Development of a Multiplexed Activity-Based Protein Profiling Assay to Evaluate Activity of Endocannabinoid Hydrolase Inhibitors

Abstract

Endocannabinoids, an important class of signaling lipids involved in health and disease, are predominantly synthesized and metabolized by enzymes of the serine hydrolase superfamily. Activity-based protein profiling (ABPP) using fluorescent probes, such as fluorophosphonate (FP)-TAMRA and β-lactone-based MB064, enables drug discovery activities for serine hydrolases. FP-TAMRA and MB064 have distinct, albeit partially overlapping, target profiles but cannot be used in conjunction due to overlapping excitation/emission spectra. We therefore synthesized a novel FP-probe with a green BODIPY as a fluorescent tag and studied its labeling profile in mouse proteomes. Surprisingly, we found that the reporter tag plays an important role in the binding potency and selectivity of the probe. A multiplexed ABPP assay was developed in which a probe cocktail of FP-BODIPY and MB064 visualized most endocannabinoid serine hydrolases in mouse brain proteomes in a single experiment. The multiplexed ABPP assay was employed to profile endocannabinoid hydrolase inhibitor activity and selectivity in the mouse brain.

Identification of α,β-Hydrolase Domain Containing Protein 6 as a Diacylglycerol Lipase in Neuro-2a Cells

“…Fluorophosphonate-rhodamine (FP-TAMRA) was purchased from Thermo Fisher, as well as synthesized in-house as previously described (Janssen et al, 2018). FP-Biotin was purchased from Santa Cruz Biotechnology, KT182 was purchased from Sigma Aldrich.…”

“…FP-Biotin was purchased from Santa Cruz Biotechnology, KT182 was purchased from Sigma Aldrich. Fluorophosphonate-BODIPY (FP-BODIPY) (Janssen et al, 2018), MB064 (Baggelaar et al, 2013), MB108 (Baggelaar et al, 2013), DH376 (Ogasawara et al, 2016), and LEI105 (Baggelaar et al, 2013) were synthesized as previously described. All synthesized compounds were at least 95% pure as analyzed by LC-MS, NMR, and HRMS.…”

Structure–Activity Relationship Studies of α-Ketoamides as Inhibitors of the Phospholipase A and Acyltransferase Enzyme Family

“…FP-alkyne was used as a positive control. 37 This was followed by incubation with FP-TAMRA (500 nM, 0.5 μL 30× inhibitor stock in DMSO, 20 min, rt). The final concentrations for the inhibitors are indicated in the figure legends.…”

Fluorophosphonates on‐Demand: A General and Simplified Approach toward Fluorophosphonate Synthesis

“…We next compared the in‐gel fluorescence intensity signals of a panel of 36 recombinant serine hydrolases of FP‐rhodamine ( 3 u ) freshly prepared via our reaction‐to‐assay protocol versus those observed from historically generated data from 3 u prepared via traditional methods [6f] (Figure 3D1). Here, the loading control normalized intensities for each enzyme are plotted against each other.…”