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.
2 comments:
FYI--The PI involved in this work is widely known to employ bullying and harrassment of students to obtain the results mentioned above. As a former lab memberand first author of a paper from his lab, I can attest to the inhuman treatment i observed. Its all fun and games until federal laws are broken for this research.
Great paper and agree it has loads of success stories! Not sure I agree with their claim that only 17% of the proteins they pull out have been previously liganded. They talk about the need for chemical probes and then use DrugBank as a reference database... Surely an 'active' subset of ChEMBL would have been more informative. Still impressive.
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