Electrophilic Warhead-Based Design of Compounds Preventing NLRP3 Inflammasome-Dependent Pyroptosis

Abstract

Pyroptosis is a caspase-1-dependent pro-inflammatory form of programmed cell death implicated in the pathogenesis of autoinflammatory diseases as well as in disorders characterized by excessive cell death and inflammation. Activation of NLRP3 inflammasome is a key event in the pyroptotic cascade. In this study, we describe the synthesis and chemical tuning of α,β-unsaturated electrophilic warheads toward the development of antipyroptotic compounds. Their pharmacological evaluation and structure-activity relationships are also described. Compound 9 was selected as a model of this series, and it proved to be a reactive Michael acceptor, irreversibly trapping thiol nucleophiles, which prevented both ATP- and nigericin-triggered pyroptosis of human THP-1 cells in a time- and concentration-dependent manner. Moreover, 9 and other structurally related compounds, inhibited caspase-1 and NLRP3 ATPase activities. Our findings can contribute to the development of covalent, multitarget antipyroptotic compounds targeting molecular components of the NLRP3 inflammasome regulatory pathway.

Fumaric acid esters prevent the NLRP3 inflammasome-mediated and ATP-triggered pyroptosis of differentiated THP-1 cells

“…Moreover, it was accompanied by a decreased intracellular activation of caspase-1. Thereby, FAEs antagonise the effects of ATP by preventing the activation of the pyroptotic cascade leading to cell death, thus mimicking (at least in part) the effects of not only parthenolide, but also Bay 11-7082 [9], resatorvid, 5Z-7-oxozeaneol, and bromoxone [10], compound 9 and related derivatives [11]. FAEs exerted anti-pyroptotic effects at low micromolar concentrations.…”

“…Interestingly, albeit structurally unrelated, most of the anti-pyroptotic compounds so far identified have electrophilic substructures (Fig. S1A), and Michael-like reactions with free thiol groups in proteins of the pyroptotic cascades have been postulated to underlie their activity [9][10][11]. FAEs even are Michael acceptors (Fig.…”

“…c o m / l o c a t e / i n t i m p interest due to their therapeutic potential [8]. A group of structurally unrelated small molecules, including parthenolide and Bay 11-7082 [9], resatorvid/TAK-242, 5Z-7-oxozeaneol, and bromoxone [10], compound 9 and related derivatives [11] (Fig. S1A) have been shown to prevent pyroptosis in in vitro experiments.…”

“…Caspase-1 activity was measured by a spectrophotometric assay as previously described [11]. Reactions were carried out in 96-well plates in 100 μl final volume at 37°C by diluting enzyme (50 U) in the reaction buffer (100 mM HEPES, 10% sucrose, 0.1% CHAPS, pH 7.5, 10 mM DTT) and monitoring liberation of the chromophore from a specific labelled substrate [Ac-Tyr-Val-Ala-Asp-p-nitroaniline (Ac-YVAD-pNA; 200 μM final concentration); Vinci-Biochem; Vinci, Italy] in a Victor X4 (PerkinElmer, Waltham, MA, USA) at a wavelength of 405 nm.…”

“…Caspase-1 activity was measured by a cell-free system assay, as previously described [11]. As shown in Fig.…”