Fragments in Cell(s)

Last year we highlighted work out of Ben Cravatt’s group at Scripps on screening covalent fragments in cells. Now, in a new Cell paper, his group and collaborators at the École Polytechnique Fédérale de Lausanne, Bristol-Myers Squibb, and the Salk Institute have gone further by screening for non-covalent fragments in cells.

The researchers started by synthesizing a collection of just 14 “fully functionalized” fragments (FFF). In addition to the variable fragment (averaging 176 Da), each FFF probe contains a diazirine group, which, when exposed to UV light, generates a highly reactive species that can covalently react with proteins (or anything else) in close proximity. (In contrast, covalent fragments typically work via chemistries in which bonds form without requiring UV exposure.) The FFF probes in the current study also contain a "clickable" tag: an alkyne moiety that can react with azide-containing molecules using copper-catalyzed azide alkyne cycloaddition.

Cells were incubated with 20 µM of each fragment for 30 minutes, then exposed to UV light for 10 minutes on ice to capture non-covalent interactions. The cells were then lysed, treated with an azide-containing flurorescent dye in the presence of copper, and analyzed by gel electrophoresis to visualize those proteins with bound fragments. The results were striking: lots of proteins were labeled, and each fragment labeled a different set of proteins. This is what you would expect for low-complexity molecules, but it is nice to see reality match predictions.

Not content to look at gels, the researchers switched to mass spectrometry for a more global analysis using “stable isotope labeling with amino acids in cell culture” (SILAC). In this approach, one population of cells grown under normal conditions was treated with one of the FFF probes, while a second population of cells containing isotopically labeled proteins was treated with a control probe containing just a methyl group instead of the variable fragment. The resulting cell lysates could then be proteolyzed and analyzed by mass spectrometry; most peptides would show two peaks of similar intensities, one from each isotopically distinct population of cells. However, if an FFF probe bound preferentially to a protein compared with the control probe, the resulting peptide would be enriched.

Examining 11 FFF probes at 200 µM concentration allowed the researchers to identify an impressive 2000 different protein targets. Both soluble and membrane proteins were found, with expression levels ranging over 100,000-fold (i.e. the technique seems to work for both rare and abundant proteins). Remarkably, only about 17% had known ligands. There was also little overlap with the proteins targeted by the researchers’ previously described covalent fragments.

Where did the FFF probes bind? An analysis of 186 proteins whose crystal structures had previously been reported showed that about 80% of the modified peptides were close to a computationally predicted pocket, as might be expected.

Next, the researchers made or purchased analogs of some of their FFF probes. When added to screens, these decreased labelling of hundreds of targets; this competition assay both suggests the FFF probes make specific interactions while also providing more potent analogs. Two proteins – the enzyme PTGR2 and the transporter SLC25A20 – were studied in some detail. Probe 8 modified two peptides near the active site of PTGR2 and could be competed by compound 20. Compound 20 was also a modest inhibitor of the enzyme. Further modification led to compound 22, with nanomolar activity against the isolated enzyme and in cells. Since this protein previously lacked any good chemical probes, this could be useful.

This approach also lends itself well to phenotypic screening, so the researchers expanded their FFF probes to 465 members, increasing the size of the variable fragment portion to an average of 267 Da. They also made competitor molecules for most of these, which contained the fragment but not the alkyne or photoreactive group.

The researchers screened about 300 of their new FFF probes (at 50 µM each) to look for molecules that would increase the differentiation of mouse preadipocytes to adipocytes. This led to nine hits, one of which was active at 10 µM. SILAC experiments revealed the target of this to be a somewhat obscure membrane protein called PGRMC2. Subsequent experiments suggested that PGRMC2 is a positive regulator of adipogenesis, and that the identified compound is an activator.

This is a remarkable paper, and it is impressive that the researchers have described not just the approach but several success stories – each of which could well form a stand-alone publication. The covalent platform described last year has already led to a company - Vividion - which recently raised $50 million, and I’m sure the new approach will find use in both academia and industry.