29 July 2019

SAR by WaterLOGSY?

Among ligand-based NMR methods, WaterLOGSY is nearly as popular as STD NMR. Normally the information obtained is limited: does a given small molecule bind to a protein or not? In a new paper in J. Enzyme Inhib. Med. Chem., Isabelle Krimm and collaborators at the Université de Lyon and University of York try to wring more data from this common experiment.

In WaterLOGSY, magnetization is transferred from water, to protein, and then to bound ligand. This can happen through multiple mechanisms, and even talented NMR spectroscopists have told me they have trouble understanding exactly what is going on. In short, the WaterLOGSY spectra of molecules bound to proteins show a change in sign compared to molecules that don’t bind. Examining ligands in the presence and absence of protein can thus provide evidence for whether a ligand binds.

The researchers go beyond this simple qualitative approach and look at changes in peaks corresponding to specific hydrogen atoms in each ligand. They define a “WLOGSY factor,” which shows an inverse correlation to solvent exposure. In other words, a smaller WLOGSY factor means that a given hydrogen atom in a ligand is more exposed to water, and thus less exposed to protein. If all the hydrogen atoms in a bound ligand have the same WLOGSY factor, this suggests either multiple binding modes, or that the ligand is completely enclosed by the protein. If, on the other hand, different hydrogen atoms in a bound ligand have different WLOGSY factors, this could provide information on the binding mode. This analysis is conceptually similar to the STD epitope mapping the Krimm lab described several years ago, and STD experiments were also run on the proteins here for comparison.

To validate the approach, the researchers tested six proteins (with molecular weights ranging from 22 to 180 kDa) for which fragment ligands had been previously identified with affinities from 50 µM to worse than 1 mM. Screens were done using 400 µM fragment and 5 to 20 µM protein. (NMR aficionados, please see the paper for details on the effects of mixing times and ligand exchangeable protons.)

The results look pretty impressive: for PRDX5, HSP90, Bcl-xL, Mcl-1, and glycogen phosphorylase, the ligand hydrogen atoms previously shown to be solvent exposed from crystallographic or two-dimensional NMR structures do in fact show reduced WLOGSY factors. In the case of human serum albumin, a ligand showed uniform WLOGSY factors, suggesting multiple binding modes, as expected given the multiple promiscuous binding sites on this protein.

To a non-NMR spectroscopist such as myself, this seems like a useful approach for obtaining binding information in the absence of crystallographic data. It also seems easier to run than the LOGSY titration we highlighted a couple years ago. But the first word of this blog is “Practical.” We recently discussed work demonstrating that STD NMR data is perhaps not as easily interpretable as many assume. Have you tried anything like this yourself, and if so how well does it actually work?


Dave Fry said...

re: "even talented NMR spectroscopists have told me they have trouble understanding exactly what is going on" - Agreed; but underappreciated is that when you hit water with these techniques, you are also hitting the really-fast-exchanging protons of the protein (Ser, Thr, Tyr OH's; and Lys and Arg side-chain NH's) and thereby getting NOEs and spin-diffusion to the ligand, which must be near some of these. There's actually no need to invoke "bound water".

Anonymous said...

Using LOGSY data to orient a ligand in an active site is a very seductive idea, particularly when no other structural information is available.

We first tried correlate LOGSY intensities (as a free-bound difference, normalised by the number of protons, not the free LOGSY) to solvent exposure in the late 1990s. This involved looking at a number of ligands bound to a protein with a deep & narrow binding site. We had X-ray structures for a number of ligands, but found it was not even possible to decide which way they inserted themselves into the site using their LOGSY data. My belief is that the distribution of water-correlation times in and around the site, along with the exchange of water protons with NH and OH groups of the protein and ligand, modulated the LOGSY effects to an extent that proximity to bulk solvent became a minor factor.

When STD and LOGSY became routine screening tools, Mark Howard (then at the University of Kent) and I had a PhD student, Nathan Ley, who tried to orient bound ligands for HSP90 by combining STD and LOGSY data, while also making allowance for ligand relaxation. It was clear that, to be useful for drug design, detailed knowledge of the positions and lifetimes of bound water molecules would be needed which severely limits the value. The thesis is online here: https://kar.kent.ac.uk/50197/

Glyn Williams said...

Apologies - I'm not the well-known Anonymous.