Membrane proteins such as GPCRs account for something like half of all drug targets, but they present a serious challenge for fragment-based approaches. This is partly because the biophysical methods usually used for fragment screening often don’t work for membrane proteins, and partly because, in the absence of structural information, it’s hard to know what to do with fragment hits. But there is progress. We’ve highlighted a couple papers that use functional screens or TINS to find fragments against membrane proteins, and in a recent issue of Biochem. Pharmacol. U. Helena Danielson and colleagues at Beactica and two academic institutes show how surface-plasmon resonance (SPR) can also be applied.
The researchers were interested in GABAA receptors, a class of ion channels involved in multiple physiological functions. The receptors normally form hetero-pentamers, but for simplicity the researchers used a homo-oligomeric receptor, consisting of five β3 subunits. Each β3 subunit carried a tag containing eight histidine residues that could be recognized by antibodies immobilized to the surface of the SPR chip. The GABAA receptors were detergent-solubilized; control channels contained antibodies and detergent with no receptors. The resulting GABAA-modified chips were quite stable; the researchers report being able to run roughly 200 samples over the course of 20 hours with a single chip. (The specific detergents and conditions are critical, so if you’re interested in pursuing this yourself the experimental section is invaluable.)
A set of 51 histaminergic and 15 GABAergic ligands were tested for binding, resulting in nearly two dozen hits with dissociation constants (KDs) between 13 and 300 micromolar. Some of these are exceptionally small: for example, histamine, with a molecular weight of 111 g/mol and just 8 heavy atoms showed a KD of 98 micromolar, which is consistent with published results using different methods. A number of other ligands were also identified, some for the first time, though other previously reported ligands did not repeat in this system.
It will be fun to see the screening results of a larger, unbiased library. Of course, finding fragment hits against a membrane protein is only the first step to developing drugs – one still needs to figure out how to improve potency, most likely in the absence of structure. But, to paraphrase Churchill, at least this paper and related research represent the end of the beginning.