The tendency of fragments found in one assay to reproduce – or not – in another assay is a frequent topic at Practical Fragments. In a recent paper in Bioorg. Med. Chem. Lett., researchers at Sanofi describe their experience screening the oncology-associated kinase MEK1.
The researchers were interested in the ATP-binding site of MEK1, and they started with a virtual screen (using Glide-SP) of a 10,000 compound library. The top 196 hits were then tested experimentally by differential scanning fluorimetry (DSF) and surface plasmon resonance (SPR), leading to 30 and 44 hits, respectively, with 12 in common. A subsequent biochemical assay of the same 10,000 compound library yielded 106 hits, only 13 of which were in common with the virtual screen. 158 different fragments were identified by one or more of the three experimental methods.
Of 13 hits selected for follow-up experiments, crystallography ultimately yielded structures for 7 of them, of which 5 had been identified in the virtual screen. Interestingly, SPR had only confirmed 2 of these molecules, while DSF had confirmed all of them. Thus, in contrast to some reports, the Sanofi folks are quite sanguine about DSF and advocate using it widely and early in a project (as indeed many people do seem to be doing). The technique is fast and easy, and in this case the researchers were able to run the DSF screen before they had finished developing their biochemical assay.
The paper includes detailed comparisons between virtual screening, DSF, SPR, biochemical, and X-ray approaches, and is well worth examining if you are putting together a screening cascade.
The researchers conclude:
There is no gold-standard method for screening fragments. The general approach is to conduct a primary screen and then follow this up with at least another method to confirm hits, which are subsequently prioritised for structure determination. Different groups adopt different methods based on availability of materials, in-house expertise and prior experiences screening fragments.
In other words, multiple methods can find fragments. Ultimately, you’ll probably find real hits whatever methods you use, as long as you’re careful.