Evaluation of Site-Diversified, Fully Functionalized Diazirine Probes for Chemical Proteomic Applications

Photoaffinity probes combined with the chemical proteomic platform have emerged as versatile tools for ligand and target discovery. However, photoaffinity probes with retained activity cannot always label the known target, indicating that it is challenging to profile a ligand’s targets based on its photoaffinity probe modified at a single site. Herein, we construct a series of site-diversified probes (P1-P6) of 4-anilinoquinazoline, a scaffold shared by several marketed EGFR-targeted drugs, via attaching a “fully functionalized” diazirine tag to six different sites, respectively. Chemical proteomic analysis revealed that these probes show different proteome-wide profiles and distinct competition patterns by erlotinib. Remarkably, low activity P4 towards EGFR inhibition has better EGFR labelling efficiency than the higher one, P5, which highlights the dominance of labelling accessibility of diazirine over probe affinity. In addition, the integrated analysis of protein targets of site-diversified probes can also help distinguish false positive targets. We anticipate that site-diversification of the probes of a given scaffold is an indispensable strategy to truly harness the power of photoaffinity-based chemoproteomics in drug discovery.

Evaluation of Site-Diversified, Fully Functionalized Diazirine Probes for Chemical Proteomic Applications

Photoaffinity probes combined with the chemical proteomic platform have emerged as versatile tools for ligand and target discovery. However, photoaffinity probes with retained activity cannot always label the known target, indicating that it is challenging to profile a ligand’s targets based on its photoaffinity probe modified at a single site. Herein, we construct a series of site-diversified probes (P1-P6) of 4-anilinoquinazoline, a scaffold shared by several marketed EGFR-targeted drugs, via attaching a “fully functionalized” diazirine tag to six different sites, respectively. Chemical proteomic analysis revealed that these probes show different proteome-wide profiles and distinct competition patterns by erlotinib. Remarkably, low activity P4 towards EGFR inhibition has better EGFR labelling efficiency than the higher one, P5, which highlights the dominance of labelling accessibility of diazirine over probe affinity. In addition, the integrated analysis of protein targets of site-diversified probes can also help distinguish false positive targets. We anticipate that site-diversification of the probes of a given scaffold is an indispensable strategy to truly harness the power of photoaffinity-based chemoproteomics in drug discovery.

New Trends in Diaziridine Formation and Transformation (a Review)

This review focuses on diaziridine, a high strained three-membered heterocycle with two nitrogen atoms that plays an important role as one of the most important precursors of diazirine photoaffinity probes, as well as their formation and transformation. Recent research trends can be grouped into three categories, based on whether they have examined non-substituted, N-monosubstituted, or N,N-disubstituted diaziridines. The discussion expands on the conventional methods for recent applications, the current spread of studies, and the unconventional synthesis approaches arising over the last decade of publications.

Structure, bonding, and photoaffinity labeling applications of dialkyldiazirines

Dialkyldiazirine photoaffinity probes are unparalleled tools for the study of small molecule-protein interactions. Here we summarize the basic principles of structure, bonding, and photoreactivity of dialkyldiazirines, current methods for their synthesis, and their practical application in photoaffinity labeling experiments. We demonstrate the unique utility of dialkyldiazirine probes in the context of our recent photoaffinity crosslinking-mass spectrometry analysis to reveal a hidden cholesterol binding site in the Hedgehog morphogen proteins.

Evaluation of fully-functionalized diazirine tags for chemical proteomic applications

The chemical proteomic properties of five diazirine-based, fully-functionalized photoaffinity tags, including a newly developed, minimal tag, were compared. This study provides guidance for the development of new photoaffinity probes.

Clickable gold-nanoparticles as generic probe precursors for facile photoaffinity labeling application

Clickable photoreactive gold nanoparticles have been developed to facilitate one-step preparation of photoaffinity probes for bioactive small molecules and their application to target protein analysis.

Harnessing affinity-based protein profiling to reveal a novel target of nintedanib

We identified tripeptidyl-peptidase 1 (TPP1) as one of the direct targets of nintedanib (NDNB) employing clickable photoaffinity probes, which provides insights into the functional meaning of the well-known IPF therapeutic drug.