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.