Dan and I don't see every fragment paper and so it's nice when people point out papers to us. Its typically their recently published paper and they are looking for some sort of recognition/validation from us. Sometimes its a paper we have on our radar, sometimes it isn't. Recently, I received an email pointing out this paper from someone I met at FBLD2014. Well, this is a follow up paper to a paper I discussed a few years back. The title of the post sums up my thoughts: "Another (Impractical) NMR Method." One comment from that post (by a co-author from Astex on the current paper) was
This is a relaxation filtered ligand based method (like T1rho or selective T1). You therefore may consider using it in competition mode if you find one suitable "spy" molecule: this would allow with a single point experiment screening mixtures and/or ranking for low affinity hits(especially if solubility is limiting). I would actually give it a try.
So, it looks like she did. The advantages of Long Lived States (LLS) NMR is that the the dynamic range is wider and it works at low protein concentrations (3 uM or roughly the same as STD or WaterLOGSY) or with very weak affinities. Against the workhorse HSP90 system, they screened mixtures using LLS and a spy molecule.
I still don't think this is a very practical method because it still requires a lot of tailoring to individual systems; in particular the compounds need pairs of protons which are suitable for excitation to the LLS. Using it in "spy" mode gets around this. I would still hold that this is an impractical NMR method. I like to see people developing new methods and trying to improve them. We also need people doing good comparisons of "standard" experiments to new ones. (Here is how NOT to do a method comparison.) I won't dismiss this out of hand, but there is still a lot of work to be done here to move it into a "front line" screening technique.
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Actually, this looks like it may be pretty useful for finding very weak binders. Are any other NMR techniques capable of detecting fragments with Kd > 10 mM?
I would argue that >10 mM is really a regime no one need go to. To have a decent lig eff (pIC50/HAC) of 0.3, the fragment would have to be 6 atoms or less. So, the requirement for two protons that can be excited to LLS may be difficult to achieve for fragments this small. In a very specific instance, it may be the ONLY way to find that weak, very small fragment, but I still wouldn't call this practical.
A 9 atom fragment with Kd = 0.01 mM would have LE = 0.3 kcal/mol/atom. 9 atoms is small, but vemurafenib started from a fragment this size, and if you're going after tougher targets like some PPIs you may need to look for very weak binders.
See, Folks, Dan and I don't always agree.
But, Vem& was a >200uM hit, which at least an order of activity more potent than what we are looking at.
There is a bit of confusion here! For an LE of 0.3, a 9-atom fragement would require a Kd/IC50 of 10mM (and a 6-atom fragment a Kd/IC50 of 50mM).
There is also a strong argument that,at the screening stage, LE requirements should be relaxed. Even the best fragemnt libraries only cover a small proportion of the available chemical space so it is unlikely that the library will contain the best example of a given chemotype. A more ligand-efficient example should be sought in follow-up screening.
We have found plenty of useful hits which have Kd > 10mM when screening PPI targets. These can be detected using conventional NMR screening (eg LOGSY) but only at a cost of using higher protein concentration than when screening for 1mM hits. However there are limited methods available for measuring the Kd's of these hits (e.g. ITC, HSQC-NMR) and these are protein-hungry.
When the first LLS paper was published, I too was suspicious of its wider applicability. It turns out that the NMR requirements are not so strict (two non-degenerate spins which couple more strongly to each other than to the rest of the molecule) and we have many fragements that satisfy this.
If one of these fragments is a hit against your target, then the LLS method can be used to measure its Kd. Once a suitable hit has been detected, competition methods can be used to screen for other hits.
However, there is no easy way to use it as a general screening method, since the experiment contains a frequency-selective spin-lock. This would have to be optimised for each ligand, prior to screening.
Glyn,
Interesting. Can you post some data on what fraction of your library (or A library) would fit the spin requirements?
That's a good question and one that I had been meaning to find the answer to... The compounds for the paper with Geoffery Bodenhausen's group were generated by looking at our weak HSP90 hits and then selecting ones that were potentially suitable for LLS.
The ideal LLS fragment would contain two non-degenerate, coupled spins, isolated from the rest of the molecule. About 12% of our fragment library contains 6-membered aromatic rings with two adjacent protons isolated from other spins, and so could in principle be screened or used as 'spy' molecules with LLS detection. Another 4% of the library contains 5-membered aromatic rings with 2 isolated spins (although the couplings are small and would require a long spin lock). In addtion there will be a number of chiral fragments with diasterotopic CH2 groups which might also be suitable.
These %ages are comparable to our NMR hit rates for many projects, so its likely that we will often find at least one fragment hit suitable for LLS. So, its a useful tool in the workshop.
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