Have you ever had that feeling your assays are picking up multiple false positives, spikey SAR repeating itself through the screening cascade. Well it may not be the assays causing the problems. There has been some discussion recently about inorganic contaminants in screening decks causing false positives. Last year a group from Roche published a paper (1) (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027514/) from their experience that shines a little more light on this problem.
This group were running a project for Pad4 and their initial screen (a high throughput enzyme assay) produced a number of hit series. These were confirmed in the conformational screening using an ELISA (enzyme-linked immunosorbant assay) and binding was also demonstrated in a ForteBio and Biacore assay with IC50 and Kd values being in the low µM range. The trouble was all three series ‘lacked conclusive SAR’ and upon re-synthesis all displayed varying activities depending upon batch. The group then did a little detective work and found that the different routes of synthesis had a strong impact on the resulting compound potency. In short those batches that used zinc in their synthesis were positive in the downstream assays, those that didn’t were negative. This hypothesis was tested by screening ZnCl2, which was found to have an IC50 of 1 µM and a Kd of 1 µM.
There are regularly false positives in screening cascades, but these are commonly identified in orthogonal assays. What is interesting in this case is the hits were positive in three separate screens, demonstrating simply screening compounds in multiple assays is no guarantee of identifying false positives due to contamination.
To see how common zinc contamination was as a source of false positives the Roche group looked back at 175 past HTS campaigns. 41 showed a high hit rate of zinc-probing compounds (>25%), with the expected hit rate being <0.01%, demonstrating the extent of the problem. The important thing to note is they were only looking at zinc contamination; it is likely the total hit-rate for other inorganic containing compounds would be much greater.
The highest hit rate observed was in a fragment screen, which was performed at 250 µM. The group postulated since fragment screens are performed at higher concentrations they are likely to be a source of high false-positives due to inorganic contamination.
The trouble with these impurities is they are not flagged by purity checks on organic material and, as demonstrated by the Roche group, they can often maintain activity in downstream assays. It may be possible to run screening assays in the presence and absence of a non-selective chelator such as EDTA, however, the only way to be fully confident of the hits would be to re-purify, or re-synthesise hits. This is especially important when compounds are obtained from external sources and the route of synthesis is unknown.
Finally it is important to congratulate the Roche group for not only the investigation, but for publishing the results. It is something many of us will have come across, but mostly we will just move on rather than fully explore the frequency of the issue and share the information with the rest of the scientific community.
1. Hermann JC, Chen Y, Wartchow C, Menke J, Gao L, Gleason SK, Haynes N, Scott N, Petersen A, Gabriel S, Vu B, George KM, Narayanan A, Li SH, Qian H, Beatini N, Niu L, Gan Q. Metal Impurities Cause False Positives in High-Throughput Screening Campaigns. ACS Med. Chem. Lett. Chem. Lett. 4: 197–200, 2012.