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

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.

Fig. 1. Identification of new necroptosis inhibitors.

a Schematic overview of the drug screen workflow. b Chemical structure of GSK’067 and GSK’074. c Mouse smooth muscle cell line MOVAS were treated with 30 ng/ml TNFα plus 60 μM zVAD and indicated compounds for 6 h. Cell viability was detected by CellTiter-Glo. Data were normalized to DMSO treated control cells and presented as mean ± S.D. n = 3. d Mouse fibroblast cell line L929 were treated with 20 ng/ml TNFα plus 40 μM zVAD and GSK’067, GSK’074, or GSK’843 for 3 h. Cell viability was detected by CellTiter-Glo. Data were normalized to DMSO treated control cells and presented as mean ± S.D. of three independent experiments

Fig. 2. GSK’074 inhibits necroptosis in various cell types.

a–c MOVAS cells were treated with 30 ng/ml TNFα plus 60 μM zVAD and compounds indicated for 6 h. Cells were then stained with 7-AAD and analyzed by flow cytometry. Necrotic cells were identified as 7-AAD⁺. d Mouse primary smooth muscle cells were treated with 100 ng/ml TNFα plus 60 μM zVAD and 10 nM GSK’074 for 24 h. Cells were then stained with 7-AAD and analyzed by flow cytometry. e Mouse bone marrow derived macrophages (BMDM) were treated with 10 ng/ml LPS plus 50 µM zVAD and 10 nM GSK’074 for 24 h. Cell viability was detected by CellTiter-Glo. f Human colorectal adenocarcinoma cell line HT-29 cells were treated with 20 ng/ml TNFα plus 200 nM Smac mimetic, 20 μM zVAD, and 10 nM GSK’074 for 24 h. Cells were then stained with 7-AAD and analyzed by flow cytometry. Data were presented as mean ± S.D. n = 3. *P < 0.05

Fig. 3. GSK’074 blocks necroptosis signaling pathway.

a–c MOVAS cells were treated with 30 ng/ml TNFα plus 60 μM zVAD for 3 h in the presence or absence of GSK’074. RIP1 and RIP3 complex formation were detected by co-immunoprecipitation with anti-IgG or anti-RIP3 antibodies followed by immunoblot analysis with the indicated antibodies (a) or by in situ PLA assay (b). Scale bar = 50 µm. Cells were stained with anti-MLKL serine345 phosphorylation (p-MLKL S345) and representative pictures were shown (c). Scale bar = 50 µm. d MOVAS cells were treated with indicated concentrations of GSK’074 for 24 h, whole-cell lysates were subjected to immunoblot analysis with the indicated antibodies

Fig. 4. GSK’074 directly binds to RIP1 and RIP3.

a, b The kinase domains of recombinant human RIP1 and RIP3 were used in the competitive binding assay of GSK’074. Data were presented as mean ± S.D. n = 2. c, d In vitro kinase activity was performed with recombinant RIP3 (c) or the kinase domain of RIP1 (1–327) (d). Levels of phosphorylation in the presence of compounds indicated were determined by ADP-Glo. Data were presented as mean ± S.D. n = 3. e, f Molecular docking of GSK’074 and kinase domain of human RIP3 (e) or RIP1 (f) in a DFG-out conformation. g L929 cells were primed with IFNβ (50 units/mL) for 24 h, then treated with 10 μg/ml poly(I:C) plus 40 μM zVAD and different concentrations of compounds indicated. Cells were stained with 7-AAD and analyzed by flow cytometry. Data represent mean ± S.D. of three independent experiments. h Mouse primary aortic smooth muscle cells were pretreated with indicated compounds for 2 h, followed by 10 ng/ml TNFα for additional 2 h. Levels of mRNA were determined by Real-time PCR. Data represent mean ± S.D. of three independent experiments. *P < 0.05

Fig. 5. GSK’074 does not induce apoptosis.

a, b, d, e L929 cells were treated with indicated compounds for 24 h, then stained with Annexin V-PE and 7-AAD and analyzed by flow cytometry. Apoptotic cells were identified as PE Annexin V⁺/7-AAD⁻. c L929 cells were treated with 10 μM GSK’843 or GSK’074 for 24 h, cell lysates were immunoprecipitated with anti-RIP1 antibody followed by immunoblot analysis with the indicated antibodies. *P < 0.05

Fig. 6. Selectivity profile of GSK’074.

Binding of GSK’074 (100 nM) against 468 human kinases (403 non-mutant kinases, 65 mutant kinases) was assessed by DiscoveRx KINOMEscan™. a Selectivity scores of GSK’074. Selectivity score was defined as the number of non-mutant kinases bound to GSK’074 <35% of control (S(35)), 10% of control (S(10)), or 1% of control (S(1)). b TREEspot map of kinases potentially targeted by GSK’074. Binding with RIP1 (0% of control) was shown by the blue circle, red circles indicated other kinases that displayed similar binding as RIP1 (S(1))

Fig. 7. GSK’074 attenuates cell death and inflammation in calcium phosphate injured aortae.

a Experimental design of the calcium phosphate aneurysm model. Mice were treated with 200 µl vehicle (8% DMSO) or GSK’074 at 0.93 mg/kg/day immediately following aneurysm induction. Three male mice were in each group. Mice were euthanized 4 days after. b, e Representative photographs of immunostaining for markers smooth muscle cell (SM-αActin) or macrophages (CD68). Scale bar = 50 μm. c Mice were injected with propidium iodide (PI) 2 h before euthanization. DAPI was used to stain nuclei. Quantification of PI-positive cells in the medial layer (indicated by white dashed line) was shown on the right. Scale bar = 50 μm. d Cross-sections of aneurysm prone tissue were immunostained with anti-MLKL serine345 phosphorylation (p-MLKL S345) as a marker of necroptosis. Scale bar = 50 μm. f Representative photographs of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in aortic cross-sections. Quantification of TUNEL-positive cells percentage was shown on the right. Scale bar = 50 μm. *P < 0.05

Fig. 8. GSK’074 inhibits aneurysm formation in mouse models of aneurysms.

a, d Experimental design. Abdominal aortic aneurysm was induced in male C57BL/6J mice (8–10 weeks) by the calcium phosphate aneurysm model (a) or in Apoe^−/− female mice (9–10 months) by the Angiotensin II infusion (1000 ng/kg/min) (d). 200 µl vehicle (8% DMSO) or GSK’074 at 0.93 mg/kg/day was administered daily via IP. Mice were euthanized 14 (a) or 28 days (d) after aneurysm induction. b, e Representative photos of perfused abdominal aortae with indicated treatments. Arrows indicate aneurysm formation. c, f Percentage increase of maximal external aortic diameter. An AAA is defined as a percentage increase in aortic diameter ≥50% (red dashed line) compared to the aortic diameter before calcium phosphate treatment (c) or compared to the external diameter of infrarenal diameter (f). Data were presented as mean ± S.D. *P < 0.05