11 April 2010

Getting misled by NMR: ILOE artifacts

We’ve pointed out potential pitfalls with crystallography (here, here, and here) as well as with biochemical screening, but NMR has escaped attention– until now.

NMR has of course been a mainstay of fragment discovery methods since the original SAR by NMR paper. There have been plenty of developments since, but one that is particularly intriguing relies on the “interligand nuclear Overhauser effect,” or ILOE. In the “SAR by ILOE” approach, a 2D NMR experiment is used to detect when two small molecule ligands bind to a protein next to one another. There are some attractive features of this method. First, only ligands that bind in relatively close proximity to each other will generate a signal, thereby allowing researchers to identify fragments close enough to allow productive linking. Second, the technique can be applied to proteins that are too large to study by other NMR methods. In fact, it can be used even in the complete absence of structure. So what’s the problem?

In a new paper in J. Am. Chem. Soc., Chris Abell and colleagues at the University of Cambridge applied the approach to pantothenate synthetase (PtS) from M. tuberculosis. They previously did rigorous fragment screening followed by both linking and growing on this enzyme, which we discussed last year. Initial NMR experiments with compounds 1 and 2 (see figure) in the presence of PtS showed strong ILOE signals; the problem was that signals were seen between all the protons of compound 1 and all the protons of compound 2. This suggests non-specific binding: if the two molecules were binding next to each other in a single orientation you would expect that some protons from compound 1 would be closer to some protons in compound 2 than others, and there would thus be differences in signal intensities.

Adding a methyl group to compound 1 to give compound 4 didn’t help. In fact, there were ILOE signals from both the methyl groups of compound 4 to all the aromatic protons of compound 2, again suggesting non-specific binding. Even more damning, adding the substrates ATP and pantoate failed to significantly diminish the ILOE signals as expected; because crystallography showed these fragments bind in the active site, they should have been readily displaced by substrates.

Reasoning that the hydrophobic nature of compound 4 might be causing it to aggregate at high concentrations, the researchers appended a carboxyl group to give compound 5. NMR experiments with this compound in the presence of compound 2 and the protein now revealed specific ILOE signals between the 2-methyl group of compound 5 and H2 of compound 2. Moreover, this signal could be competed by adding ATP and pantoate.

Happily, linking these two fragments together resulted in compound 6, which bound to the enzyme three orders of magnitude more tightly than either of the starting fragments. The compound was also well-behaved mechanistically, showing competitive inhibition with ATP, and a crystal structure revealed that it binds as expected given the structures of the individual fragments.

Overall then this is a success story. However, it does suggest that the ILOE method may be more prone to aggregation artifacts than other biophysical methods. In particular, had the researchers not been able to do competition experiments (if, for example, they did not have another small molecule inhibitor available) they would have had a harder time sorting things out. Also, the researchers actually had crystal structures of both compounds 1 and 2 bound to PtS, so it is not clear how valuable the ILOE data really were for linking. Still, the potential advantages of an NMR-based method that doesn’t require structure are appealing. Hopefully we will see more applications of SAR-by-ILOE, now that people are more aware of the dangers.

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