Celastrol: A lead compound that inhibits SARS-CoV-2 replication, the activity of viral and human cysteine proteases, and virus-induced IL-6 secretion

Abstract

The global emergence of coronavirus disease 2019 (COVID-19) has caused substantial human casualties. Clinical manifestations of this disease vary from asymptomatic to lethal, and the symptomatic form can be associated with cytokine storm and hyperinflammation. In face of the urgent demand for effective drugs to treat COVID-19, we have searched for candidate compounds using in silico approach followed by experimental validation. Here we identified celastrol, a pentacyclic triterpene isolated from Tripterygium wilfordii Hook F, as one of the best compounds out of 39 drug candidates. Celastrol reverted the gene expression signature from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells and irreversibly inhibited the recombinant forms of the viral and human cysteine proteases involved in virus invasion, such as M^pro (main protease), PL^pro (papain-like protease), and recombinant human cathepsin L. Celastrol suppressed SARS-CoV-2 replication in human and monkey cell lines and decreased interleukin-6 (IL-6) secretion in the SARS-CoV-2-infected human cell line. Celastrol acted in a concentration-dependent manner, with undetectable signs of cytotoxicity, and inhibited in vitro replication of the parental and SARS-CoV-2 variant. Therefore, celastrol is a promising lead compound to develop new drug candidates to face COVID-19 due to its ability to suppress SARS-CoV-2 replication and IL-6 production in infected cells.

Keywords: COVID-19; SARS-CoV-2; celastrol; cysteine protease inhibition; molecular docking.

Figure 1

Assessment of gene signature in SARS‐CoV‐2‐infected human bronchial epithelial cells (NHBE) for drug discovery to treat COVID‐19. (a) Enriched biological pathways associated with COVID‐19 from Reactome annotations (p _adj < .05) from DEGs identified in NHBE‐infected cells transcriptome data. The genes that enriched each pathway (left) are indicated together with statistical results and pathway description (right). (b) Heatmap of genes from the 50 best‐ranked compound signatures that reversed the genetic signature of SARS‐CoV‐2‐infected NHBE cells in decreasing order of Q _score. The signature map annotations are related to up‐ and downregulated genes, and cell lines are indicated in different colors. (c) Compounds in decreasing order of Q _score following the output of L1000CDS.² A dashed line indicates the mean Q _score (0.26) threshold. Equal Q _score values are displayed over the bars. (d) B _score for each compound, considering the enrichment analysis and reverse signature. The dashed line corresponds to the mean B _score (0.49). *Genetic signature that justified the biological validation of celastrol. COVID‐19, coronavirus disease 2019; DEGs, differentially expressed genes; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 2

Docking analysis of molecular interactions between M^pro and molecules capable of reversing SARS‐CoV‐2 genetic signature. (a) Boxplots with the affinity binding energies (kcal. mol⁻¹) were obtained from docking analysis between several structural conformations of 39 molecules and each viral site of 83 M^pro structures. The reverser compounds were sorted based on decreasing order of Q _score that indicated their potential to revert the genetic signature of SARS‐CoV‐2 infection. Dotted lines indicate median affinity binding energy (−7.3 kcal. mol⁻¹) considering all investigated drugs. (b) Representative structure of M^pro where atoms are represented as lines and secondary structures as a cartoon with helices highlighted in magenta and sheets in yellow. Colored line circles in brown indicate the binding site region used for docking. (c) 3D and (d) 2D representations detailing configuration and chemical interactions between the best‐ranked pose for celastrol in M.^pro 2D target‐drug interaction was constructed using the Discovery Studio® software (version‐2020). The distance between the B‐ring C6 and the sulfur atom of the Cys145 residue, which may be related to a possible Michael adduct formation for the best energy poses in each M^pro structure, ranged from 0.43 to 1.33 nm, with an average value of 0.63 nm (black dashed line). M,^promain protease; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 3

Time‐dependent inhibition of M^pro (a) and papain (b) activity by celastrol, expressed as IC₅₀ value. M,^pro main protease.

Figure 4

Persistent inhibition of M^pro and papain activity by celastrol. (a) Assay procedure to determine the irreversibility of M^pro and papain inhibition by celastrol. The enzymes were incubated for 30 min with buffer (control) or 22 mM celastrol, as indicated in the Methods section. Part of the incubation mixture was kept on ice and the other part was submitted to three ultrafiltration cycles to remove free celastrol and concentrate the enzymes. The M^pro and papain activity was determined in the concentrate. (b) M^pro and (c) papain activity before and after repeated dilution/filtration cycles. M,^pro main protease.

Figure 5

Celastrol suppresses SARS‐CoV‐2 in vitro propagation in nonhuman and human cell lines. RT‐PCR quantification of SARS‐CoV‐2 RNA in supernatants from the infected cell lines (a) Vero CCL‐81, (b) Caco‐2, and (c) Calu‐3, and (d) Calu‐3 infected with SARS‐CoV‐2 gamma variant treated with celastrol at concentrations of 125, 250, 500, and 1000 nM. DMSO solution (0.05%; vehicle) was used as a negative control. Cells were infected using MOI = 1.0 for 2 h and then treated with celastrol for 48 h. The detection levels of SARS‐CoV‐2 RNA were performed in the supernatants of cultures and expressed in viral load using a standard curve described in Section 2. Statistical differences between celastrol treatments and the negative control were analyzed by ANOVA followed by Tukey's posttest. The significance levels were indicated as *p < .05, **p <.01, ***p < .001, and ****p < .0001. ANOVA, analysis of variance; MOI, multiplicity of infection; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.

Figure 6

Celastrol does not affect cell viability. Alamar Blue assay was used to measure the potential cytotoxic effect of celastrol. The monkey cell line (a) Vero CCL‐81 and the human cell lines (b) Caco‐2 and (c) Calu‐3 were treated with celastrol at 250 and 1000 nM, for 48 h. DMSO solutions at 0.05% and 5.0% were used as the negative and positive controls of cell death, respectively. Statistical analysis was performed using ANOVA followed by Tukey's posttest to compare treatment with celastrol and 5.0% DMSO. ****p < .0001. ANOVA, analysis of variance.

Figure 7

Celastrol inhibits IL‐6 production in SARS‐CoV‐2‐infected human cell lines. ELISA quantification of IL‐6 in the culture supernatants from the human cell lines (a) Caco‐2 and (b) Calu‐3, infected with SARS‐CoV‐2 (MOI = 1.0) for 2 h. Then, after the washing step, infected cells were treated with celastrol for 48 h. DMSO solution at 0.05% (vehicle) was used as the negative control. Statistical differences between celastrol treatment and control were analyzed by ANOVA followed by Tukey's posttest. The significance levels were indicated as **p < .01, ***p < .001, and ****p < .0001. The results are expressed as mean ± standard error of the mean (SEM) of IL‐6 (pg/ml). They are representative of two independent experiments with Caco‐2 cells with three replicates and one experiment with Calu‐3 cells with four replicates. ANOVA, analysis of variance; IL‐6, interleukin‐6; MOI, multiplicity of infection; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2.