Discovery and Evaluation of New Activity‐Based Probes for Serine Hydrolases

Abstract

Serine hydrolases play crucial biological roles and are important therapeutic targets in many clinical applications. Activity‐based protein profiling of serine hydrolases by using fluorophosphonate probes, pioneered by Cravatt and co‐workers, has been a powerful tool for interrogating serine hydrolases in various biological systems. Herein, we present new phenyl phosphonate probes with an azide handle for click chemistry that offer remarkable improvements over the classical fluorophosphonate serine hydrolase activity‐based probes including ease of preparation, excellent cell permeability, and distinct reactivity profiles, as controlled by the phenolate leaving group. Thus, these new activity‐based serine hydrolase probes are valuable tools to further interrogate this important class of enzymes.

Chemoproteomics‐Enabled De Novo Discovery of Photoswitchable Carboxylesterase Inhibitors for Optically Controlled Drug Metabolism

“…We first optimized the labeling condition using various broad activity‐based probes to observe most optimal competition of CES1. In this context, our recently reported para ‐nitrophenyl phosphonate activity‐based probe 17 (30 n m ) provided the best visualization of 16 binding to CES1 in Hep G2 lysate (Figure S14) [11] . Under the same conditions, we performed an MS experiment to identify proteins competed by sulfone 16 in Hep G2 lysate.…”

“…Here we sought to identify differences in the gel band pattern of lysates pretreated with the E ‐isomers of our compounds as competitors, treated with light at 365 nm or not, and labeled with a serine hydrolase‐specific activity‐based probe. Human colorectal adenocarcinoma Caco‐2 cell lysate was first treated with 100 n m of the indicated E ‐isomer, with or without concomitant UV irradiation for 5 minutes, for one hour at 37 °C and then with our recently published serine hydrolase‐directed activity‐based protein profiling (ABPP) probe 10 (Figure 3 A) for one hour at RT [11] . A carboxytetramethylrhodamine (TMR) fluorescent dye was then installed on 10 using copper(I)‐catalyzed azide‐alkyne cycloaddition (CuAAC), the probe‐labeled proteins were separated by SDS‐PAGE, and the resulting fluorescent bands were visualized using in‐gel fluorescence scanning.…”

“…To identify the 60 kDa serine hydrolase, we performed a competitive mass spectrometry‐based experiment in Caco‐2 lysate again using probe 10 [11] . The lysate was treated with E ‐ 4 or DMSO for one hour at 37 °C and then with 10 μ m probe 10 followed by streptavidin enrichment, digestion, and MS‐based analysis.…”

“…C) Quantification of the competitive gel‐based assay where gel filtration reduces competition by E ‐ 16 but not E ‐ 4 of CES1 labeling by probe 14 in Hep G2 lysate (see Figure S13 for gel images). D) Chemical structure of the activity‐based serine hydrolase probe 17 [11] and competitive MS‐based identification of E ‐ 16 (80 μ m ) protein targets using 17 (30 n m ) in Hep G2 lysate ( n= 6). Error bars are SD *** indicates p <0.001 by Student's t‐test.…”

Chemoproteomics‐Enabled De Novo Discovery of Photoswitchable Carboxylesterase Inhibitors for Optically Controlled Drug Metabolism

“…[10] Here we sought to identify differences in the gel band pattern of lysates pretreated with the Eisomers of our compounds as competitors,t reated with light at 365 nm or not, and labeled with aserine hydrolase-specific activity-based probe.H uman colorectal adenocarcinoma Caco-2 cell lysate was first treated with 100 nm of the indicated E-isomer,w ith or without concomitant UV irradiation for 5minutes, for one hour at 37 8 8Ca nd then with our recently published serine hydrolase-directed activity-based protein profiling (ABPP) probe 10 ( Figure 3A)f or one hour at RT. [11] Ac arboxytetramethylrhodamine (TMR) fluorescent dye was then installed on 10 using copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), the probe-labeled proteins were separated by SDS-PAGE, and the resulting fluorescent bands were visualized using in-gel fluorescence scanning.I ndeed, we observed disappearance of Scheme 1. Synthesis of arylazopyrazole urea inhibitors;a )12MHCl, NaNO 2 (1.2 equiv), AcOH/H 2 O, 0 8 8C, 1hthen acetylacetone (1.17 equiv), NaOAc (3.0 equiv),E tOH/H 2 O, 0 8 8Ct oRT, 1h,9 5%;b)hydrazine monohydrate (1.0 equiv),E tOH, reflux, 3h,quantitative yield;c)corresponding carbamoyl chloride, NaH (2.0 equiv), THF, RT,1h.…”