As noted last week, Practical Fragments has been on something of a crystallography binge. But according to polling, NMR is the most common fragment-finding method. And, according to a different poll, saturation transfer difference (STD) is the most popular NMR technique. Familiarity breeds complacency, and widespread assumptions go untested. A new paper in Front. Chem. by Jonas Aretz and Christoph Rademacher (Max Planck Institute and Freie Universität Berlin) suggests that this is a mistake.
In STD NMR, a protein is saturated by specific electromagnetic pulses, and the resulting magnetization transfers to bound ligands. Assuming that the bound ligands are in rapid equilibrium with ligands free in solution, this “saturation transfer” results in a reduction of NMR signal for the small molecule in the presence of protein compared to no protein. High affinity ligands will remain bound to the protein and thus be missed by STD NMR, but this is usually not relevant in FBLD, where most fragments bind with dissociation constants weaker than 10 µM.
A common assumption with STD NMR is that the strength of an STD signal increases with the affinity of the ligand (again, in affinity ranges between about 10 µM and 10 mM). Indeed, when STD NMR is used as part of a screening cascade, molecules showing the strongest effect are generally prioritized as hits. But is this assumption correct?
To find out, the researchers retrospectively analyzed a fragment screen against langerin, a carbohydrate-binding protein we discussed last year. When they plotted the STD amplification factor against the affinity (measured by SPR) for several dozen fragments, the resulting scatter plot showed no correlation.
Recognizing that experimental errors could obscure a true correlation, the researchers ran virtual STD experiments using COmplete Relaxation and Conformational Exchange MAtrix (CORCEMA) theory. They used well-characterized fragments with published crystal structures and affinities for some dozen diverse proteins. As they conclude, “varying saturation time, receptor size, binding kinetics, and interaction site… there were no conditions in which the STD NMR amplification factor correlated unambiguously with affinity.”
But it gets worse. When the researchers explored the effects of binding kinetics, they found that ligands with slower on-rates or off-rates also had lower STD signals. Several groups have advocated prioritizing compounds with slower-off rates, yet these are the very compounds STD is most likely to miss.
All in all this paper could go some way toward explaining the sometimes poor correlation between different fragment-finding methods.
That said, I’m no NMR spectroscopist, so I’m certainly not as qualified to comment on the importance of this paper as someone like Teddy, who co-wrote this how-to guide for STD NMR. I’d be interested to hear what NMR folks think, and whether we should rethink use of STD. In any case, this work is a useful reminder that skepticism is a scientific virtue.