Dual-Color Real-Time Chemosensing of a Compartmentalized Reaction Network Involving Enzyme-Induced Membrane Permeation of Peptides.

The design of synthetic systems with interrelated reaction sequences that model incipient biological complexity is limited by physicochemical tools that allow the direct monitoring of the individual processes in real-time. To mimic a simple digestion-resorption sequence, the authors have designed compartmentalized liposomal systems that incorporate extra- and intravesicular chemosensing ensembles. The extravesicular reporter pair consists of cucurbit[7]uril and methylene blue to monitor the enzymatic cleavage of short enkephalin-related peptides by thermolysin through a switch-off fluorescence response ("digestion"). Because the substrate is membrane-impermeable, but the dipeptide product is permeable, uptake of the latter into the pre-formed liposomes occurs as a follow-up process. The intravesicular chemosensing ensemble consists of i) cucurbit[8]uril, 2-anilinonaphthalene-6-sulfonic acid, and methyl viologen or ii) cucurbit[7]uril and berberine to monitor the uptake ("resorption") of the enzymatic products through the liposomal membranes by i) a switch-on or ii) a switch-off fluorescence response. The dyes are designed to allow selective optical excitation and read-out of the extra- and intravesicular dyes, which allow for dual-color chemosensing and, therefore, kinetic discrimination of the two sequential reactions.

A Label-Free Continuous Fluorescence-Based Assay for Monitoring Ornithine Decarboxylase Activity with a Synthetic Putrescine Receptor.

Polyamines play an important role in cell growth, differentiation, and cancer development, and the biosynthetic pathway of polyamines is established as a drug target for the treatment of parasitic diseases, neoplasia, and cancer chemoprevention. The key enzyme in polyamine biosynthesis is ornithine decarboxylase (ODC). We report herein an analytical method for the continuous fluorescence monitoring of ODC activity based on the supramolecular receptor cucurbit[6]uril (CB6) and the fluorescent dye trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (DSMI). CB6 has a significantly higher binding constant to the ODC product putrescine (>107 M-1) than to the substrate L-ornithine (340 M-1). This enables real-time monitoring of the enzymatic reaction through a continuous fluorescence change caused by dye displacement from the macrocycle by the formed product, which allowed a straightforward determination of enzyme kinetic parameters ( kcat = 0.12 s-1 and KM = 24 µM) and inhibition constants of the two ODC inhibitors α-difluoromethylornithine (DFMO) and epigallocatechin gallate (EGCG). The potential for high-throughput screening (HTS) was demonstrated by excellent Z' factors (>0.9) in a microplate reader format, and the sensitivity of the assay is comparable to or better than most established complementary methods, which invariably have the disadvantage of not being compatible with direct implementation and upscaling to HTS format in the drug discovery process.