Four years ago we highlighted
work out of Ben Cravatt’s lab describing “fully functionalized fragments,” or
FFFs (see here for a schematic). In addition to the variable fragment portion, FFFs
contain a photoaffinity tag that can react with proteins plus an alkyne moiety that
allows the labeled proteins to be captured for identification by Western
blots or mass spectrometry. In the 2017 paper FFFs were used to identify
fragments binding to proteins in cells, and more recently other researchers
have used the same approach to find fragment hits against isolated proteins and
even RNA. In all these cases the photoaffinity tag used has been a diazirine.
But how do diazirines react with proteins?
As more researchers work with
FFFs, it is important to consider what affects the reactivity of the probes. This
question is addressed in two new papers.
The first, by Christina Woo and
collaborators at Harvard, Dana-Farber Cancer Institute and Jnana appears in J.
Am. Chem. Soc. The researchers looked at alkyl diazirines (exemplified by
LD below – the most commonly used photoaffinity tag) as well as aryl fluorodiazirines
(exemplified by Ar below). When treated with ultraviolet light, both moieties
form diazo intermediates that can lose nitrogen to generate highly reactive
carbenes. However, alkyl diazo intermediates can also react with carboxylic
acids, while aryl diazo intermediates do not.
The researchers assessed the reactivity
of alkyl and aryl diazirines with individual amino acids. In aqueous solution,
the specific aryl diazirine tested did not react with any amino acid, while the
alkyl diazirine reacted with Glu, Asp, and – at higher concentrations – Tyr and
Cys. Moreover, reaction with Glu and Asp was pH-dependent; at higher pH, when the
carboxylic acids were deprotonated, the reaction did not occur. A similar pH
effect was observed with the model protein bovine serum albumin and the alkyl
diazirine probe.
Next, the researchers tested a
panel of 32 FFFs in intact cells or cell lysates (typically at 10 µM, with 60 s
photoirradiation). Positively charged probes tended to give better labeling,
suggesting that these FFFs were binding near acidic Glu and Asp residues on
proteins. Positively charged fragments yielded an average of 50 unique binding
sites, while neutral FFFs produced an average of 14 and negatively charged FFFs
gave only 5. A closer look at some of the specific proteins revealed patches of
electronegativity in the regions labeled. Interestingly, and consistent with earlier
work, membrane proteins were particularly enriched, possibly because Glu and
Asp residues in membrane proteins often have elevated pKa values and are thus
likely to be more reactive with the diazo intermediates.
The second paper, in Chem Sci. by Christopher
Parker and collaborators at Scripps Research Institute in Jupiter, FL and Bristol
Myers Squibb, explores five different diazirine tags. In addition to the
conventional LD probe, they examined an aryl and three alternative alkyl
diazirines. All five (LD, Ar, BD, DF, and Tm) were appended to either a simple
phenyl substituent (controls), a positively charged lipophilic fragment (FFFs),
the promiscuous kinase inhibitor staurosporine, or the bromodomain ligand JQ1.
All the controls and FFFs could
modify proteins in cells in a dose dependent manner after treatment with UV
light. The Ar control appeared to give more non-specific binding, and the LD,
Ar, and BD-tagged fragments produced more robust labeling than the Df and Tm
fragments.
As for the target-specific
probes, LD-, BD-, and Tm-tagged staurosporine each labeled between 529 and 836
proteins, but only 10-21 kinases – far fewer than the hundreds of kinases staurosporine inhibits.
(To be fair, the photoaffinity tag did reduce the affinity of the probes for
protein kinase A and abolished it entirely for the Ar probe.) Among the
JQ1-derived probes, only the BD- and Tm-tags pulled down one or two
bromodomains.
There are many critical but subtle
details in both papers. For example, the DF and Tm tags require shorter wavelength
illumination (300-310 nm) than the more conventional tags (~360 nm). Changing
the wavelength can shift the balance between diazo intermediates versus carbenes.
The diazirines can also react with water and buffers or generate olefins,
though the latter reaction can be disfavored by deuterating the methylenes on
either side of the diazirine.
So what does all this mean? To
state the obvious, this is complex stuff, and small changes to the tag or
conditions can completely change the outcome of the experiment. As the kinase
and bromodomain examples show, a negative result does not mean your ligand does
not bind to a given target, while a positive result says nothing about the
strength of the interaction. Perhaps the “understanding” promised in the title of
this post has some way to go. But photoaffinity tagging and chemoproteomics
make a powerful combination, and these papers contribute to helping us figure
out how to use this tool.
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