Quantitative native MS identifies a new zinc binder

Last week we highlighted a case where undetected zinc contamination turned out to be completely responsible for the observed activity of a fragment hit. But zinc plays many essential roles in biology, and several groups have sought fragments that target metals; drugs such as vorinostat derive most of their affinity from such interactions. In a recent paper in J. Med. Chem., Thomas Peat, Sally-Ann Poulsen, and collaborators at Griffith University and CSIRO have identified a new zinc-binding fragment. 

The researchers previously screened human carbonic anhydrase II (hCA II) against a library of 720 fragments, which yielded seven hits that bind to the catalytic zinc, as described here. Most of these fragments were either known zinc binders or had modest (high micromolar) affinities. In the new paper, the researchers reveal an eighth fragment that is both novel and potent.

Surface plasmon resonance (SPR) and native electrospray ionization mass spectrometry (ESI-MS) identified compound 10, which has an affinity and ligand efficiency approaching that of sulfonamides such as compound 3, a well-known class of zinc binder.

The researchers determined the crystal structure of compound 10 bound to hCA II, which revealed an interaction between the catalytic zinc and the deprotonated nitrogen, whose pKa is ~5.5. The oxazolidinedione core of the fragment has previously been used as a carboxylic acid bioisostere, but a search of the protein data bank (pdb) revealed no precedents as a zinc binder. In addition to the primary interaction with the metal, the fragment also formed a couple hydrogen bonds with the protein, helping to explain the high affinity.

Next the researchers made or purchased a series of 18 analogs to assess the SAR using both SPR and MS. Native ESI-MS results are usually assessed qualitatively, but the researchers were able to get quantitative data by holding protein concentration constant (at 14.5 µM) and varying the fragment concentration from 0.5 to 120 µM. Plotting the percentage of protein bound and curve-fitting revealed dissociation constants remarkably similar to those determined using SPR.

Nine of the new fragments showed at least some activity, though none were significantly more potent than compound 10. Crystal soaking experiments led to seven new structures, with all the fragments binding in a similar manner as compound 10. (There was one surprise: a bit of extra electron density in one structure led the authors to reexamine the fragment by high-resolution MS, revealing that about 5% had oxidized, and that this was in fact the bound species.)

Combing through the PDB revealed that some of the SAR compounds had not previously been reported as zinc binders. Interestingly, the key pharmacophore in one of the inactive molecules – hydantoin 15 – has been reported to be a zinc binder. The fact that it was inactive against hCA II augers well for achieving selectivity with metal-binding moieties. It will be fun to watch this story develop.