It is widely accepted that activating the transcription factor NRF2 will blast the physiological anti-inflammatory mechanisms, which will help combat pathologic inflammation. Much effort is being put in inhibiting the main NRF2 repressor, KEAP1, with either electrophilic small molecules or disrupters of the KEAP1/NRF2 interaction. However, targeting β-TrCP, the non-canonical repressor of NRF2, has not been considered yet. After in silico screening of ∼1 million compounds, we now describe a novel small molecule, PHAR, that selectively inhibits the interaction between β-TrCP and the phosphodegron in transcription factor NRF2. PHAR upregulates NRF2-target genes such as Hmox1, Nqo1, Gclc, Gclm and Aox1, in a KEAP1-independent, but β-TrCP dependent manner, breaks the β-TrCP/NRF2 interaction in the cell nucleus, and inhibits the β-TrCP-mediated in vitro ubiquitination of NRF2. PHAR attenuates hydrogen peroxide induced oxidative stress and, in lipopolysaccharide-treated macrophages, it downregulates the expression of inflammatory genes Il1b, Il6, Cox2, Nos2. In mice, PHAR selectively targets the liver and greatly attenuates LPS-induced liver inflammation as indicated by a reduction in the gene expression of the inflammatory cytokines Il1b, TNf, and Il6, and in F4/80-stained liver resident macrophages. Thus, PHAR offers a still unexplored alternative to current NRF2 activators by acting as a β-TrCP/NRF2 interaction inhibitor that may have a therapeutic value against undesirable inflammation.

人们普遍认为，激活转录因子 NRF2 会破坏生理抗炎机制，从而有助于对抗病理性炎症。人们正在努力用亲电子小分子或 KEAP1/NRF2 相互作用的干扰剂来抑制主要的 NRF2 阻遏蛋白 KEAP1。然而，尚未考虑针对 NRF2 的非典型阻遏蛋白 β-TrCP。在对约 100 万种化合物进行计算机筛选后，我们现在描述了一种新型小分子 PHAR，它选择性抑制 β-TrCP 与转录因子 NRF2 中磷酸降解子之间的相互作用。PHAR 以不依赖 KEAP1 但依赖 β-TrCP 的方式上调 NRF2 靶基因，如Hmox1、Nqo1、Gclc、Gclm和Aox1，破坏细胞核中 β-TrCP/NRF2 相互作用，并抑制 β-TrCP - 介导的 NRF2 体外泛素化。PHAR 减弱过氧化氢诱导的氧化应激，并且在脂多糖处理的巨噬细胞中，它下调炎症基因Il1b、Il6、Cox2、Nos2的表达。在小鼠中，PHAR 选择性地靶向肝脏，并极大地减轻 LPS 诱导的肝脏炎症，炎症细胞因子Il1b、TNf和Il6以及 F4/80 染色的肝脏巨噬细胞的基因表达减少表明了这一点。因此，PHAR 通过充当 β-TrCP/NRF2 相互作用抑制剂，为当前 NRF2 激活剂提供了一种尚未探索的替代品，可能对不良炎症具有治疗价值。