Mass spectrometry does not come up frequently in the context of fragment-based lead discovery (though see here, here, and here). A new paper in Bioorg. Med. Chem. Lett. from Matthew Carson and colleagues at Lilly, with collaborators from Scripps, seeks to change that by describing a technique that can elucidate binding sites for fragment hits.
The researchers were interested in the vitamin D receptor (VDR), an osteoporosis target. Upon binding to ligands such as the D vitamins, this nuclear hormone receptor changes conformation and binds to another receptor, retinoid X receptor (RXR), to control gene expression. The biology quickly gets complicated, but suffice it to say that there is a need for ligands that behave differently than the D vitamins. Enter fragments.
The researchers assembled a collection of about 10,000 compounds, most of which had fewer than 23 non-hydrogen atoms. These were screened at 0.1 or 1 mM concentration in a fluorescence polarization binding assay, resulting in 417 hits. These were then tested in an AlphaScreen assay for compounds that would enhance or decrease binding to RXR (ie, agonists or antagonists). The screen came up empty for agonists. VDR is a member of the same family as the PPARs, for which fragment screens have delivered agonists, so this result was a bit disappointing. The researchers speculate that the fragments may not be large enough to induce the required conformational changes in VDR.
The researchers were more successful finding antagonists: 247 fragments with “lean values” > 0.25 (corresponding to ligand efficiencies > 0.35 kcal/mol/atom). About 2000 analogs of these were then tested, leading to more hits, some of which were quite potent, and 13 of which are shown in the paper. Although some of these look structurally reasonable, one is a toxoflavin-type molecule with a catechol attached that looks disconcertingly similar to a molecule I proposed as an April Fools’ joke. Presumably they are keeping the more interesting structures confidential.
The ultimate goal is to find agonists. Antagonists could potentially be grown into agonists, and to do so it would be helpful to know how they bind. Unfortunately, co-crystallography proved unsuccessful, so the researchers turned to hydrogen deuterium exchange mass spectrometry (HDX MS).
In HDX MS, a protein-ligand complex is exchanged into D2O for seconds to minutes, allowing exchangeable protons (such as those in the amide backbone of the protein) to exchange with deuterium. The reaction is stopped by lowering the pH, the protein is digested into individual peptides, and these are analyzed by mass spectrometry to assess the extent of exchange. If an amide makes a hydrogen bond in a highly structured region of the protein it will be less prone to exchange than if it is in an unstructured region of the protein. Therefore, if a ligand induces structural changes in the protein these should manifest themselves by altering the exchange rates, and if the crystal structure is known this provides a rough map of which regions of the protein are affected by ligand binding.
The 13 fragment hits were tested in HDX MS with a 200-fold excess of fragment to protein. Of these, 6 stabilized the protein, as assessed by decreased H-D exchange. The stabilized regions overlap with the regions stabilized by the natural ligand, vitamin D3, though the extent of the regions and degree of stabilization is considerably less, consistent with the fragments binding within a smaller footprint.
Of course, since we don’t have co-crystal structures, it is difficult to interpret the HDX data precisely. Still, it is nice that fragments can produce a signal in this type of assay. It will be interesting to apply this technique to better characterized systems to see how general it is.