20 May 2019


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.


Dr. Teddy Z said...

Hi Folks. I don't do this anymore, so I just skimmed the paper right now after being prompted by Paul Belcher. What I couldn't find in a cursory inspection was what protons were used for STD rank ordering. However, they do cite the Yan et al paper from 2003 which means that they tried to control, or atleast understand that you can't rank order different types of protons, for different T1 times of the observed protons. I would be curious to see if this is a one off. i.e. due to the nature of this particular target. I believe others have (IIRC) found decent correlations with SPR and STD NMR in the past.

Yun Shi said...

Hi there, I think many NMR folks won't find this as news, and might be a little surprised that this Front Chem paper on something commonly known (for NMR folks) gets published. But as the blog indicated it has definitely been mis-assumed for non-NMR folks.

Similar to line-broadening effects, STD effects only exist for binding parameters in certain ranges. Non-binding parameters such as rotational correlation time would also influence. What's useful, though, would be competitive assays with a positive control (known good binder). We routinely use competition STD NMR to verify fragment hits from 19F NMR screenings.

CORCEMA calculations would be nice if accurate estimations can be obtained for parameters like rotational correlation times and kinetic constants of association/dissociation.

Ben Davis said...

Agreed - there's no real reason why STD intensity should correlate with a single parameter such as affinity; there are many factors which contribute to the observed signal. As Yun Shi says, competition (displacement by a known potent ligand) definitely improves the correlation between observed STD signal and "true" binding, and I'd also strongly advocate looking for consistency of behavior in multiple binding experiments (LOGSY, STD, T2 filtered) to prioritise the most probable hits.

Kevin G said...

This is certainly not surprising. However, it is nice to have a clearly documented rebuttal the next time someone suggests triaging by STD intensity instead of doing the appropriate follow-up experiments.