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.
Highlights
Receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) are threonine/serine protein kinases that share a conserved kinase domain
Additional to its role in necroptosis, RIP1, through its death domain, binds to death receptors including tumor-necrosis factor receptor 1 (TNF-R1) and triggers the formation of various signaling complexes that promote cell survival or apoptosis depending on the cell type and cellular contents[6,7]
In agreement with the literature, we showed that IFNβ plus poly(I:C) and zVAD (IPZ) significantly increased 7-AAD positive cells (DMSO control = 3.98 ± 0.56% vs. IPZ = 63.91 ± 3.21%). 100 nM GSK’074 or 10 μM GSK’843 significantly inhibited IPZinduced necroptosis, reducing 7-AAD+ cells to 23.90 ± 0.56% and 7.55 ± 1.36%, respectively (Fig. 4g and Supplementary Figure 3)
Summary
Receptor interacting protein kinase-1 and -3 (RIP1 and RIP3) are threonine/serine protein kinases that share a conserved kinase domain. The protein–protein interaction between RIP1 and RIP3 is an essential signaling step to initiate necroptosis in most cell types[1,2]. Necroptosis or programmed necrosis is increasingly recognized as a major cell death mechanism in conditions when the apoptotic. Official journal of the Cell Death Differentiation Association. Additional to its role in necroptosis, RIP1, through its death domain, binds to death receptors including tumor-necrosis factor receptor 1 (TNF-R1) and triggers the formation of various signaling complexes that promote cell survival or apoptosis depending on the cell type and cellular contents[6,7]. RIP1 and RIP3 regulate expression of proinflammatory cytokines through mechanisms independent of necrosis and the subsequent release of danger-associated molecular pattern molecules (DAMPs)[8].
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have