Discovery of potent necroptosis inhibitors targeting RIPK1 kinase activity for the treatment of inflammatory disorder and cancer metastasis

Jue Hou,Yangxin Li,Jie Ju,Xiaoliang Yu,Linkun Hu,Hongjian Zhang,Jiyue Zheng,Meng Wu,Tian Sheng,Haikuo Ma,Wei Zhang,Zhanhui Li,Cong Zhao,Xiaohu Zhang,Zili Zhang,Sudan He

doi:10.1038/s41419-019-1735-6

Abstract

Necroptosis is a form of regulated necrosis controlled by receptor-interacting kinase 1 (RIPK1 or RIP1), RIPK3 (RIP3), and pseudokinase mixed lineage kinase domain-like protein (MLKL). Increasing evidence suggests that necroptosis is closely associated with pathologies including inflammatory diseases, neurodegenerative diseases, and cancer metastasis. Herein, we discovered the small-molecule PK6 and its derivatives as a novel class of necroptosis inhibitors that directly block the kinase activity of RIPK1. Optimization of PK6 led to PK68, which has improved efficacy for the inhibition of RIPK1-dependent necroptosis, with an EC50 of around 14–22 nM in human and mouse cells. PK68 efficiently blocks cellular activation of RIPK1, RIPK3, and MLKL upon necroptosis stimuli. PK68 displays reasonable selectivity for inhibition of RIPK1 kinase activity and favorable pharmacokinetic properties. Importantly, PK68 provides strong protection against TNF-α-induced systemic inflammatory response syndrome in vivo. Moreover, pre-treatment of PK68 significantly represses metastasis of both melanoma cells and lung carcinoma cells in mice. Together, our study demonstrates that PK68 is a potent and selective inhibitor of RIPK1 and also highlights its great potential for use in the treatment of inflammatory disorders and cancer metastasis.

Highlights

Necroptosis is a form of regulated necrosis that is tightly controlled by the activation of receptor-interacting protein kinases (RIPKs)[1]
Pre-treatment of PK68 powerfully blocks tumor necrosis factor (TNF)-induced lethal shock and inflammatory responses as well as tumor metastasis in vivo. These findings suggest that PK68 can be used in the development of new therapies for the treatment of necroptosis-activated pathologies including inflammatory disorders and cancer metastasis
We evaluated the effect of PK6 on TNF-induced necroptosis in mouse cells and found that PK6 efficiently inhibited TNF-induced necroptosis in both mouse embryonic fibroblasts (MEFs) with EC50 of ~0.95 μM and mouse fibroblast L929 cells with EC50 of ~0.76 μM (Fig. 1e, f)

Summary

Introduction

Necroptosis is a form of regulated necrosis that is tightly controlled by the activation of receptor-interacting protein kinases (RIPKs)[1]. Extensive studies have shown that inhibition of RIPK1 kinase activity by the chemical compound necrostatin-1 or by knock-in of a kinase-dead form of RIPK1 can ameliorate necroptosis-associated pathologies in mouse models of various diseases, including inflammatory diseases (e.g. colitis and dermatitis) and in neurodegenerative diseases (e.g. multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS))[24,25]. It has been shown that suppression of RIPK1 kinase activity improves anti-tumor immunity by modulating tumor-associated macrophages[26]; this occurs independently of RIPK3-mediated necroptosis, demonstrating that the kinase activity of RIPK1 can be viewed as a new immunomodulatory target for the development of new anti-cancer therapies

Methods

Results

Conclusion