15 May 2017

NMR structures without protein assignments

Our latest poll asks how much structural information you need to advance a fragment (please vote on the right hand side of the page). On this subject, a recent paper by Marielle Wälti, Roland Riek, and Julien Orts in Angew Chem. demonstrates a new NMR method.

Researchers typically begin an NMR structure campaign by examining the chemical shift perturbations (CSPs) of proton-nitrogen or proton-carbon crosspeaks from an isotopically labeled protein in the presence and absence of a ligand. If you know which crosspeaks correspond to which specific atoms in an amino acid residue, you can deduce the ligand binding site by looking for the residues with the largest CSPs. Next comes the measurement of nuclear Overhauser effects (NOEs) between atoms in the ligand and atoms in the protein; these are exquisitely dependent on distance, so if you have enough measurements you can use these to accurately dock your ligand into the binding site of your protein.

This is how SAR by NMR was done more than twenty years ago, and it still works well today, but it is neither fast nor easy. In particular, the initial step of assigning the hundreds of protons, nitrogens, and carbons in a typical protein can be daunting.

To streamline the process, the researchers developed NMR molecular replacement (NMR2), first published last year (here) and presented by Julien at FBLD 2016. Rather than requiring knowledge of which peaks correspond to which specific protein atoms, NMR2 relies on the increasing power of computers to run large numbers of complex calculations. Various docking poses will generate different NOEs, so exhaustively and iteratively examining these possibilities and comparing them with the experimental data should generate an optimal model. (NMR2 does require that the structure of the protein is known, so you know the residues surrounding a ligand-binding pocket, even if you don’t know their chemical shifts. Also, the protons of the ligand are assigned, and in fact the intramolecular NOEs of the ligand itself are an important input.)

In the new paper the researchers apply NMR2 to a complex between the onocology target MDMX and a previously disclosed high nanomolar binder and find good agreement (1.35 Å RMSD) with the crystal structure.

The researchers then turn to “ligand #845,” which binds with millimolar affinity to the oncology target HDM2. A total of 33 intramolecular NOEs (from ligand #845) and 21 intermolecular NOEs (between ligand #845 and HDM2) were fed into NMR2 and used to crank through 54,000 structure calculations in a few hours to produce a binding model. No crystal structure was available, but conventional NMR methods support the model.

This seems like a rapid and powerful approach, but readers of this blog are probably wondering how well it will apply to fragments. Clearly NMR2 is sufficiently sensitive to weak binders. However, with 24 non-hydrogen atoms and a molecular weight of 354 Da, ligand #845 is too large to be called a fragment. Smaller molecules will have fewer hydrogen atoms and thus fewer intramolecular and intermolecular NOEs, decreasing the information content of the model. It will be fun to plumb the lower ligand size limits for this technique – leave a comment if you’ve done so!

1 comment:

Unknown said...

Interesting technique. I hope we see more of such 'marriage' between computational and experimental approaches...

FWIW I think that with a 'true' fragment (a dozen CNO, less than ten H) the NMR2 method will help 'funnel' down possibilities - ideally resulting in a significantly paired-down list of poses similar to what an advanced MD/D simulation could provide (but with the distinct advantage of having experimental data as part of the picture). The smaller the fragment, the more ambiguity in the final results. With fragments one also has to be fairly agnostic regarding the 'binding site' residues since fragments are known to bind 'all over the place' (not really but you get my drift).

Practically speaking, if one is interested in a specific binding site and one already has a (literature) compound that is known to bind, NMR can be used to map out perturbations in N15/H spectra such that many of the active site residues become known (not individually assigned, but known as such). When binding fragments are discovered it becomes possible to map them w.r.t. known resonances of active site residues - this should significantly decrease computational burden as well as ambiguity of output.

In the case of unknown structure, this still helps determine the overall nature of binding.

Or we can solve the structure :)