As illustrated by our poll, surface plasmon resonance (SPR) is one of the most widely used techniques for finding fragments. However, as commonly practiced, SPR – like all techniques – has drawbacks. For one thing, despite impressive recent advances, it is still not particularly high throughput. Also, the protein is typically immobilized on a sensor chip, and the detection of binders depends on the mass ratio of the ligand to the protein. With larger proteins and smaller fragments, this can quickly push the signal below the noise.
A seemingly simple solution is to reverse the experiment: immobilize the small molecule and add the (comparatively large) protein to get a whopping signal. Indeed, this is the approach that Graffinity (now part of NovAliX) takes, and is conceptually similar to work done at RIKEN. However, both these techniques require dedicated surfaces functionalized with fragments.
In a recent paper in J. Med. Chem., Stefan Geschwindner, Jeffrey Albert, and colleagues at AstraZeneca sought to simplify matters. Their idea is to immobilize a single high-affinity molecule to a chip. Protein in solution should give a good signal when the protein binds to the surface, and adding competitor to the solution should decrease protein binding to the immobilized target compound, thereby reducing the signal. They call this the “inhibition in solution assay”, or ISA.
The researchers used the protein PDE10A as a test case and attached a previously characterized small molecule to the surface; this modified small molecule has an IC50 of 991 nM for the target. They then used two different approaches for detecting interactions, SPR (GE/Biacore) and a 384-well plate-based optical waveguide grating (OWG) from SRU Biosystems. Both formats work and give comparable results for a set of molecules ranging in affinities from 40 nM to 0.5 mM.
One nice feature of this approach is that, as a competition assay, it should only identify molecules that are competitive with a known binder. On the flip side, ISA does require a reasonably potent binder for your protein, and you must be able to modify this molecule such that it can be immobilized to the surface. And of course, there are still problems at high concentrations; the researchers mention that high loading of immobilized small molecule can cause other molecules to stick to the surface. Still, this is an interesting approach that should be easily applied to many systems. I’d be curious to know whether you’ve tried it or a variant, and how it compares to more conventional SPR methods.