20 March 2012

Practical Application of NMR

I am sometimes harsh on academic papers, especially those that purport to describe drug discovery. However, Isabelle Krimm and colleagues have continued their excellent work, previously discussed on this blog in a this paper. This paper reads like a "How To" on prosecuting an NMR-based screen. In this work, they have two goals in mind: studying fragments interacting with targets with multiple hotspots and determining the utility of fragments for allostery.

Glycogen phosphorylase is an interesting system to work in: it has an active site and six regulatory sites, including an allosteric site with a variety of positive homotropic and negative heterotropic effects between the various sites. They took 19 known inhibitors that bind to the active site (1-6), inhibitor site (7-9), allosteric site (10-12), and the "new" allosteric site (13-15) and deconstructed them.
Then they screened these fragments against GPa and GPb using both STD and WaterLOGSY. A compound was only deemed a hit if it was observed to bind via both methods. I find this approach very interesting. STD works via NOE from the saturated target to the bound compound. WaterLOGSY works via NOE from bound water to the compound. Each experiment has advantages and disadvantages, but are they truly orthogonal experiments. As a third experiment, the authors use transfer NOE to confirm binding. I would expect to see at least one truly orthogonal method to confirm binding, such as SPR. They then used competition screening against known inhibitors to bucket their fragments based upon the site they are binding to.

While I don't think the results here are not similar to results achieved in industry many times over, this is an excellent paper that shows the power of NMR in screening and how to apply that to drive answers to target validation and compound bucketing.

This paper leads I think to interesting academic musings. Does ontogeny recapitulate phylogeny for compounds derived from fragments? Does it matter if the fragment is 3D or not? Is there a floor below which a fragment will not bind? Does this floor move if you are using 3D fragments vs. highly planar ones?

1 comment:

Pete said...

When de-constructing ligands into fragments it’s really important that the fragments represent the molecular recognition characteristics of the parent compounds as accurately as possible. I’ll make some comments on the de-construction based on what I can see in the scheme shown in the blog post although please be warned that I can’t actually see the article. When deconstructing molecules one should, in the first instance, aim not create or destroy charged centers, hydrogen bond donors or hydrogen bond acceptors. This particularly important if you are going to draw inferences from the failure of fragments to bind (I don’t know if the authors have done this).

The doubly-connected oxygens in the chromones 7 and 8 will be a very weak hydrogen bond acceptor so replacing it with a hydroxyl (compound 24) really doesn’t make a lot of sense since this introduces a relatively strong hydrogen bond donor. Methoxy or fluorine would make a lot more sense than hydroxyl if you’re thinking about molecular recognition. The (triply-connected) nitrogens in 11 and 12 will not be hydrogen bond acceptors because the electron density that they would otherwise offer to a donor gets sucked into the π-systems that they are in (like amides and pyrroles). I would argue that these nitrogens ‘look’ a lot more like sp2 carbon than the doubly connected nitrogens in 29 and 30.

I’m reasonably sure (although not currently got access to a pKa database) that neither piperazine nitrogen in 15 will be predominantly protonated at normal physiogical pH. If this is indeed the case, deconstructing 15 to 34 will introduce a cationic center where there was none before and it would probably be more appropriate to deconstruct the piperazine to morpholine or piperidine.