Discovery of a new class of RIP1/RIP3 dual inhibitors with anti‐cell death and anti‐inflammatory properties

Background Receptor interacting protein kinase‐1 and ‐3 (RIP1 and RIP3) are essential mediators of cell death processes and participate in inflammatory responses. Our group recently demonstrated that gene deletion of Rip3 or pharmacological inhibition of RIP1 attenuate the pathogenesis of abdominal aortic aneurysm (AAA), a life‐threatening, degenerative vascular disease characterized by depletion of smooth muscle cells, inflammation, negative extracellular matrix remodeling, and progressive expansion of the aorta. The goal of this study is to develop a potent RIP1/RIP3 inhibitor applicable to treating disease conditions such as AAA that involve cell necrosis and inflammation. Methods and Results We screened 1,141 kinase inhibitors available in the Small Molecule Screening Facility at UW‐Madison. The compounds were selected for their ability to block necroptosis more potently than Necrostatin‐1s (Nec‐1s, 20 μM, a widely used RIP1 inhibitor), followed by cytotoxicity tests and a virtual screen for RIP3 binding affinity. Among the top hits, compound C9 displayed structural similarity to the established RIP3 inhibitor GSK’843. In multiple cell types including mouse smooth muscle cells, fibroblast cells (L929), and human colon cancer cells (HT29), C9 inhibited necroptosis with an IC50 of 3 nM, which was significantly lower than Nec‐1s (802 nM) and GSK’843 (993 nM). Furthermore, C9, but not Nec‐1s, blocked cytokine production by smooth muscle cells. Biochemical studies and molecular docking revealed that C9 directly targeted RIP1 and RIP3 as a type II kinase inhibitor. C9 inhibited necroptosis signaling including necrosome formation and downstream activation of mixed lineage kinase domain like (MLKL). Unlike GSK’843 which caused apoptosis at high doses (>3 μM), C9 showed no cytotoxicity at even 20 μM. Of note, C9 did not rescue the apoptotic response induced by GSK’843 or ER stress. Encouraged by the minimum cytotoxicity and relative high stability (half‐life in liver microsome ~1 hour), we tested C9 in mice using two AAA models: the calcium phosphate model and angiotensin II (AngII) model. Daily intraperitoneal injection of C9 at 2 μmol/kg significantly blocked aortic expansion in both models (aortic expansion in calcium phosphate model: DMSO 66.06±9.17% vs C9 27.36±8.25%, P<0.05; AngII model: DMSO 85.39±15.76% vs C9 36.28±5.76%, P<0.05). Histologically, C9 treatment diminished necrosis and macrophage infiltration in aneurysm‐prone aortae. Conclusion Together, our data suggests that C9 is a new class of type II kinase inhibitor with dual targeting ability to both RIP1 and RIP3. Its high potency and minimum cytotoxicity make C9 a desirable drug candidate for pharmacological attenuation of aneurysm progression and other necroptosis related pathologies. Support or Funding Information This study was supported by NIH R01HL088447 (Bo Liu), American Heart Associationpostdoctoral fellowship 17POST33680095 (Ting Zhou). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .