Regorafenib as a potential drug for severe COVID-19: inhibition of inflammasome activation in mice

Abstract

SARS-CoV-2 infection can lead to severe COVID-19, particularly in elderly individuals and those with compromised immunity. Cellular senescence has been implicated as a key pathogenic mechanism. This study investigated the therapeutic potential of regorafenib, a previously characterized senomorphic drug, for severe COVID-19. SARS-CoV-2 virus-infected K18-hACE2 mice, overexpressing the human ACE2 receptor, exhibited 100% mortality by 10 days post infection. Regorafenib treatment significantly improved survival rates, approximately 43% remaining alive. Mechanistically, regorafenib effectively suppressed type I and II interferon and cytokine signaling. Notably, regorafenib inhibited NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, a key driver of the cytokine storm associated with severe COVID-19. Our findings elucidate the molecular mechanisms underlying therapeutic effects of regorafenib and suggest its potential use as a promising treatment option for severe COVID-19.

Keywords: K18‐hACE2; SARS‐CoV‐2; infection; inflammasome; regorafenib.

Fig. 1

Regorafenib increases the survival rates of SARS‐CoV‐2 infected K18‐hACE2 transgenic mice. (A) Experimental scheme to define the therapeutic effect of regorafenib (Reg). hACE2 mice were infected with 5 MLD50 of SARS‐CoV‐2 (β‐CoV/Korea/KCDC03/2020, S clade) and treated with Reg (5 mg·kg⁻¹) three times once daily 3 dpi to 5 dpi. (B) Survival curve. Kaplan–Meier plot of the survival of three groups (N = 7 for each group). Two independent experiments exhibited a similar survival curve. (C) Body weights of mice were measured daily for 14 days (N = 7 for each group). Mice exhibited loss ≥25% of their initial body weight were euthanized. Error bars represent standard error of the mean (SEM) for each group of mice.

Fig. 2

Transcriptome analysis for effect of regorafenib in SARS‐CoV‐2 infected mice. (A) Experimental scheme for RNA sequencing (RNA‐seq) analysis (B) Representative immune‐modulating genes, Stat1 and Stat2, critical transcription factors that mediate IFN signaling, TNF‐α, and Ccl3 were subjected to RT‐qPCR. Naïve; SARS, SARS‐CoV‐2‐infected; SARS + Reg, Reg‐treated following SARS‐CoV‐2 infection. N = 3 for each group. Student's t‐test, *P < 0.05; **P < 0.01; ***P < 0.001. Error bars represent standard error of the mean (SEM) for each group of mice. (C) Heat map of regorafenib‐modulated inflammasome‐ and pyroptosis‐related genes at 4 dpi following SARS‐CoV‐2 infection. N = 5 for each group. (D) Validation of RNA‐seq analysis by RT‐qPCR. Nlrp3 and its downstream effectors Casp1 and Gsdmd were analyzed. Naïve, non‐infected; SARS, SARS‐CoV‐2‐infected; SARS + Reg, Reg‐treated following SARS‐CoV‐2 infection (N = 3 for each group). Student's t‐test, *P < 0.05; **P < 0.01; ****P < 0.0001. Error bars represent standard error of the mean (SEM) for each group of mice.

Fig. 3

Validation for effect of regorafenib by immunofluorescence staining in SARS‐CoV‐2 infected lungs. (A) Experimental scheme for immunofluorescence (IF). (B) Representative lung immunofluorescence images for NLRP3, Caspase 1 and GSDMD. Mice were exposed to three different conditions: Naïve, SARS‐CoV‐2 virus, and SARS‐CoV‐2 combined with Reg. Lungs were harvested from the treated mice at 5 dpi, and subsequently analyzed by immunofluorescence. Scale bars, 100 μm. (C) Quantification of staining intensity. Naïve, non‐infected; SARS, SARS‐CoV‐2‐infected; SARS + Reg, Reg‐treated following SARS‐CoV‐2 infection (N = 3 for each group). Student's t‐test, *P < 0.05. Error bars represent standard error of the mean (SEM) for each group of mice.

Fig. 4

Effect of regorafenib on the NLRP3 inflammasome activation following SARS‐CoV‐2 infection. (A) Experimental scheme. THP‐1 monocytes were differentiated into macrophages by PMA (100 nm) incubation for 2 days, then infected with SARS‐CoV‐2 (0.5 MOI), treated with increasing concentrations of Reg, and finally harvested. (B) Cell lysates and supernatants were subjected to western blot analysis for the indicated proteins. Representative blots are shown from three independent experiments.