In Silico Prediction of Off Target Activities of Chemical Probes


Chemical probes are frequently described as important tools in the validation of new drug targets, yet many of them are of little value due a number of important factors frequently including poor selectivity. This issue was highlighted by Frye in 2010 (Frye, Nature Chemical Biology 2010, 6(3), 159-161) where he illustrated the problem with the case of staurosporine, a promiscuous kinase inhibitor and the subject of 8,000 publications, many of which make conclusions which are not valid due to the compounds promiscuity. Frye defines a set of criteria which a probe should have in order for it to be a valuable tool to the drug discovery scientist. One key criterion is selectivity and he states that it is important that ‘a quality chemical probe has sufficient in vitro potency and selectivity data to confidently associate its in vitro profile to its cellular or in vivo profile,’ Which is valid suggestion.
Determination of selectivity is difficult as the majority of groups including pharmaceutical companies only have access to a limited range of selectivity assays and establishing selectivity against targets related to the protein of interest is relatively straightforward; however assessing broader selectivity is an issue both with respect to choosing targets to screen against and accessing suitable assays.
A recent publication highlights the importance of assessing broad selectivity and describes an in silico approach capable of predicting off target liabilities (Antolín and Mestres, ACS Chemical Biology 2014, ahead of print). The authors apply their software to the full set of chemical probes from the NIH Molecular Libraries Program (MLP) with some very interesting findings, illustrated by the examples below.
Table 1 shows the activity at the canonical (original target) of four probes in the collection together with off target activity identified through in silico testing and subsequently demonstrated in a biological assay. In each case the probes are more potent at the newly discovered target than the original published target.


ML006 was published as a weak probe for the S1P3 shingosine receptor but has micromolar potency for mTOR kinase. ML124 and ML204 were originally identified as probes for TRP channels, TRPML3/2 and TRPC4/5 respectively but show off target activity as acetylcholinesterase inhibitors (ML204) and sigma receptor ligands (ML124). ML141 was identified as a highly potent carbonic anhydrase inhibitor having originally been described as a selective inhibitor of Cdc42 GTPase. The latter discovery regarding ML141 should possibly have been identified earlier as the template in question is a well documented carbonic anhydrase inhibitor chemotype (Weber et al J. Med. Chem. 2004, 47(3),550-7). These results thus pose significant questions as to the usefulness of this set of compounds as probes.
The publication from Antolin et al not only highlights the caution that workers need to exercise when using chemical probes but also offer a potential strategy for checking the validity of the probe before drawing conclusions regarding experiments in which the probes have been used.

Compound contaminants; a story of false positives

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) ( 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.