Design and Synthesis of Quenched Activity‐based Probes for Diacylglycerol Lipase and α,β‐Hydrolase Domain Containing Protein 6

Abstract

Diacylglycerol lipases (DAGL) are responsible for the biosynthesis of the endocannabinoid 2-arachidonoylglycerol. The fluorescent activity-based probes DH379 and HT-01 have been previously shown to label DAGLs and to cross-react with the serine hydrolase ABHD6. Here, we report the synthesis and characterization of two new quenched activity-based probes 1 and 2, the design of which was based on the structures of DH379 and HT-01, respectively. Probe 1 contains a BODIPY-FL and a 2,4-dinitroaniline moiety as a fluorophore-quencher pair, whereas probe 2 employs a Cy5-fluorophore and a cAB40-quencher. The fluorescence of both probes was quenched with relative quantum yields of 0.34 and 0.0081, respectively. The probes showed target inhibition as characterized in activity-based protein profiling assays using human cell- and mouse brain lysates, but were unfortunately not active in living cells, presumably due to limited cell permeability.

ABHD6: Its Place in Endocannabinoid Signaling and Beyond

“…In addition, triazole urea featuring chiral hydroxylated 2-benzylpiperidines have recently been synthesized to act as dual inhibitors of both DGL and ABHD6, citing the chirality of the carbon at the C2 substituent and position of the C5 hydroxyl to dictate inhibitory activity on the enzymes in mouse brain extracts [54]. Finally, the recent chemical biology tools based on fluorescence activity-based probes that are being developed to try and differentiate the enzymatic activity of these enzymes will certainly help develop more select inhibitors [55]. Thus, much effort has been dedicated to developing selective inhibitors of ABHD6 to unravel the role of this enzyme in regulating cell functions and explore the possibility of targeting this enzyme for therapeutic purposes.…”

“…The remarkably high expression of ABHD6 in astrocytes tallies well with the recent studies establishing a dominant role for eCB signaling in this glial subtype and raises the question whether other glial cell types might express this enzyme and whether ABHD6 expression changes as a function of reactive gliosis associated with select neurological diseases [82]. The tools that were recently developed to follow ABHD6 expression and activity, which include specific antibodies that were validated using genetic controls and fluorescence activity-based probes that differentiate serine hydrolase enzymatic activities, will certainly help answer these questions [55,83].…”

Fluorescent Triazole Urea Activity‐Based Probes for the Single‐Cell Phenotypic Characterization of Staphylococcus aureus

“…[9a] Selective release of the leaving group adds additional versatility to this chemotype allowing for the design of quenched fluorescent ABPs. [10] Initially, we screened a library of ~150 TU compounds for competition with FP-tetramethylrhodamine (FP-TMR) labeling of serine hydrolases active in live S. aureus ATCC35556 grown on Tryptic Soy Agar supplemented with MgCl 2 (TSAMg), as used in our previous screen [7a] and reported as biofilm-promoting. [11] Library compounds had high activity against S. aureus serine hydrolases with 53 hit compounds showing >50% competition of FP-TMR labeling of at least 1 hydrolase target at a final concentration of 1 μM (8 compounds exceeding this threshold at 100 nM; Figure S1).…”

Strategies for Tuning the Selectivity of Chemical Probes that Target Serine Hydrolases

“…For example, conjugation of a BODIPY dye to the carbamoylating group of an optimized triazole urea enabled single-cell imaging of FphE in S. aureus (Chen et al, 2019a(Chen et al, , 2019b(Chen et al, , 2019c. In addition, the van der Stelt group converted a triazole urea inhibitor for DAGL-a into a quenched fluorescent probe by attaching a fluorophore on the carbamoylating moiety and a quencher on the triazole leaving group (van Rooden et al, 2018). These examples serve to highlight how synthetic chemistry has enabled the discovery of diverse probes using the triazole urea scaffold.…”