Multicolor Imaging of Endoplasmic Reticulum-Located Esterase As a Prodrug Activation Enzyme

Abstract

The carboxylesterase families of enzymes are key participants in phase I drug metabolism processes. Carboxylesterase families 1 and 2 are of particular clinical relevance. These enzymes produce endoplasmic reticulum localization signals, are primarily localized in the endoplasmic reticulum, and hydrolyze a wide range of ester-containing prodrugs into an activated form. In order to detect enzymes belonging to both families, we developed an optical multicolor imaging technique, which provides a distinct color window for multicolor imaging. This technique required the design and synthesis of three new mechanistic colored probes that fluoresce red, green, or blue and are based on the quinone methide cleavage process. These activity-based probes allow rapid and clear visualization with high specificity against the endoplasmic reticulum in cultured cells based on endoplasmic reticulum localized esterases including both families of carboxylesterase enzymes.

Cellular Protection of SNAP-25 against Botulinum Neurotoxin/A: Inhibition of Thioredoxin Reductase through a Suicide Substrate Mechanism

“…These inhibitors were utilized for the sole purpose of esterase profiling of the proteome. (40) In light of TrxR’s newly-found acyltransfer activity, it is worth mentioning that such probes might have selected TrxR; however, TrxR’s selection was dismissed because of its previously unknown hydrolase properties. Regardless of BoNT serotype, all botulinum neurotoxins require translocation of the neurotoxin’s protease.…”

Prolonged bioluminescence imaging in living cells and mice using novel pro-substrates forRenillaluciferase

“…In summary, the prodrug or caged-luciferin strategy affords an excellent platform for persistent bioluminescence imaging. [45][46][47][48][49][50][51] By introducing ester protecting groups of different sizes into the carbonyl group of the free luciferin, we designed and synthesized ten novel pro-substrates for Renilla luciferase. These precursors could be effectively activated by intracellular esterases, lipases or nucleophilic substances, leading to a bioluminescence enhancement.…”

Enzyme-Activated Fluorogenic Probes for Live-Cell and in Vivo Imaging

“…Probe 31 is one of a family of probes tuned to report esterase activity in the endoplasmic reticulum selectively, with potential for application as a drug release trigger. 74 Probe 32 contains a phosphatidylglycerol moiety, allowing it to embed in the outer surface of cells and monitor endocytic events. 20 The internalization of probe 32 allows intracellular esterases to unmask fluorescence, a feature that was used to demonstrate fundamental differences in rates of endocytosis in matched cancer and non-cancer cell lines.…”