PhABits: photoaffinity-based fragment screening

Three years ago we highlighted work out of Ben Cravatt’s lab describing “fully-functionalized fragments” that – in addition to a variable portion – contain a photoreactive diazirine moiety and an alkyne moiety. These were incubated with cells and irradiated with UV light to crosslink the fragments to bound proteins. The alkyne was then used in click chemistry to isolate and identify the bound proteins. Cell-based screening is not for the faint of heart, but as demonstrated in a paper recently posted on ChemRxiv by Jacob Bush and collaborators at GlaxoSmithKline and University of Strathclyde, the functionalized fragments can also be used in biophysical screening. (Emma Grant presented a nice poster on some of this work at FBLD 2018.)

A small library of 556 fragments, rebranded as PhotoAffinity Bits (or PhABits), was synthesized by coupling the alkyne- and diazirine-containing carboxylic acid with a diverse set of amines (each with < 16 heavy atoms). These were then screened at 200 µM against six pure recombinant proteins, irradiated with UV light, and analyzed using intact protein mass spectrometry as in Tethering and other forms of covalent FBLD. Hit rates varied tremendously, from less than 3% for myoglobin to 47% for lysozyme. It would be interesting to see whether this approach, like other fragment finding methods, is able to assess protein ligandability.

Most of the PhABits did not react with the proteins tested, though 58 crosslinked to at least four, and 10 crosslinked to all six. For one of the proteins screened, the bromodomain BRD4-BD1, a known high-affinity ligand could compete 68 of the 89 fragment hits, suggesting a specific interaction at the acetyl lysine pocket. Of the 21 fragments that were not competed, 19 bound to at least three other proteins. Interestingly, the physicochemical properties and solubilities of these fragments were not notably different from the rest, and the researchers speculate that their non-specificity may be due to a longer-lived reactive intermediate generated after UV irradiation.

Several of the BRD4-BD1 fragments were confirmed as binders using a TR-FRET assay, some with low micromolar affinities, though the tighter ones tended to contain known bromodomain binding motifs such as isoxazoles. A couple of these were successfully used to generate PROTACs, as suggested here. Protein digestion and LC-MS/MS sequencing revealed that the fragments crosslinked residues near the acetyl lysine binding site, and this binding mode was confirmed using X-ray crystallography for one of the fragments.

In addition to BRD4-BD1, another target the researchers highlight is KRAS4B^G12D. Of the 11 unique hits, some resembled previously reported molecules, and LC-MS/MS studies suggested that they do in fact bind in the same pocket. Competition studies confirmed this, and the resulting IC₅₀ values were similar to those previously determined using HSQC NMR.

As the researchers point out, this photoaffinity-based screening approach is limited to homogenous proteins that are suitable for mass spectrometry. Also, the crosslinking efficiency is not necessarily related to the affinity of the fragment. Still, this is an interesting approach to both find fragments and identify their binding sites. It will be fun to see how it develops.