Recent Applications of Diazirines in Chemical Proteomics

The elucidation of substrate–protein interactions is an important component of the drug development process. Due to the complexity of native cellular environments, elucidating these fundamental biochemical interactions remains challenging. Photoaffinity labeling (PAL) is a versatile technique that can provide insight into ligand-target interactions. By judicious modification of substrates with a photoreactive group, PAL creates a covalent crosslink between a substrate and its biological target following UV-irradiation. Among the commonly employed photoreactive groups, diazirines have emerged as the gold standard. In this Minireview, recent developments in the field of diazirine-based photoaffinity labeling will be discussed, with emphasis being placed on their applications in chemical proteomic studies.     

Design and Synthesis of Matrix Metalloprotease Photoaffinity Trimodular Probes

To explore the molecular mechanism of the matrix metalloproteinases (MMPs) in tumor processes, two photoaffinity trimodular probes were designed and synthesized based on the structure activity relationship and the following photoaffinity labelling experiments afforded positive results.     

Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes

Histone deacetylases (HDACs) regulate the function and activity of numerous cellular proteins by removing acetylation marks from regulatory lysine residues. We have developed peptide‐based HDAC probes that contain hydroxamate amino acids of various lengths to replace modified lysine residues in the context of known acetylation sites. The interaction profiles of all human HDACs were studied with three sets of probes, which derived from different acetylation sites, and sequence context was found to have a strong impact on substrate recognition and composition of HDAC complexes. By investigating K382 acetylation of the tumor suppressor p53 as an example, we further demonstrate that the interaction profiles reflect the catalytic activities of respective HDACs. These results underline the utility of the newly established probes for deciphering not only activity, but also substrate selectivity and composition of endogenous HDAC complexes, which can hardly be achieved otherwise.     

一种含光亲和标记异戊烯侧链探针的合成与初步表征

经5步反应、以9%的总收率合成了一种含生物素修饰和光亲和标记的异戊烯侧链功能探针分子, 初步考察了反应条件下该探针分子与Saccharomyces cerevisiae AS 2.399粗蛋白的相互作用; 生物素印迹分析结果表明, 酵母中多种蛋白被探针分子修饰, 为进一步开展化学蛋白组学研究奠定了基础.     

Redesign of a Fluorogenic Labeling System To Improve Surface Charge,Brightness, and Binding Kinetics for Imaging the Functional Localization of Bromodomains

Protein labeling with fluorogenic probes is a powerful method for the imaging of cellular proteins. The labeling time and fluorescence contrast of the fluorogenic probes are critical factors for the precise spatiotemporal imaging of protein dynamics in living cells. To address these issues, we took mutational and chemical approaches to increase the labeling kinetics and fluorescence intensity of fluorogenic PYP‐tag probes. Because of charge‐reversal mutations in PYP‐tag and probe redesign, the labeling reaction was accelerated by a factor of 18 in vitro, and intracellular proteins were detected with an incubation period of only 1 min. The brightness of the probe both in vitro and in living cells was enhanced by the mutant tag. Furthermore, we applied this system to the imaging analysis of bromodomains. The labeled mutant tag successfully detected the localization of bromodomains to acetylhistone and the disruption of the bromodomain–acetylhistone interaction by a bromodomain inhibitor.     

Cross-linking chemistry and biology: development of multifunctional photoaffinity probes

An efficient method of photoaffinity labeling has been developed based on rationally designed multifunctional photoprobes. Photoaffinity techniques have been used to elucidate the protein structure at the interface of biomolecules by the photochemical labeling of interacting sites. However, the identification of labeled sites within target proteins is often difficult. Novel biotinyl bioprobes bearing a diazirine photophore have contributed significantly to the rapid elucidation of ligand binding sites within proteins, thereby extending conventional photoaffinity methods. This article discusses the synthesis and applications of various photoprobes bearing a biotin, including strategies using cleavable linkages between photophores. The combination of photoaffinity methods with chip technology is also described as a novel entry to rapid affinity-based screening of inhibitors. This review focuses on a rapid and reliable photoaffinity method utilizing diazirine-based multifunctional photoprobes with numerous potential applications in functional proteomics of biomolecular interactions.     

Exploring the Binding Proteins of Glycolipids with Bifunctional Chemical Probes

Glycolipids are important structural components of biological membranes and perform crucial functions in living systems, including signaling transduction and interaction with extracellular environment. However, the mechanistic exploration of glycolipids in vivo is challenging because they are not genetically encoded. Herein, we designed and synthesized a series of bifunctional monogalactosyldiacylglycerol (MGDG) probes as a model by introducing diazirine and terminal alkyne moieties on an aliphatic chain. In combination with proteome profiling and molecular modeling, we have demonstrated that MGDG alleviates inflammation by antagonizing TLR4.     

携异戊烯基光亲和化学探针的合成和应用

应用“一锅法”耦连反应为关键步骤合成了3个同时含有异戊烯链和叠氮基团的光亲和探针分子。在光照条件下这些化合物和酿酒酵母总蛋白反应后,经过点击反应与含有生物素的报告分子连接,再进行亲和素印迹分析,初步表明它们可作为钓取与异戊烯链相互作用蛋白的化学探针。     

AgHalo: A Facile Fluorogenic Sensor to Detect Drug-Induced Proteome Stress

Drug-induced proteome stress that involves protein aggregation may cause adverse effects and undermine the safety profile of a drug. Safety of drugs is regularly evaluated using cytotoxicity assays that measure cell death. However, these assays provide limited insights into the presence of proteome stress in live cells. A fluorogenic protein sensor is reported to detect drug-induced proteome stress prior to cell death. An aggregation prone Halo-tag mutant (AgHalo) was evolved to sense proteome stress through its aggregation. Detection of such conformational changes was enabled by a fluorogenic ligand that fluoresces upon AgHalo forming soluble aggregates. Using 5 common anticancer drugs, we exemplified detection of differential proteome stress before any cell death was observed. Thus, this sensor can be used to evaluate drug safety in a regime that the current cytotoxicity assays cannot cover and be generally applied to detect proteome stress induced by other toxins.