Development of Fluorescent and Biotin Probes Targeting NLRP3

Abstract

Extracellular signals drive the nucleation of the NLRP3 inflammasome which leads to the release of cytokines and causes inflammatory events. Hence, the inflammasome has gained enormous momentum in biomedical basic research. The detailed mechanisms of inflammasome generation and regulation remain to be elucidated. Our study was directed toward the design, convergent synthesis, and initial biochemical evaluation of activity-based probes addressing NLRP3. For this purpose, probes were assembled from a CRID3/MCC950-related NLRP3-binding unit, a linker portion and a coumarin 343 fluorophore or biotin. The affinity of our probes to NLRP3 was demonstrated through SPR measurements and their cellular activity was confirmed by reduction of the interleukin 1β release from stimulated bone marrow-derived macrophages. The initial characterizations of NLRP3-targeting probes highlighted the coumarin probe 2 as a suitable tool compound for the cellular and biochemical analysis of the NLRP3 inflammasome.

Structure of the NLRP3 decamer bound to the cytokine release inhibitor CRID3

“…The system was flushed with running buffer (10 mM HEPES pH 7.4, 200 mM NaCl, 0.5 mM ADP, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), 2 mM MgCl2, 1 g/L carboxymethyl dextran hydrogel (CMD), 0.05% Tween20, 2% DMSO) at 25°C. A streptavidin functionalized sensor chip (Series S Sensor Chip SA, Cytiva) was conditioned with three consecutive injections of 1 M NaCl in 50 mM NaOH (10 μL/min) for 1 min as described 34 . Biotinylated NLRP3 NACHT-trLRR protein from HEK cell expression was immobilized at 2 μL/min for 900 s. The flow system was washed using 50% isopropanol in 1 M NaCl and 50 mM NaOH.…”

A novel optical imaging probe for targeted visualization of NLRP3 inflammasomes in a mouse model of age-related macular degeneration

“…Due to the association between NLRP3 inflammasomes, activated macrophages and CNV, ( 15 ) we hypothesized that an NLRP3-targeted fluorescent probe would enable visualization of pro-inflammatory macrophages in CNV. Recently, a group of researchers synthesized an NLRP3-targeted fluorescent probe by conjugating a fluorophore, coumarin 343, to the NLRP3 inhibitor, MCC950 ( 17 ). Although this is a promising new probe that enabled visualization of NLRP3 in cells, it is not suitable for ophthalmic in vivo applications.…”

New Azido Coumarins as Potential Agents for Fluorescent Labeling and Their “Click” Chemistry Reactions for the Conjugation with closo-Dodecaborate Anion

“…Some commercially available and commonly used for labeling fluorescent coumarins are shown in Figure 1 . Functionalized with carboxylic groups or with activated esters, coumarins ( Figure 1 ) have been aimed for detection of amino acids, small sized proteins, and antibodies through the peptide bond formation [ 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 ]. 7-Diethylamino-coumarin-3-carbohydrazide is used for fluorescence labeling of carbonylated proteins and lipids [ 44 , 45 , 46 , 47 , 48 ].…”