10 Common Mistakes in Fragment Screening

There is an excellent review paper from Dan Erlanson and Ben Davis that came out last year detailing some of the more common mistakes and artifacts that can arise in fragment-based screening campaigns (so-called “unknown knowns”).  I encourage readers to go read the original paper.  I have summarized some of the key points below:

1) Not checking compound identity to make sure what you think you purchased is what you actually have.

2) Low-level impurities in compound stocks can cause problems at the high concentrations used in fragment screens.

3) DMSO, commonly used to store fragments in plates, can act as a mild oxidant and is also hygroscopic.

4) Pan-assay interference compounds (PAINS) are common in many libraries and are found to give false positives to many targets.

5) Reactive functional groups in fragment hits can cause covalent binding or aggregation of the target.

6) Many fragments can show binding or inhibition while acting as aggregators rather than reversible binders.  Including a small % of detergent can help eliminate these kinds of fragments from giving positive signals.

7) STD-NMR is very sensitive to weak binders, but because it relies on a relatively fast disassociation rate for the ligand, tight binders (<1 uM) can be missed by this method.

8) X-ray crystallographic structures are often taken as the “truth” when they are in fact a model of an electron density.  Fragments can often be modeled into the density in incorrect orientations or in place of solvent atoms.

9) SPR methods are very sensitive to fragment binding, but can be confounded by non-specific binding of fragment to the target or chip, as well as compound-dependent aggregation.

10) Fragment hits should be validated by more than one method before embarking on optimization.  They should also be screened for being aggregators by DLS or other methods.

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