Recent Advances in Target Characterization and Identification by Photoaffinity Probes

Jitapa Sumranjit,Sang Chung

doi:10.3390/molecules180910425

Abstract

Target identification of biologically active molecules such as natural products, synthetic small molecules, peptides, and oligonucleotides mainly relies on affinity chromatography, activity-based probes, or photoaffinity labeling (PAL). Amongst them, activity-based probes and PAL have offered great advantages in target identification technology due to their ability to form covalent bonds with the corresponding targets. Activity-based probe technology mainly relies on the chemical reactivity of the target proteins, thereby limiting the majority of the biological targets to enzymes or proteins which display reactive residues at the probe-binding site. In general, the probes should bear a reactive moiety such as an epoxide, a Michael acceptor, or a reactive alkyl halide in their structures. On the other hand, photoaffinity probes (PAPs) are composed of a target-specific ligand and a photoactivatable functional group. When bound to the corresponding target proteins and activated with wavelength-specific light, PAPs generate highly reactive chemical species that covalently cross-link proximal amino acid residues. This process is better known as PAL and is widely employed to identify cellular targets of biologically active molecules. This review highlights recent advances in target identification by PAL, with a focus on the structure and chemistry of the photoaffinity probes developed in the recent decade, coupled to the target proteins identified using these probes.

Highlights

Photoreactive molecules are being continuously developed for a variety of applications
During the photoaffinity labeling (PAL) process, a ligand is covalently attached to a photoreactive group, which generates a reactive species upon irradiation that covalently cross-links the ligand to its target macromolecule (Scheme 1)
The applied multiple-component reaction (MCR), the Ugi reaction, employs the following components: (1) an isoxazole carboxylic acid decorated with an aldehyde, which mimics pTyr and binds to the Protein tyrosine phosphatases (PTPs) primary active site; (2) an amine-component which potentially targets the secondary binding site on PTPs; (3) an isonitrile-functionalized benzophenone motif to initiate cross-linking of PTP and the probe upon UV

Summary

Introduction

Photoreactive molecules are being continuously developed for a variety of applications. During the PAL process, a ligand is covalently attached to a photoreactive group, which generates a reactive species upon irradiation that covalently cross-links the ligand to its target macromolecule (Scheme 1). The typical components of a PAP are a photoreactive group, a target binding ligand, and a reporter tag. Diazirine derivatives are the smallest molecular size PAPs amongst the common photoreactive units These derivatives are quite stable at room temperature, highly reactive upon photoactivation and relatively inert towards nucleophilic attack, acidic- and alkaline conditions. Diazirine generates a reactive carbene (Scheme 4) which rapidly forms a covalent bond with the nearest target molecule via C–C, C–H, O–H, or X–H insertion. Typical reporter tags include radioactive isotopes, biotin, epitope tags, or fluorophores These reporting units are usually covalently attached to the probes; their bulkiness and/or instability could perturb the protein binding process. Fluorophores can be detected by in-gel fluorescence emission, while the conjugated biotin probe is subjected to western blot analysis

Benzophenone-Based Photoaffinity Labeling

Arylazide-Based Photoaffinity Labeling

Diazirine-Based Photoaffinity Labeling

Natural Photoaffinity Probes

Conclusions