Structure-Based Design of Novel Alkynyl Thio-Benzoxazepinone Receptor-Interacting Protein Kinase-1 Inhibitors: Extending the Chemical Space from the Allosteric to ATP Binding Pockets

Abstract

Systemic inflammatory response syndrome (SIRS), characterized by severe systemic inflammation, represents a major cause of health loss, potentially leading to multiple organ failure, shock, and death. Exploring potent RIPK1 inhibitors is an effective therapeutic strategy for SIRS. Recently, we described thio-benzoxazepinones as novel RIPK1 inhibitors and confirmed their anti-inflammatory activity. Herein, we further synthesized novel thio-benzoxazepinones by introducing substitutions on the benzene ring by an alkynyl bridge in order to extend the chemical space from the RIPK1 allosteric to ATP binding pockets. The in vitro cell and kinase assays found that compounds 2 and 29 showed highly potent activity against necroptosis (EC50 = 3.7 and 3.2 nM) and high RIPK1 inhibitory activity (K d = 9.7 and 70 nM). Prominently, these two analogues possessed better in vivo anti-inflammatory effects than the clinical candidate GSK′772 and effectively blocked hypothermia and deaths in a TNFα-induced SIRS model.

Insight from Linker Investigations: Discovery of a Novel Phenylbenzothiazole Necroptosis Inhibitor Targeting Receptor-Interacting Protein Kinase 1 (RIPK1) from a Phenoxybenzothiazole Compound with Dual RIPK1/3 Targeting Activity

“…To evaluate the antinecroptotic activity of target compounds, a chemiluminescence assay using TNF-α, Smac mimetic (SM-164), and a caspase inhibitor (z-VAD-FMK), abbreviated as TSZ, was conducted on human HT-29 cells. ,,− ,, The reference compound SZM-610, which contains the original ether linker (see Figure ), exhibits an antinecroptotic activity (EC 50 ) of 0.082 μM. , …”

“…A heteroaryl compound DNL758/SAR443122 was initiated by Denali and Sanofi to conduct a clinical trial on cutaneous lupus erythematosus (CLE) patients (JXHL2200324, JXHL2200325). Additionally, various potent RIPK1 inhibitors reported by different research groups − and comprehensive reviews are available for reference. ,, …”

Discovery of a Novel Benzimidazole Necroptosis Inhibitor from an In-House Compound Library

“…4 Compound 1 blocked necroptosis through the inhibition of RIPK1 phosphorylation. (A) HT-29 cells were pretreated with compound 1 at specified concentrations(20,40, and 80 μmol/L) and GSK'843 (10 μmol/L) for 30 minutes before a 6-hour exposure to TSZ. Cells were lysed, and the resulting lysates were analyzed by immunoblotting with the corresponding antibodies.…”