Investigation of Specific Binding Proteins to Photoaffinity Linkers for Efficient Deconvolution of Target Protein

Abstract

Photoaffinity-based target identification has received recent attention as an efficient research tool for chemical biology and drug discovery. The major obstacle of photoaffinity-based target identification is the nonspecific interaction between target identification probes and nontarget proteins. Consequently, the rational design of photoaffinity linkers has been spotlighted for successful target identification. These nonspecific interactions have been considered as random events, and therefore no systematic investigation has been conducted regarding nonspecific interactions between proteins and photoaffinity linkers. Herein, we report the protein-labeling analysis of photoaffinity linkers containing three photoactivatable moieties: benzophenone, diazirine, and arylazide. Each photoaffinity linker binds to a different set of proteins in a structure-dependent manner, in contrast to the previous conception. The list of proteins labeled by each photoaffinity linker was successfully used to eliminate the nonspecific binding proteins from target candidates, thereby increasing the success rate of target identification.

Tetrazine ligation for chemical proteomics

“…Photoaffinity based target ID overcomes those limitations by UV induced covalent bond generation between small molecule and interacting proteins [68]. The covalent bonding secure small molecule-protein interaction in various experimental conditions [69, 70]. Moreover, weak binding or low abundant target proteins can be tractable from huge amount of other non-target proteins in cell lysate [71].…”

Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs)

“…36 top). They reported [102] the PAL of photo-affinity linkers containing three photoactivatable moieties, DA, BP and AA. They found that each photo-affinity group binds to a different set of proteins in a structure-dependent manner, in contrast to previous beliefs.…”

“…Figure reproduced from ref. [102] with permission from ACS publication. ( Bottom ) Schematic illustration of the molecular shape-dependence of protein labeling.…”

Current advances of carbene-mediated photoaffinity labeling in medicinal chemistry

“…The concept of photoaffinity labeling (PAL) was originally introduced by Frank Westheimer in the early 1960s, 1 and since then it has gradually emerged as a powerful tool for the identication and localization of targets and corresponding interaction sites in complicated biological systems, 2 especially in some cases such as low-abundance proteins and low-affinity interactions that fail to survive disruptive washing steps. 3,4 Four common types of photoreactive group are used in PAL, namely, aryl azides (AZs), benzophenones (BPs), diazirines (DAs) 5,6 (New) and 2-aryl-5-carboxytetrazoles (ACTs), 7,8 which are classied in accordance with their photochemically generated reactive species, namely, nitrenes, diradicals, carbenes and carboxy-nitrile imines. In addition, some photoreactive components of natural molecules that can undergo photolysis to form highly reactive intermediates, such as steroid enones (e.g., pyrones and pyrimidones 9 ), various aryl chlorides, and several thioethers, have been utilized as probes to avoid complications associated with fully synthetic probes.…”