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.
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