直接结合 NLRP3 吡喃结构域作为一种预防 NLRP3 驱动的炎症和痛风性关节炎的新策略。

Direct Binding to NLRP3 Pyrin Domain as a Novel Strategy to Prevent NLRP3-Driven Inflammation and Gouty Arthritis.

机构信息

College of Pharmacy, The Catholic University of Korea, Bucheon, Republic of Korea.

St. Mary's Hospital, Uijeongbu, Republic of Korea, and The Catholic University of Korea, Seoul, Republic of Korea.

出版信息

Arthritis Rheumatol. 2020 Jul;72(7):1192-1202. doi: 10.1002/art.41245. Epub 2020 May 27.

DOI:10.1002/art.41245

PMID:32134203

Abstract

OBJECTIVE

The NLRP3 inflammasome is closely linked to the pathophysiology of a wide range of inflammatory diseases. This study was undertaken to identify small molecules that directly bind to NLRP3 in order to develop pharmacologic interventions for NLRP3-related diseases.

METHODS

A structure-based virtual screening analysis was performed with ~62,800 compounds to select efficient NLRP3 inhibitors. The production of caspase 1-p10 and interleukin-1β (IL-1β) was measured by immunoblotting and enzyme-linked immunosorbent assay to examine NLRP3 inflammasome activation. Two gouty arthritis models and an air pouch inflammation model induced by monosodium urate monohydrate (MSU) crystal injection were used for in vivo experiments. Primary synovial fluid cells from gout patients were used to determine the relevance of NLRP3 inflammasome inhibition in human gout.

RESULTS

Beta-carotene (provitamin A) suppressed the NLRP3 inflammasome activation induced by various activators, including MSU crystals, in mouse bone marrow-derived primary macrophages (P < 0.05). Surface plasmon resonance analysis demonstrated the direct binding of β-carotene to the pyrin domain (PYD) of NLRP3 (K = 3.41 × 10 ). Molecular modeling and mutation assays revealed the interaction mode between β-carotene and the NLRP3 PYD. Inflammatory symptoms induced by MSU crystals were attenuated by oral administration of β-carotene in gouty arthritis mouse models (P < 0.05), correlating with its suppressive effects on the NLRP3 inflammasome in inflamed tissues. Furthermore, β-carotene reduced IL-1β secretion from human synovial fluid cells isolated from gout patients (P < 0.05), showing its inhibitory efficacy in human gout.

CONCLUSION

Our results present β-carotene as a selective and direct inhibitor of NLRP3, and the binding of β-carotene to NLRP3 PYD as a novel pharmacologic strategy to combat NLRP3 inflammasome-driven diseases, including gouty arthritis.

摘要

目的

NLRP3 炎性小体与广泛的炎症性疾病的病理生理学密切相关。本研究旨在鉴定直接与 NLRP3 结合的小分子，以开发用于 NLRP3 相关疾病的药物干预措施。

方法

使用约 62800 种化合物进行基于结构的虚拟筛选分析，以选择有效的 NLRP3 抑制剂。通过免疫印迹和酶联免疫吸附试验测量 caspase 1-p10 和白细胞介素-1β（IL-1β）的产生，以检查 NLRP3 炎性小体的激活。使用尿酸单钠一水合物（MSU）晶体注射诱导的两种痛风性关节炎模型和气囊炎症模型进行体内实验。使用来自痛风患者的原代滑液细胞来确定 NLRP3 炎性小体抑制在人类痛风中的相关性。

结果

β-胡萝卜素（维生素 A 前体）抑制了包括 MSU 晶体在内的各种激活剂诱导的小鼠骨髓来源的原代巨噬细胞中 NLRP3 炎性小体的激活（P <0.05）。表面等离子体共振分析表明β-胡萝卜素直接与 NLRP3 的吡喃结构域（PYD）结合（K = 3.41×10-5 M）。分子建模和突变分析揭示了β-胡萝卜素与 NLRP3 PYD 之间的相互作用模式。在痛风性关节炎小鼠模型中，通过口服给予β-胡萝卜素可减轻 MSU 晶体诱导的炎症症状（P <0.05），这与其在炎症组织中对 NLRP3 炎性小体的抑制作用相关。此外，β-胡萝卜素可减少从痛风患者分离的人滑膜液细胞中 IL-1β 的分泌（P <0.05），表明其在人类痛风中的抑制作用。