Screening


shoulder or second peak suggesting that very few of the hits were binding the target in a manner similar to the orthosteric reference ligand (Figure 3). Taken together, the unique thermal stability fingerprint, the unusually shallow Hill slopes, the limited range of potencies despite considerable physicochemical and structural diversity and the unprecedented con- firmation rates across multiple orthogonal assay formats raised the suspicion that there was some common contaminant affecting the hit samples. One area of investigation became assessing what common materials were likely to be present in preparation of the various hit structures, which led to the suggestion that oxalic acid would be worth investigating. Indeed, when oxalic acid was tested in the thermal shift assay it produced the exact same stabilisation profile as the hit compounds (Figure 4). It also inhibited the target in the bio- chemical assays with a Hill slope of ~0.6. The tar-


get was a large multidomain enzyme so a truncated construct lacking the reference compound binding domain was tested in the thermal shift assay. The orthosteric reference inhibitor and the six com- pounds that mimicked the thermal shift profile of the reference inhibitor in the full-length enzyme had no effect against the truncated protein. By contrast, oxalic acid and the remainder of the screening hits resulted in the appearance of a second peak with a concomitant reduction in the size of the main peak. Unfortunately, at this stage of the triage, the limited quantity of compound due to the nature of the ELF, and the sheer number of hits, prevented desalting and/or retesting from solid, which would have answered the question as to whether the activity we observed was solely driven by oxalic acid or some other common salt in the screening samples. Indeed, some of the hits may genuinely interact with the target in a manner similar to oxalic acid,


Figure 4 Thermal shift data showing the concentration-dependent appearance of a second peak caused by oxalic acid


Drug Discovery World Fall 2019


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