17 May 2021

Understanding fully-functionalized diazirine tags

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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