Bifunctional Molecular Probes for Activity‐Based Visualization of Quinone‐Dependent Amine Oxidases

Abstract

The design, synthesis, and evaluation of two bifunctional molecular probes that can be used to visualize quinone-dependent amine oxidase enzymes in an activity-dependent manner are described. These probes use alkylhydrazines to irreversibly bind the target enzymes, which can then be visualized with either Western blotting or in-gel fluorescence. The results show that the Western blotting readout, which utilizes commercially available anti-nitrophenyl antibodies to detect a simple dinitrophenyl antigen, provides a stronger readout than the fluorescein-based fluorescence readout. This visualization strategy can be used to measure the potency of enzyme inhibitors by selectively visualizing the active enzyme that remains after treatment with an inhibitor. Looking forward, this probe molecule and visualization strategy will enable activity-based protein-profiling experiments, such as determining inhibitor selectivity values within full proteome mixtures, for this family of amine oxidase enzymes.

Activity‐Based Protein Profiling of Oxidases and Reductases

“…The group of Jakobsche has developed ABPP probe 30 with a hydrazine nucleophile to visualize lentil seedling diamine oxidase enzyme (LSDAO) in in vitro experiments (Scheme 8). [74] The hydrazine group in 30 attacks one carbonyl of the 1,2‐benzoquinone group formed from the tyrosine residue, which results in hydrazine condensation. Tautomeric shift ultimately produces the azophenol conjugate 31 .…”

Activity‐Based Protein Profiling (ABPP) of Oxidoreductases

“…Quinone‐dependent amine oxidases utilize a 1,2‐benzoquinone cofactor together with copper ions to oxidize aliphatic amines into aldehydes. The group of Jakobsche has developed ABPP probe 25 to visualize lentil seedling diamine oxidase enzyme (LSDAO; Scheme 5), [47] which is described as a typical representative of the topoquinone subfamily. Fluorescein and 2,4‐dinitrophenyl were used as readout domains.…”

A Class of Valuable (Pro-)Activity-Based Protein Profiling Probes: Application to the Redox-Active Antiplasmodial Agent, Plasmodione

“…In the case of the flavin-dependent oxidoreductase family, the design of clickable ABPP probes was limited to the cytochrome P450, 8 2-oxoglutarate oxygenases, 9 and amine oxidases. 10 , 11 According to the literature, several proteomics studies have been conducted with ABPP probes for the detection of drug targets in P. falciparum . 12 − 16 …”

“…An additional advantage in the ABPP field is the enlargement of the variety of chemical probes that trap representatives of various enzyme classes and can be utilized in proteome studies. In the case of the flavin-dependent oxidoreductase family, the design of clickable ABPP probes was limited to the cytochrome P450, 2-oxoglutarate oxygenases, and amine oxidases. , According to the literature, several proteomics studies have been conducted with ABPP probes for the detection of drug targets in P. falciparum . − …”

“…In the case of the flavin-dependent oxidoreductase family, the design of clickable ABPP probes was limited to the cytochrome P450, 8 2-oxoglutarate oxygenases, 9 and amine oxidases. 10,11 According to the literature, several proteomics studies have been conducted with ABPP probes for the detection of drug targets in P. falciparum. 12−16 NAD(P)H-dependent flavoenzymes from the malarial parasites have been proposed as possible targets of plasmodione, an early antimalarial lead compound (PD, 3-[4-(trifluoromethyl)benzyl]-menadione, 1, Figure 1).…”