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.
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3 comments:
Do you think the fragment screening concentration plays a significant role in hit identification? I noticed that the authors use 500 uM for the DSF and 200 uM for SPR.
Generally, yes, but Id say this is more a reflection of the techniques used.
2 Things:
1)
SPR is VERY sensitive for bad behaviour and you really dont want to challange solubility. DSF will generally not give false positives as easily and higher conc is more feasible.
2) sensitiivity. SPR, when it works well can handle partial occupancy just fine (i.e. no need to be at or close to saturating affinities) while DSF typically do benifit significantly of higher concentration (as long as cpds stay soluble that is). Id say 500uM is quite conservative, generally i prefere 1mM for DSF (unless its a high hit rate target as a kinase) where dropping down a bit can make sense.
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