Discovery of a Potent RIPK3 Inhibitor for the Amelioration of Necroptosis-Associated Inflammatory Injury

Abstract

Necroptosis is a form of regulated necrosis that requires the activation of receptor-interacting kinase 3 (RIPK3 or RIP3) and its phosphorylation of the substrate MLKL (mixed lineage kinase domain-like protein). Necroptosis has emerged as important cell death involved in the pathogenesis of various diseases including inflammatory diseases, degenerative diseases, and cancer. Here, we discovered a small molecule Zharp-99 as a potent inhibitor of necroptosis through blocking the kinase activity of RIPK3. Zharp-99 efficiently blocks necroptosis induced by ligands of the death receptor and Toll-like receptor as well as viral infection in human, rat and mouse cells. Zharp-99 strongly inhibits cellular activation of RIPK3, and MLKL upon necroptosis stimuli. Zharp-99 directly blocks the kinase activity of RIPK3 without affecting RIPK1 kinase activity at the tested concentration. Importantly, Zharp-99 exerts effective protection against TNF-α induced systemic inflammatory response syndrome in the mouse model. Zharp-99 displays favorable in vitro safety profiles and in vivo pharmacokinetic parameters. Thus, our study demonstrates Zharp-99 as a potent inhibitor of RIPK3 kinase and also highlights its potential for further development of new approaches for treating necroptosis-associated inflammatory disorders.

Inhibition of Pseudomonas aeruginosaLPS‐Induced airway inflammation by RIPK3 in human airway

“…Lung sections were processed with anti‐RIPK3 (A) or phospho‐RIPK3 (C) antibody, stained with Deep‐Red, and images were obtained for each core. Five separate images of each section were given a score 1–10 by three independent scientists. (B, D) The expression density was used to assess stained differences between cells.…”

“…Lung sections were processed with either anti-RIPK3 antibody, phosphor-RIPK3 antibody (Ser277) or goat anti-rabbit IgG (Alexa Fluor 546), stained with Deep-Red, and images were taken for each core. Five separate images of each section were given a score [1][2][3][4][5][6][7][8][9][10] by three independent scientists.…”

“…Receptor‐interacting serine/threonine‐protein kinase (RIPK) 3 is a major protein of necroptosis that can be initiated by various signals including the activation of death receptors, Toll‐like receptors, interferon receptors and pathogen infection 8,9 . Necroptosis is a system of regulated cell death characterized by necrotic features including cell swelling and disrupted cell plasma membrane 10 . Necroptosis is tightly regulated by the activation of RIPK1 and RIPK3 10 .…”

“…8,9 Necroptosis is a system of regulated cell death characterized by necrotic features including cell swelling and disrupted cell plasma membrane. 10 Necroptosis is tightly regulated by the activation of RIPK1 and RIPK3. 10 RIPK3 is regulated by the proteins related to post-translational modification, such as protein phosphatase 1B, 11 deubiquitylating enzyme A20, 12 C terminus of Hsp-70-interacting protein (CHIP) 13 and Pellino E3 ubiquitin protein ligase 1 (PELI1).…”

“…10 Necroptosis is tightly regulated by the activation of RIPK1 and RIPK3. 10 RIPK3 is regulated by the proteins related to post-translational modification, such as protein phosphatase 1B, 11 deubiquitylating enzyme A20, 12 C terminus of Hsp-70-interacting protein (CHIP) 13 and Pellino E3 ubiquitin protein ligase 1 (PELI1). 14 Although RIPK3 is a pivotal protein in necroptosis, post-translational processes that may control RIPK3 activity and stability during P. aeruginosa infection in the airway remain poorly understood.…”