Fragments vs human Adensoine 2a Receptor using SPR

Last week we highlighted the use of surface plasmon resonance (SPR) to find ligands against RNA. Although RNA is not a typical protein target, it is at least normally free in solution. Targets such as GPCRs are more technically challenging because they are bound within membranes. Challenging, but not impossible, as illustrated by this post from 2012. A new ACS Med. Chem. Lett. paper by Reid Olsen, Iva Navratilova, and colleagues at Exscientia, University of Dundee, and AstraZeneca provides the latest example.

 

Navratilova and colleagues previously described using SPR to screen the β2 adrenergic receptor. In the new paper, the researchers studied the human adenosine 2a receptor (hA_2AR), a “rheostat for energy homeostasis” that also plays a role in cancer immunotherapy. hA_2AR is one member of a small family of adenosine receptors, and the researchers expressed all four of them, each with a polyhistidine tag that could be captured in the SPR instrument using a nickel-NTA sensor chip. Other labs (such as Heptares) have used mutant, stabilized forms of GPCRs, but here the researchers used native proteins and stabilized them by crosslinking them to the surface of the chip. They confirmed that these GPCRs bound known ligands with similar affinities to those reported in the literature.

 

Next the researchers screened a library of 656 fragments, each at 50 µM, against hA_2AR. This led to 72 potential hits taken into dose-response experiments, of which 17 confirmed with affinities ranging from 1.1 to 410 µM. All the sensorgrams are shown, as are the structures of the fragment hits. These confirmed hits were also screened against A₁, A_2B, and A₃; most of the fragments bound to all the receptors, though two were selective for hA_2AR.

 

To assess where the fragments bind, the researchers added a known high-affinity ligand; ten of the fragments could be competed, while seven showed less or no competition, suggesting that they may bind to an allosteric site.

 

GPCRs biology is complicated, and just because a ligand binds does not mean it will have any effect on signaling. In cell experiments, none of the fragments behaved as agonists, but five fragments could act as antagonists of a known agonist. Another fragment seemed to increase the signal, suggesting it is an allosteric modulator. As the researchers conclude, “while SPR can screen fragment-like molecules that allow for extrapolation of extremely large and diverse chemical spaces, it cannot predict the biological activity of these binders."

 

Nonetheless, this paper provides a nice guide on how to use SPR, with its low protein requirements, to screen GPCRs. And the fragments disclosed could be interesting starting points for medicinal chemistry.