Still Impractical, but Getting Better...

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.

Another NMR Tool...

There are many things which aid in the successful prosecution of fragments.  Most people would agree that structural information is one of those things.  However, in many cases there is no structure, nor any hope of obtaining one.  Many different methods have been developed to try to address this gap.  Oftentimes they are impractical, sometimes they are useful.  In this paper, Gregg Siegal, Marcellus Ubbink, and co-workers from his academic lab present a new NMR-based structural tool.  [Editor's Note: I used to have a business relationship with Gregg's commercial side.]  So, is this a practical or impractical tool?  You can skip down to the bottom for the answer, or keep reading and follow me down the rabbit hole.

Their approach is not to generate high-resolution structures, but low resolution models of how initial fragments bind to the target. To accomplish this, the use pseudocontact shifts  (PCS)induced by paramagnetic ions.  To those of you whose eyes just glazed over, let me explain.  We typically only use diamagnetic atoms in NMR, because paramagnetic atoms cause line broadening, sometimes to extinction.  For ease of explanation, the PCS is similar to any dipolar coupling, it is a way to relax between atoms, like the NOE, but with a longer distance dependence r^-3 (PCS), vs. r^-6 (NOE).  However, with good decisions like the choice of the ion, the placement of the ion, and so on, you can get subtle effects on your ligand, rather than wiping it out. In the end, you need to know a few things: the actual fraction of ligand bound, the structure of the target (or a good homology model), and the PCS tensor (see below).  This work used rigid, paramagnetic ion binding tags attached to the target via engineered disulfide linkages (CLaNP).

 In total, they made three different tagged proteins and used Yb3+ as the paramagnetic ion and Lu3+ as the diagmagnetic ion. 

This data represents a mixture of bound and free ligand, so using the experimentally determined Kd and the known concentrations of ligand and target, the % bound ligand can be determined.  This can then be converted into PCS of only the bound state. 

However, the authors then tried to calculate the tensor, which is necessary to calculate the orientation of the PCS tensor.  When compared to the orientation of the ligand determined by NOE, there was an 4.7 A RMSD.  This approach only gives the relative location of the binding site.  When they formally calculated the PCS tensors they were able to get a better match of the PCS-derived orientation compared to the NOE-derived, but still not perfect agreement.  That is expected for different methods which can be considered orthogonal.  There ends up being a lengthy discussion of the shortcomings of this method and why it could be possibly better than NOE-based methods, in particular it does not need labeled protein.  However, I would argue if you are not producing your protein in E. coli it is likely being made in insect cells or mammalian cells.  In the case of insect cells, why would you wait two months, to get ligand orientation information on an initial hit?  The project has come and gone on the initial screen hits by that time.

While this is a interesting approach academically, it is really impractical.  Why?  As the authors state, this method is best for ligands with high micromolar to low millimolar affinity.  This positions it firmly in the very early stages of FBHG.  You need to have the structure of the target, or a good homology model.   You need to generate multiple mutants (they do state you can get by with only two positions, but three is better).  You need to do some seriously involved computation; something that is not routine at all.  This would be a much better tool if it could be robustly used at late hit expansion/early lead generation, but that doesn't seem likely.  So, you have what is largely an academic tool for generating models of ligand-target binding with fragments, but not something that would be routinely used.  

NMR as an Impractical Tool, Again

When I was in grad school, I was faced with the choice of two NMR labs to join (after starting as a organic chemist and flirting with enzymology).  Both used NMR, but with very different goals.  One lab used NMR and found systems to study using NMR.  The other studied interesting problems.  The PI would say if NMR is most appropriate than using, but don't be a slave to it.  I have taken that attitude my entire career.  In industry, it also has to be the mantra: best tool for the problem.  Academics tend to have the opposite mindset: let's make my tool work for anything.  

The Krimm lab has been cited here, here, here, here, and here on the blog and I hold their approach to academic tool creation for drug discovery in good regard.  In this paper, they present a combination computational/NMR method for determining if a fragment induces conformational changes in the target.  In their own words: 

The approach relies on the comparison of experimental fragment-induced Chemical Shift Perturbation (CSP) of amine protons to CSP simulated for a set of docked fragment poses, considering the ring-current effect from fragment binding.

Sometimes good people do bad things. 

I am not going to get into the details, but rest assured the science is sound.  Their approach is to evaluate H-N (you could also use H-C, why not) chemicals shifts from titration data to simulated CSPs.  When they did compare experimental with calculated CSPs they could not explain some of these shifts, even when they included ring current-induced shifts.  To further investigate this phenomenon, they used Residual Dipolar Couplings (RDCs) to further explore these unexplained CSPs.  It does.  

What are my problems with this paper?  Practicality, primarily.  Is this another anti-compchem rant?  Nope.  The problem here is that everything they propose to do, and they do it well, relies upon a whole sh!tpile of a priori knowledge: 1. the structure of the protein (typically from X-ray) and 2. the assignments of the protein (not trivial).  Additionally, the RDCs require acquiring two sets of data, aligned and unaligned.  RDCs are wholly impractical.  All of this should red flag this paper as a "Impractical" approach.  It also does not present a method for interrogating structural changes induced by ligands that is any better or more robust than the current standard of analysis.