As Teddy just mentioned, biophysics provides multiple methods to find fragments, and it's best to use several. They may not always agree, but fragments that hit in several assays are more likely to be real. Although this requires using different skills, using diverse methods does not take a village, as illustrated by a recent paper in Proc. Nat. Acad. Sci. USA by Hernani Silvestre, Tom Blundell, Chris Abell and Alessio Ciulli at the University of Cambridge.
The researchers were interested in the enzyme pantothenate synthetase (Pts) from the bacterium that causes tuberculosis (a target we’ve covered previously here and here). They used a thermal shift assay to screen 1250 fragments from Maybridge, each at a whopping 10 mM concentration, resulting in 39 hits (3.1%) that stabilized the enzyme by at least 0.5 ˚C. Another 17% of the fragments slightly stabilized Pts, while 73% of the fragments destabilized the protein (a poorly understood phenomenon that does not seem to reflect specific binding).
Despite being relatively small, the fragment library had some scaffolds that were over-represented, and some of these were enriched among the hits, providing early SAR. Perhaps not surprisingly given the anionic character of the enzyme’s ATP cofactor and pantoate substrate, about half the hits were carboxylic acids.
All 39 hits were analyzed by WaterLOGSY and STD NMR, and only 17 showed evidence of binding, although the NMR experiments were done at a much lower concentration of fragment (0.5 mM). Competition experiments revealed that all except one of the 17 fragments bound at either the substrate or cofactor sites.
Next, isothermal titration calorimetry (ITC) was used to measure the dissociation constants of the 17 validated fragments. Measurements could not be obtained for three of the fragments; values for the rest ranged from 0.5 to 17 mM. There seemed to be a rough correlation between affinity and the extent of stabilization in the thermal shift assay, and binding was enthalpically-driven.
The 17 fragments were then soaked into crystals of Pts, resulting in 8 co-crystal structures. Most of the fragments that did not produce structures also had low affinities as assessed by ITC. Four fragments bound in a pocket normally occupied by the adenine ring of the cofactor ATP, while the other four bound in the substrate pantoate pocket. One of these bound quite deeply in this pocket and caused a conformational change in the protein. The researchers were able to obtain a structure of Pts bound to both this fragment and ATP.
There’s lots of nice data in this paper, and all the new structures have been deposited in the protein data bank. More generally, the “integrated biophysical approach” provides a practical template for applying FBLD. The paper has just four authors, and only two of them actually performed experiments according to the author contributions section. That short list provides more evidence that a very small but dedicated team can successfully find and validate fragments.