RIPK3 Inhibitor-AZD5423 Alleviates Acute Kidney Injury By Inhibiting Necroptosis And Inflammation

Acute kidney injury affects 13.3 million people worldwide each year and causes1.7 million deaths,with an incidence of about 10 to 15%in hospitalized patients and occurring in more than 50%of patients in intensive care.Acute kidney injury（AKI）has become a global health problem due to the frequent adverse clinical outcomes and the heavy economic burden.Acute kidney injury（AKI）is defined as a clinical syndrome caused by a rapid decline in renal function in a short period of time caused by various reasons,the most common causes include renal toxicity,ischemia-reperfusion injury,etc.Clinically,AKI is usually characterized by the decrease of glomerular filtration rate,the retention of serum creatinine and urea nitrogen,and the pathophysiological process related to AKI mainly includes the necroptosis of renal proximal tubular epithelial cells and inflammatory response.Necroptosis,also known as"necroptosis",plays a key role in renal injury models induced by ischemia-reperfusion and cisplatin.Necroptosis depends on three key regulators-RIPK1,RIPK3 and MLKL.Knockout or inhibition of these regulators can alleviate renal injury.A growing body of literature suggests that RIPK3 can regulate necroptosis independently of RIPK1 activation.RIPK3 can be independently involved in protecting cells from a wider range of necrotic pathologies.For example,infection with mouse cytomegalovirus m45 mutant strain（MCMV）and herpes simplex virus 1（HSV1）ICP6 protein can directly activate RIPK3.Toll-like receptors TLR3 and TLR4,as well as ZBP1 proteins,do not require RIPK1 to participate in mediating necroptosis.Therefore,finding novel and safe inhibitors targeting RIPK3 is considered to be one of the promising strategies for the prevention and treatment of AKI.On this basis,based on the crystal structure of RIPK3,we used a computerized hybrid virtual screening strategy to screen small molecules capable of inhibiting RIPK3 from a small molecule database containing approximately 350,600 compounds,resulting in 68 candidates.Subsequently,these 68 compounds were tested for their protective effects on cisplatin-stimulated HK2 cells by MTT assay,and AZD5423 was found to have the ability to rescue cell death without drug toxicity within the dose used.However,whether it can be used as a drug for the prevention and treatment of AKI is still unclear.In an in vitro model of HK-2 cell injury stimulated by cisplatin and H/R,AZD5423 treatment significantly reduced RIPK3 phosphorylation.In the specific necroptosis model induced by TNF-αand z VAD,AZD5423 also inhibited the activation of RIPK3.The possible binding interaction between AZD5423 and RIPK3was also suggested by CETSA assay.In addition,the results of experiments in RIPK3knockdown cells showed that AZD5423 could directly target RIPK3 and inhibit RIPK3 kinase activity.Functionally,AZD5423 could also effectively alleviate cisplatin and H/R-induced injury and inflammatory response in HK-2 cells,as shown by the down-regulation of KIM1 and the improvement of inflammatory cytokines TNF-a and IL-6.Similarly,AZD5423 inhibited the phosphorylation and membrane translocation of MLKL to ameliorate necroptosis.To assess potential binding sites of AZD5423 to RIPK3,docking simulations revealed strong hydrogen bond binding of AZD5423 to LYS⁵⁰and Arg³¹³.Using site-directed mutagenesis,we generated a K50A/R313A mutant of RIPK3,which further indicated that K50 and R313 residues may be responsible for the interaction between AZD5423 and RIPK3.In vivo,we evaluated the preventive and therapeutic effects of AZD5423 using different regimens（two prophylactic regimens and one therapeutic regimen）in cisplatin and ischemia-reperfusion injury-induced mouse models.Firstly,in cisplatin-induced AKI mouse model,AZD5423 ameliorated inflammation by reducing the phosphorylation of RIPK3 and membrane translocation of MLKL,as well as glycogen deposition and tubular injury.The therapeutic effect of AZD5423 was then evaluated in an established AKI mouse model induced by cisplatin,which also attenuated cisplatin-induced pathological injury and inflammation.Finally,we further evaluated the preventive effect of AZD5423 on ischemia-reperfusion（I/R）-induced acute kidney injury by inhibiting RIPK3 phosphorylation and inflammatory response.In summary,in this thesis,we have found that AZD5423 binds to the key amino acid residues Lys⁵⁰and Arg³¹³of RIPK3 kinase and protects kidney function by inhibiting necroptosis and inflammation;AZD5423 pretreatment has a preventive effect on cisplatin and ischemia-reperfusion induced AKI mouse model and a therapeutic effect on the established AKI mouse model.It is revealed that AZD5423 is a promising clinical candidate for AKI prevention and treatment.