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Cell Death Dis. 2019 Mar 6;10(3):226. doi: 10.1038/s41419-019-1468-6.

Identification of a novel class of RIP1/RIP3 dual inhibitors that impede cell death and inflammation in mouse abdominal aortic aneurysm models.

Author information

1
Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI, 53705, USA.
2
Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, 02215, USA.
3
Department of Radiation Oncology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA.
4
School of Pharmacy, Medicinal Chemistry Center, University of Wisconsin, Madison, WI, 53705, USA.
5
UW Carbone Cancer Center, School of Medicine and Public Health, University of Wisconsin, Madison, WI, 53705, USA.
6
Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI, 53705, USA. liub@surgery.wisc.edu.
7
Department of Cellular and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin, Madison, WI, 53705, USA. liub@surgery.wisc.edu.

Abstract

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 attenuated pathogenesis of abdominal aortic aneurysm (AAA), a life-threatening degenerative vascular disease characterized by depletion of smooth muscle cells (SMCs), inflammation, negative extracellular matrix remodeling, and progressive expansion of aorta. The goal of this study was to develop drug candidates for AAA and other disease conditions involving cell death and inflammation. We screened 1141 kinase inhibitors for their ability to block necroptosis using the RIP1 inhibitor Necrostatin-1s (Nec-1s) as a selection baseline. Positive compounds were further screened for cytotoxicity and virtual binding to RIP3. A cluster of top hits, represented by GSK2593074A (GSK'074), displayed structural similarity to the established RIP3 inhibitor GSK'843. In multiple cell types including mouse SMCs, fibroblasts (L929), bone marrow derived macrophages (BMDM), and human colon epithelial cells (HT29), GSK'074 inhibited necroptosis with an IC50 of ~3 nM. Furthermore, GSK'074, but not Nec-1s, blocked cytokine production by SMCs. Biochemical analyses identified both RIP1 and RIP3 as the biological targets of GSK'074. Unlike GSK'843 which causes profound apoptosis at high doses (>3 µM), GSK'074 showed no detectable cytotoxicity even at 20 µM. Daily intraperitoneal injection of GSK'074 at 0.93 mg/kg significantly attenuated aortic expansion in two mouse models of AAA (calcium phosphate: DMSO 66.06 ± 9.17% vs GSK'074 27.36 ± 8.25%, P < 0.05; Angiotensin II: DMSO 85.39 ± 15.76% vs GSK'074 36.28 ± 5.76%, P < 0.05). Histologically, GSK'074 treatment diminished cell death and macrophage infiltration in aneurysm-prone aortae. Together, our data suggest that GSK'074 represents a new class of necroptosis inhibitors with dual targeting ability to both RIP1 and RIP3. The high potency and minimum cytotoxicity make GSK'074 a desirable drug candidate of pharmacological therapies to attenuate AAA progression and other necroptosis related diseases.

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