FBDD is a maturing field and one sign this maturation is the publication of a volume of Methods in Enzymology devoted to the subject. The article in this collection that most interested me was the review by Anthony Giannetti on the use of Surface Plasmon Resonance (SPR) in Fragment Lead Generation. The review is described as a ‘comprehensive walk-through’ and in-depth treatment of topics such as target immobilization and buffer/compound preparation justifies this description. I’m still working my way through some of the data analysis sections...
The target is tethered to a surface in SPR and this is usually referred to as ‘immobilization’, which is an unfortunate term, albeit the one that is most commonly used in the literature. Vendors of competing assay technologies (who would naturally prefer you to use their technology instead) often present this as a weakness of SPR. One concern is that tethering will compromise the ability of the target to bind ligands and the review does cite a couple of articles which compare affinities measured with SPR to those measured using methods such as isothermal titration calorimetry.
The system in an SPR assay is heterogenous, which is another way of saying that the concentration of protein is not uniform, particularly in the direction perpendicular to the surface to which it is tethered and this creates some interesting possibilities. Tight binding occurs when the value of the ligand Kd is lower than the concentration of the protein to which it binds. We typically configure assays for measuring affinity and potency so that ligand concentration is significantly greater than protein concentration. This means that ligand binding does not affect the concentration of unbound ligand and the math is a whole lot easier if you can make this assumption. If, however, the protein concentration in your assay is 1nM and you want to measure the potency for a compound with an IC50 of 0.01nM you’re going have a problem because you’ll need the compound at a concentration of 0.5nM in order to occupy half the binding sites. In enzyme inhibition assays, the concentration of the enzyme limits sets an upper limit on the potency that you can measure and this may be an issue for attempts to estimate the maximum potency of ligands.
In a heterogeneous system, things are not quite as simple because concentration is less clearly defined and you need to think in terms of quantities (in molar terms of course) of protein and ligand. Localising a small amount of protein on the chip surface rather than having a larger amount of protein distributed evenly throughout the sample volume means less depletion of the reservoir of unbound ligand when 50% of binding sites become occupied. Also in the SPR assay, the solution of ligand flows over the chip, making depletion of unbound ligand even less of a problem.
Tight binding is not usually a problem when screening fragments and the main reason for bringing up the subject was to get you thinking a bit about assays. There are a number of technologies for detecting the binding of fragments and quantifying the affinity with which they bind. This raises a couple of questions. Firstly, to what extent do we need new screening technologies for FBDD? Secondly, which weaknesses in the current methodology should be addressed with the highest priority?
Giannetti, From experimental design to validated hits: A comprehensive walk-through of fragment lead identification using surface plasmon resonance. Methods Enzymol. 2012, 493, 169-218. DOI