A cardiotoxicity-eliminated ACE2 variant as a pan-inhibitor against coronavirus cell invasion

Abstract

Human recombinant ACE2 (hrACE2) has been highly anticipated as a successful COVID-19 treatment; however, its potential to cause cardiac side effects has given rise to many concerns. Here, we developed a cardiotoxicity-eliminated hrACE2 variant, which had four mutation sites within hrACE2 (H345L, H374L, H378L, H505L) and was named as hrACE2-4mu. hrACE2-4mu has a consistent binding affinity with the variant SARS-CoV-2 spike proteins (SPs) and an efficient ability to block SP-induced SARS-CoV-2 entry into cells. In golden hamsters, injection of purified wild-type (WT) hrACE2 rescues the early stages of pneumonia caused by the SPs of the WT, delta, and omicron variants with reduced inflammatory cell infiltration. However, long-term injection of WT hrACE2 induces undesired cardiac fibrosis, as demonstrated by upregulated fibronectin and collagen expression. Our newly developed hrACE2-4mu showed similar protective abilities against a series of coronavirus cell invasions as WT hrACE2, meanwhile it did not cause apparent cardiac side effects. Thus, we generated a cardiotoxicity-eliminated variant of hrACE2 as a pan-inhibitor against coronavirus cell invasion, providing a potential novel strategy for the treatment of COVID-19 and other coronaviruses.

Keywords: ACE2; cardiac fibrosis; coronavirus; pan-inhibitor; spike protein.

Graphical abstract

Figure 1

hrACE2 works as a pan-inhibitor against the cell entry of SPs of diverse coronaviruses (A) Exogenous overexpression of ACE2 promotes the cell entry of SPs of diverse coronaviruses. BEAS-2B cells were transfected with ACE2 adenovirus and cultured with different S protein receptor binding domains for 30 min. Western blot analysis of S protein binding to cells. The results suggest that ACE2 recruits more ACE2 into human pulmonary cells. (B) hrACE2 was cultured with S protein together to block S protein entry into cells. HrACE2 with different S protein receptor binding domains in BEAS-2B cells. Western blot analysis of S protein binding to cells. The results suggest that free hrACE2 might work as a pan-inhibitor against cell entry from diverse coronaviruses. (C and D) Different mutant S proteins incubated with hrACE2 and western blot analysis of S protein binding to cells. The results indicate that hrACE2 shows superior performance in neutralizing antibodies in the blockage of cell invasion by variant SPs. (E) Schematic overview of the nasal drop of a coronavirus S protein in a Syrian golden hamster infection model. hrACE2-mFc was injected before the SP nasal drop. The morphology and inflammatory factors of the lung, including CD3, Iba1, TNFα, and IL-6, were detected by IHC staining. (F and G) Analysis of the lung/body ratio, IHC of S protein, CD3-positive T cells, and H&E staining. The results suggested that hrACE2-mFc (1 mg/kg) blocked S protein entry into lung cells, reversed the lung/body ratio and recruitment of CD3-positive T cells and Iba 1-positive macrophages, and reduced the level of inflammatory cytokines. (H) H-score of IHC of CD3, Iba1, TNFα, and IL-6 staining in lung tissue, suggesting that ectocytic hrACE2 shows promising usefulness for preventing the infection by various SARS-CoV-2 variants. Severity increases with significant numbers from 0 to 4. For (A) and (B), n = 4. For (C)–(H), n = 3. Data are representative of three independent experiments (error bars, SE). Scale bar, 100 μm. ∗p < 0.05 (two-tailed t test). NS, no significance; 40150-D001, a neutralizing antibody for SP; SP, spike protein.

Figure 2

Four site mutations rescue ACE2-triggered cardiac fibrosis in vivo and in vitro (A and B) Alterations in fibrotic genes and GO pathway analysis of overexpression of ACE2 in cardiac cells. (A) AC16 cells overexpressed with ACE2, and the cells were detected by bulk RNA sequencing and GO pathway analysis. The results suggest that ACE2 promotes fibrosis-related gene RNA expression. (B) Gene levels, including those of ACE2, fibronectin, collagen Ia, collagen IIIa, SMA, and MLKL1, were detected with or without ACE2 overexpression. (C and D) Western blot analysis of fibronectin and collagen 1A level during overexpression of ACE2 with pathological stimulus in the human AC16 cell line and neonatal rat fibroblasts. The results suggest that ACE2 promotes fibrosis-related protein expression. (E) Western blot analysis of different mutant active sites (H374L, H378L, H374/378L, H345/374/378/505L [4mu]) on ACE2-triggered cardiac fibrotic genes in AC16 cells. The results showed that only 4mu blocked the ACE2-triggered cardiac fibrotic effect. (F–I) The effect of circulating hrACE2-mFc and its four site mutations on cardiac remodeling and fibrosis. FVB mice were injected intraperitoneally with 1 mg/kg of mouse Fc, hrACE2-mFc, or hrACE2-4mu-mFc protein per day for a week, and then the hearts were taken from the mice for further analysis. Injection of hrACE2-mFc enlarged the heart volume, reduced the number of cardiomyocytes, and increased the expression level of fibronectin. However, injection of hrACE2-4mu-mFc completely overcomes the cardiac side effects caused by hrACE2-mFc. Data are representative of three independent experiments (error bars, SE). Scale bar, 100 μm. ∗, #, & p < 0.05, ∗∗, ##, && p < 0.01. NS, no significance. For animal and cell experiments, n = 3.

Figure 3

Four site mutations in the hrACE2 protein preserve its protective role against SP pulmonary invasion in golden hamsters (A and B) Comparison of different ACE2 truncations and mutants on neutralization of the SPs. BEAS-2B cells were treated with different hrACE2 variants and truncation proteins (1 μg/mL) together with SP variants. The results showed that hrACE2 4mu showed the same performance as WT hrACE2 in blocking the cell entry of SP and its variants. (C–G) Effects of the hrACE2 protein on the lung/body ratio, SP expression, levels of CD3⁺ T cells, and H&E staining with quantitative analysis. Golden hamsters were injected with 1 μg/mL hrACE2-4mu-mFc protein before the SP nasal drop. Comparison of the protective effects of hrACE2-mFc and hrACE2-4mu-mFc on SP-invaded lungs, as analyzed by IHC staining (C), lung/body ratio, H-score of CD3⁺ cells, and levels of TNFα and IL-6 (D–G). The results showed that hrACE2 mutation did not affect the protection against SP invasion. Data are representative of three independent experiments (error bars, SE). Scale bar, 100 μm. ∗∗p < 0.01. NS, no significance; SP, spike protein; hSA, human serum albumin, treated as control. For the animal test, n = 3.