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Review
. 2015;7(2):159-83.
doi: 10.4155/fmc.14.152.

Photoaffinity labeling in target- and binding-site identification

Ewan Smith  1 , Ian Collins
Affiliations
Review

Photoaffinity labeling in target- and binding-site identification

Ewan Smith et al. Future Med Chem. 2015.

Abstract

Photoaffinity labeling (PAL) using a chemical probe to covalently bind its target in response to activation by light has become a frequently used tool in drug discovery for identifying new drug targets and molecular interactions, and for probing the location and structure of binding sites. Methods to identify the specific target proteins of hit molecules from phenotypic screens are highly valuable in early drug discovery. In this review, we summarize the principles of PAL including probe design and experimental techniques for in vitro and live cell investigations. We emphasize the need to optimize and validate probes and highlight examples of the successful application of PAL across multiple disease areas.

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Figures

Figure 1
Figure 1. General designs for photoaffinity probes
(A) Design for a PAL probe where the photogroup, pharmacophore and reporter tags are remote from one another and connected by linkers. (B) Design for a PAL probe where the photogroup is directly incorporated within the pharmacophore, but both are remote from the reporter tag. (C) Design for a two-component PAL probe where the pharmacophore and photogroup are in a separate molecule to the reporter tag. Alkyne and azide functionality on the two halves allows a conjugation step through 1,3-cycloaddition reaction (click chemistry).
Figure 2
Figure 2. The major photoaffinity functional groups and their reactive intermediates
(A) Phenylazides, phenyldiazirines and benzophenones undergo photolysis on irradiation with specific light wavelengths to produce reactive nitrene, carbene and diradical intermediates, respectively. (B) Modified PAL reagents with improved stability of the reactive intermediates.
Figure 3
Figure 3. Productive and nonproductive reactions of carbenes
The carbene species generated by photolysis of trifluoromethylphenyldiazirine (red) undergoes many possible reactions to give productive capture of targets (blue) or destructive side reactions (green). ISC: Intersystem crossing.
Figure 4
Figure 4. Overview of a typical PAL protocol.
Figure 4
Figure 4. Overview of a typical PAL protocol.
See this image and copyright information in PMC

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