Fragments vs GPCRs – virtually vs experimentally

G protein-coupled receptors (GPCRs) are common drug targets that present challenges for fragment-based approaches. Biophysical studies of these membrane proteins are often difficult. Moreover, while many fragment-finding methods reveal binders, GPCR ligands can be agonists, inverse agonists, neutral antagonists, and more – and directing a search toward desired functionality can be tough (though see here). In a paper published earlier this year in Bioorg. Med. Chem. György Keserü and colleagues at Gedeon Richter and the Hungarian Academy of Sciences describe how they have tackled this problem.

The researchers were interested in the adrenergic α_2C receptor; agonists could be useful for a variety of indications, though selectivity is challenging. No crystal structure has been reported in the literature, so the researchers investigated a radioligand displacement assay as well as a cell-based functional assay (calcium mobilization) for agonists. A test set of 160 fragments from Maybridge was screened in both assays at 250 µM, giving 3 hits in the functional assay but a whopping 48 hits in the displacement assay. A 30% hit rate in an unbiased screen generally means something’s wrong, so the researchers chose to focus on the functional assay.

For the full screen, 3071 fragments having 9-22 non-heavy atoms were tested at 250 µM in the cell-based functional assay, resulting in 318 hits – a much higher rate than the initial set. However, when these were retested, only 86 reproduced, which the researchers attribute to variability in the cell-based assay. Many of the hits were also active against an unrelated GPCR; ultimately 16 were specific for the α_2C receptor and were also active in the radioligand displacement assay (as was one of the three original Maybridge hits). The chemical structures and activities of these molecules are shown in the paper; they are all quite potent with inhibition constants from 2-220 nM in the displacement assay, with correspondingly high ligand efficiency scores.

Despite the lack of a crystal structure, the researchers also performed a virtual screen of the same set of 3071 fragments using a homology model of the α_2C receptor. Two of the top 30 hits were fragments that had been discovered in the functional assay. Although this is not as impressive as another docking study on a different GPCR, it is certainly better than chance, and not too shabby considering the lack of an actual structure for the protein.

Next, the researchers attempted to find more potent analogs by testing compounds chemically related to their best hits. Some of these did show good potency in the radioligand displacement assay, but interestingly all of these were antagonists as opposed to the desired agonists. This is further evidence that gaining affinity may be easier than maintaining functionality.

As the authors concede (and we’ve noted elsewhere), the α_2C receptor has evolved to bind fragment-sized ligands. Still, the computational discovery of agonists is encouraging. It will be interesting to see whether such approaches will work against more difficult targets, such as peptidergic GPCRs.