Ring closure strategy leads to potent RIPK3 inhibitors

Area of Science:

• Biochemistry
• Cell Biology
• Pharmacology

Background:

• Necroptosis is a regulated form of cell death.
• Receptor-interacting protein kinase 3 (RIPK3) is a key regulator of necroptosis.
• RIPK3 is a potential therapeutic target for necroptosis-associated diseases.

Purpose of the Study:

• Design, synthesize, and evaluate novel RIPK3 inhibitors.
• Identify a potent and selective RIPK3 inhibitor for therapeutic development.

Main Methods:

• Synthesis of novel compounds.
• In vitro assays to evaluate cell death inhibition (EC50).
• Biochemical assays to assess RIPK3 binding affinity (Kd) and kinase activity.
• Selectivity profiling against RIPK1.
• In vitro safety profiling (CYP, hERG).
• In vivo efficacy studies in a mouse model of systemic inflammatory response syndrome.

Main Results:

• Lead compound 38 demonstrated potent inhibition of TNFα, Smac mimetic, and z-VAD (TSZ) induced cell death in HT-29 cells (EC50 = 0.42 μM).
• Compound 38 exhibited high affinity binding to RIPK3 (Kd = 7.1 nM) and inhibited RIPK3 kinase activity.
• Compound 38 showed good selectivity over RIPK1 (Kd = 6000 nM).
• Excellent in vitro safety profiles with minimal inhibition of CYP isozymes and hERG channel.
• Compound 38 effectively blocked hypothermia and death in a mouse model of TNFα-induced systemic inflammatory response syndrome.

Conclusions:

• Novel RIPK3 inhibitors were successfully designed and synthesized.
• Compound 38 is a potent, selective, and safe RIPK3 inhibitor with in vivo efficacy.
• Compound 38 represents a promising therapeutic candidate for diseases involving necroptosis.