemical compounds. Indeed, inclusion of 2mM DTT dramatically reduced the number of compounds causing greater than 20% inhibition to 9% (Figure 1) and almost all of these were classed as redox cycling compounds (RCCs). These types of compounds react with strong reducing agents, such as DTT or TCEP to produce hydrogen perox- ide which in turn can oxidise sensitive residues such as cysteines leading to target inhibition. A third screen of the library, this time with the inclu- sion of 5mM of the weaker reducing agent cys- teine, shows an assay that has lost its sensitivity to RCCs. Identifying only two compounds that pro- duced slightly greater than 20% inhibition, albeit there is somewhat more noise in the baseline with the weaker reducing environment and the apparent effect of these two falls within that noise (Figure 1). An important caveat to the use of this robust- ness set is that it does add extra time on to assay development as any substantive changes in the buffer environment requires the reassessment of enzyme kinetics and potential adjustments to sub- strate concentration or end point read time if sig- nificant changes become apparent. We continually look for ways to improve the performance of the robustness set and we have


reported on an example of triaging the output from a screen within the European Lead Factory which illustrates how we are doing this16. A mitochondri- al enzyme linked to the proliferation and tumouri- genic capacity of some cancer types was screened against 450,000 compounds of the Joint European Compound Library. A primary hit rate of ~1% was of little concern but, somewhat unusually, almost all of the primary hits confirmed as inhibitors in two orthogonal biochemical assays and many showed shallow Hill slopes across a very limited range of potencies. One of the restrictions of the European Lead Factory is that we have a require- ment to select no more than 50 compounds from each screen17, which is a daunting task when there are ~4,000 confirmed hits to choose from and questions surrounding what the data is telling us. Usefully, we had validated a thermal shift assay, in which an orthosteric reference inhibitor showed a clear, saturable and concentration-dependent increase in stabilisation of the target protein (Figure 2). This assay provided the throughput for testing all ~4,000 compounds, which, surprisingly, almost all appeared to cause a change in the profile of protein stability. Interestingly, only six of the hits caused a thermal melting profile reminiscent of the reference inhibitor, ie a clear rightward shift of the single melting peak. The remainder all produced the appearance of a


Drug Discovery World Fall 2019


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