Affinity mass spectrometry-based fragment screening

Among the many methods to find fragments, affinity-based methods are relatively uncommon. These include ultrafiltration, in which fragments bound to a protein are retained on one side of a molecular-weight cutoff membrane (see here and here). A related approach is to immobilize a protein onto some sort of resin, incubate with fragments, wash, and then elute any bound fragments. This affinity mass spectrometry (or affinity selection-mass spectrometry, AS-MS) is used frequently in high-throughput screening to find potent binders. In an open-access ACS Chem. Biol. paper, Wenqing Shui and collaborators at ShanghaiTech University, Fudan University, and Shanghai Institute of Materia Medica apply the approach to fragments.

 

The researchers were interested in the adenosine A_2A receptor (A_2AAR), a GPCR with roles ranging from cardiovascular disease to cancer immunotherapy. The protein has also been well-studied and used as a model system for other fragment-based methods.

 

A stabilized form of A_2AAR containing a histidine tag was immobilized on nickel agarose beads. As a control, the researchers used a different GPCR, HCAR₂. Fragments were incubated with either protein for 1 hour at 4 °C. The supernatant was then removed and the beads were rinsed to remove unbound fragments. Next, the beads were washed with methanol to elute bound fragments. These were analyzed using UPLC-MS. Those that were enriched at least two-fold by A_2AAR compared to HCAR₂ were considered hits.

 

The details are interesting. In total 1100 fragments were screened in two pools of 550, with each fragment present at a mere 200 nM. The experiment was repeated four times to generate more robust data, but even so the whole process took only 10 hours. This yielded 28 hits, 17 of which were confirmed. These 17 hits also confirmed by SPR, which further revealed that 9 were quite potent (sub-micromolar).

 

The researchers were particularly interested in allosteric modulators of A_2AAR, and an extensive series of mechanistic studies involving competition with known ligands, molecular modeling, and NMR experiments suggested that one of the newly identified ligands is a negative allosteric modulator. This molecule has an IC₅₀ = 18 µM in a cell assay.

 

Overall I’m surprised the technique worked as well as it did, particularly given the low fragment concentrations, and I’d be interested to hear from readers who have had success with it. While AS-MS is likely limited to finding fairly potent binders, the simplicity and low sample requirements might make it worth investigating, particularly in cases where protein is difficult to obtain.