Targeting ACLY efficiently inhibits SARS-CoV-2 replication

Abstract

The Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the biggest public health challenge the world has witnessed in the past decades. SARS-CoV-2 undergoes constant mutations and new variants of concerns (VOCs) with altered transmissibility, virulence, and/or susceptibility to vaccines and therapeutics continue to emerge. Detailed analysis of host factors involved in virus replication may help to identify novel treatment targets. In this study, we dissected the metabolome derived from COVID-19 patients to identify key host factors that are required for efficient SARS-CoV-2 replication. Through a series of metabolomic analyses, in vitro, and in vivo investigations, we identified ATP citrate lyase (ACLY) as a novel host factor required for efficient replication of SARS-CoV-2 wild-type and variants, including Omicron. ACLY should be further explored as a novel intervention target for COVID-19.

Keywords: ACLY; COVID-19; Delta; Omicron; SARS-CoV-2; metabolomics.

Figure 1

Schematic illustration of the study.

Figure 2

GS-MS-based targeted metabolomics was used to identify metabolites in COVID-19 patient plasma samples. (A) Pie chart showing the ratio of detected and non-detected polar metabolites using the GC-MS-based targeted metabolites. (B) Pie chart showing the ratio of detected and non-detected non-polar fatty acids using the GC-MS-based targeted metabolites. (C) Overview of the upregulated and downregulated metabolites in COVID-19 patients' plasma. (D) Heatmap showing the upregulated metabolites and their belonged pathways in both COVID-19 patients and non-infected donors.

Figure 3

LC-MS-based untargeted metabolomics was used to identify perturbed metabolites in COVID-19 patient plasma samples. (A) PLS-DA model showing the metabolic profiles between the COVID-19 patients and non-infected controls were different. (B) Permutation test showing a statistically significant separation between the COVID-19 patients and non-infected donors. (C) Heatmap showing the upregulated metabolites and their belonged pathways in both COVID-19 patients and non-infected donors.

Figure 4

Combing all the identified metabolites from GC-MS and LC-MS. (A) Overview of the global metabolic pathway analysis from identified metabolites. The y-axis indicating the log10 transformed p-value after enrichment analysis, and the x-axis representing the pathway impact value calculated from the pathway topology analysis. (B) Receiver operating characteristic (ROC) curve analysis from the GC-MS and LC-MS platforms that were used in this study. (C) The top 5 perturbed metabolites from the GC-MS platform. (D) The top 5 perturbed metabolites from the LC-MS platform.

Figure 5

Inhibition of the three mini pathways within the TCA cycle reduced SARS-CoV-2 replication. (A) Schematic illustration of the three mini pathways including citrate induced de novo lipogenesis, glutamine metabolism, and malate-aspartate shuttle. (B) Caco2 cells were infected with WT SARS-CoV-2 at 0.1 MOI and treated with selective small molecule inhibitors including CTPI-2, SB 204990, Bempedoic acid, BPTES, R162, Vorasidenib, (-)-Epigallocatechin gallate, and aminooxyacetic acid hemihydrochloride at 50 µM. Supernatants were harvested at 24 hours post-infection. (C) The viabilities of all tested inhibitors in Caco2 cells without virus infection at 24 hours post-treatment. Data represented mean and standard deviation from three independent experiments. Statistical analyses in B were performed with one-way ANOVA, the differences were considered significant only when P < 0.05. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Figure 6

ACLY inhibition efficiently reduced the replication of SARS-CoV-2 wildtype and variants. (A) Schematic illustration of the citrate induced de novo lipogenesis mini pathway. (B) Caco2 cells were infected with SARS-CoV-2 WT, Delta, or Omicron BA.1 at 0.1 MOI and treated with SB 204990 or Bempedoic acid at a titration of different concentration (100, 50, 25, 12.5, 6.25, 3.125, or 1.5625 µM) or were treated with DMSO. Supernatants were harvested at 24 hours post-infection. The IC₅₀ of SB 204990 and Bempedoic acid for SARS-CoV-2 WT, Delta, or Omicron BA.1 were calculated. (C and D) The CC₅₀ of SB 204990 and Bempedoic acid in Caco2 cells were determined without virus infection at day 1. (E and F) Caco2 cells were transfected with ACLY or scrambled siRNA and infected with WT SARS-CoV-2 at 0.1 MOI. (E) The knockdown efficiency of ACLY is evaluated with qPCR. (F) Supernatants from SARS-CoV-2-infected cells treated with ACLY or scrambled siRNA were harvested at 24 hours post-infection. Virus replication was quantified with qPCR analysis. The IC₅₀ and CC₅₀ values in (B and D) were calculated with GraphPad Prism 7. Data represented mean and standard deviation from three independent experiments. Statistical differences were considered significant when P < 0.05. **P < 0.01.

Figure 7

SB 204990 suppressed WT and Omicron BA.1 SARS-CoV-2 replications in a golden Syrian hamster model. Hamsters were intranasally inoculated with 3×10³ PFU per hamster of SARS-CoV-2 WT or Omicron BA.1. At 6 hours post-infection each hamster was treated intraperitoneally with 10mg/kg of SB 204990 or 5% DMSO at a final volume of 1000 µL PBS. The infected hamsters were subsequently treated with SB 204990 or DMSO at 1, 2, and 3 days post infection for a total of 2 or 4 doses. The infected hamsters were sacrificed on day 2 (Omicron-infected hamsters) or day 4 post-infection (SARS-CoV-2 WT-infected) for virological and histological assessments. (A) Schematic illustration of the in vivo study. (B) Viral RdRp gene copies in hamster lung tissues that were infected by SARS-CoV-2 WT were quantified by qRT-PCR. (C) Viral RdRp gene copies in hamster lung tissues that were infected by Omicron BA.1 were quantified by qRT-PCR. (D) Infectious titer of SARS-CoV-2 WT in hamster lung tissues were quantified by plaque assays. (E) Viral antigen in hamster lung tissues from SARS-CoV-2 WT-infected hamsters were detected with an in-house anti-SARS-CoV-2 nucleocapsid protein antibody. Cell nuclei were identified with DAPI stain. Data represented mean and standard deviation from two independent experiments. Statistical differences were considered significant when P < 0.05. *P < 0.05 and ***P < 0.001. Scale bar in (E) represented 100µm.