Identification and Characterization of NTB451 as a Potential Inhibitor of Necroptosis.

Eun-Jung In,Jun-Hyuk Han,Tae-Bong Kang,Tae-Yeon Kim,Yuno Lee,Sushruta Koppula,Kwang-Ho Lee

doi:10.3390/molecules23112884

Abstract

Necroptosis, or caspase-independent programmed cell death, is known to be involved in various pathological conditions, such as ischemia/reperfusion injury, myocardial infarction, atherosclerosis, and inflammatory bowel diseases. Although several inhibitors of necroptosis have been identified, none of them are currently in clinical use. In the present study, we identified a new compound, 4-({[5-(4-aminophenyl)-4-ethyl-4H-1,2,4-triazol-3-yl]sulfanyl}methyl)-N-(1,3-thiazol-2-yl) benzamide (NTB451), with significant inhibitory activity on the necroptosis induced by various triggers, such as tumor necrosis factor-α (TNF-α) and toll-like receptor (TLR) agonists. Mechanistic studies revealed that NTB451 inhibited phosphorylation and oligomerization of mixed lineage kinase domain like (MLKL), and this activity was linked to its inhibitory effect on the formation of the receptor interacting serine/threonine-protein kinase 1 (RIPK1)-RIPK3 complex. Small interfering RNA (siRNA)-mediated RIPK1 knockdown, drug affinity responsive target stability assay, and molecular dynamics (MD) simulation study illustrated that RIPK1 is a specific target of NTB451. Moreover, MD simulation showed a direct interaction of NTB451 and RIPK1. Further experiments to ensure that the inhibitory effect of NTB451 was restricted to necroptosis and NTB451 had no effect on nuclear factor-κB (NF-κB) activation or apoptotic cell death upon triggering with TNF-α were also performed. Considering the data obtained, our study confirmed the potential of NTB451 as a new necroptosis inhibitor, suggesting its therapeutic implications for pathological conditions induced by necroptotic cell death.

Highlights

Programmed cell death is an important biological process in tissue homeostasis, animal development, and disease [1,2,3]
As described in the Materials and Methods Section, using L929 cells, the first-round necroptosis inhibitor screening was performed with chemical library comprising 8363 compounds and four compounds that showed higher than 50% inhibition of necroptotic cell death at a concentration of 50 μM
To investigate whether NTB451 had an inhibitory effect on cell death in cells other than L929, mouse embryonic fibroblast (MEF) cells and a human cell line (HT-29) that is often used in necroptosis studies were treated with a combination of tumor necrosis factor-α (TNF-α), zVAD, and Smac mimetic BV6 plus Nec-1, receptor interacting serine/threonine-protein kinase 1 (RIPK1) kinase inhibitor, or the indicated concentrations of NTB451

Summary

Introduction

Programmed cell death is an important biological process in tissue homeostasis, animal development, and disease [1,2,3]. 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) are known to be the key molecules in the necroptosis signaling pathway [4,5]. Many stimuli, such as tumor necrosis factor (TNF), Fas ligand (FasL), TNF-related apoptosis-inducing ligand (TRAIL), interferon (IFN), double-strand RNA (dsRNA). The internalization of complex I following cleavage of ubiquitin from RIPK1 by deubiquitinating enzymes, such as A20 or CYLD, leads to the formation of complex II, which is composed of RIPK1, caspase-8, and Fas-associated protein with death domain (FADD) [8,9,10,11]. When caspase activity is inhibited, RIPK1 and RIPK3 are recruited and activated by phosphorylation; the activated RIPK3 phosphorylates MLKL [4,5,12,13]

Methods

Results

Discussion

Conclusion