Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 is a key molecule potentially linking COVID-19 to associated metabolic defects (A) Volcano plot of differentially expressed genes after in vitro infection of SARS-CoV-2 (MOI = 0.005) in HUVECs for 24 h. (B) DisGeNET pathway enrichment of differentially expressed genes after infection (combined 685 upregulated and 774 downregulated genes from A). (C–E) Bioinformatics analysis of the mutual target disease-associated genes of diabetes mellitus (DM), hypertension (HTN), diabetic nephropathy (DN), and atherosclerosis (AS) that were queried from DisGeNET and Open Target database. The overlapping 48 shared genes from DisGeNET (C), the 72 shared genes from Open Target (D), and 20 genes identified on both DisGeNET (left) and Open Target database (right) as mutual target disease-associated genes of four diseases (E) were shown. (F) The heatmap to represent the alterations of the above 20 genes upon SARS-CoV-2 infection. Data were shown as log 2 fold change. The cross mark represents the undetectable gene from the transcriptomic study. (G) ELISA quantification of plasma angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) in COVID-19 patients, shown in Ang II against Ang-(1–7) ratio. HC, healthy control; I, infection phase; R, recovery phase. Health control group, n = 10, mean age ± SD = 42.30 ± 7.35, male/female = 5/5; non-severe group, n = 18, mean age ± SD = 51.61 ± 10.13, male/female = 9/9; severe group, n = 12, mean age ± SD = 57.75 ± 14.55, male/female = 6/6. (H and I) HUVECs were infected with SARS-CoV-2 (MOI = 0.005) for 24 h and were subjected to real-time PCR of ACE2 (H) and immunoblotting (I, blot shown on the left, quantification on the right, n = 4). (J) HUVECs were infected with SARS-CoV-2 (MOI = 0.005) for 48 h, and then the medium was removed and 100 nM Ang II in 1 mL HEPES solution was applied for the cells. After 1 h treatment in the incubator at 37°C, the supernatant containing Ang II and cleaved Ang-(1–7) was examined (n = 6). (K and L) Sixteen-week-old human ACE2 transgenic mice were intranasally challenged with 4 × 10 4 FFU SARS-CoV-2 after 10-week high-fat-diet treatment. After 7 days post-infection, all mice were fasted for 6 h and sacrificed. Livers (K) and kidneys (L) were subjected to real-time PCR (n = 3–4). (M–O) HUVECs were treated with a “cocktail” of different inflammatory factors (IFs), namely the combination of 10 or 50 ng/mL of TNF-α, IL-4, IL-6, and IFN-γ or 1 μM DX600 for 48 h and were subjected to real-time PCR of ACE2 (M) (n = 6), immunoblotting (N, blot shown on the left, quantification on the right, n = 4), and enzymatic activity assay (O, data were shown as the area under the kinetic activity curves, n = 6). DX600 was used as a negative control. Error bars represent SEM; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. HUVECs, human umbilical vein endothelial cells; AU, arbitrary unit; CoV-2, SARS-CoV-2. See also Figure S1 .

Article Snippet: The SPR assays were performed to analyze the interactions between the compounds and ACE2 protein (Sino Biological, China) by using a Biacore T100 machine with Sensor Chip CM7 (GE Healthcare, USA) at 25°C.

Techniques: In Vitro, Infection, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Western Blot, Transgenic Assay, Enzyme Activity Assay, Activity Assay, Negative Control

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 plays an important role in maintaining metabolic homeostasis (A) HUVECs were infected with 10 nM control siRNA (Con) or ACE2 siRNA (siACE2) for 24 h and subjected to real-time PCR (n = 6). (B and C) Eight-week-old male ob/ob mice were treated with intravenous injection of AAV9-CAG-hACE2-EGFP (ob/ob-ACE2) and corresponding control virus (ob/ob-Con) and their wild-type littermates with control virus (WT-Con). Glucose tolerance testing (GTT) was performed at 10 weeks (B), and insulin tolerance testing (ITT) was performed at 11 weeks (C) (n = 6). The significance of ob/ob-Con versus WT-Con was shown as ∗ ; ob/ob-ACE2 versus ob/ob-Con as #. (D–F) At 12 weeks, all mice were fasted for 6 h and sacrificed. Quantification of fasting blood glucose (D), plasma insulin (E), and homeostatic model assessment of insulin resistance (HOMA-IR) (F) were shown (n = 5). (G and H) Plasma TNF-α (G) and IL-6 (H) were measured (n = 4). (I and J) Livers were subjected to oil red O (ORO) staining (I, images shown on the left, quantification on the right; n = 4) and real-time PCR (n = 6) (J). (K) Plasma KIM-1 (n = 4). (L and M) Kidneys were subjected to periodic acid-Schiff (PAS) staining (L, images shown on the left, quantification on the right; n = 8) and real-time PCR (M) (n = 5). (N) Aortas were subjected to real-time PCR (n = 5). (O) Hearts were subjected to echocardiography (representative M-mode echocardiography images shown on the top, quantification of LVEF (%) and FS (%) on the bottom; n = 8). Error bars represent SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; # p < 0.05, ## p < 0.01, and ### p < 0.001. AUC, area under the curve; KIM-1, kidney injury molecule-1; LVEF, left ventricular ejection fraction; FS, fractional shortening. See also Figure S2 .

Article Snippet: The SPR assays were performed to analyze the interactions between the compounds and ACE2 protein (Sino Biological, China) by using a Biacore T100 machine with Sensor Chip CM7 (GE Healthcare, USA) at 25°C.

Techniques: Infection, Real-time Polymerase Chain Reaction, Injection, Staining

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib, harpagoside, and methazolamide are identified as ACE2 activators (A) Flow chart depicting the process of in silico identification of potential ACE2 activators with Tianhe-2 supercomputer virtual screening and CMAP bioinformatic analysis (top) and detailed information of the overlapped compounds (bottom, # and ∗ indicated different overlapping strategy). (B) The docking site (gray) of ACE2 protein (PDB: 1R42 ). (C) Schematic of the in vitro experimental workflow to identify the final three compounds. (D–F) Real-time PCR of gene expression in HUVECs. Log 10 fold change was calculated based on treated samples against control samples. HUVECs were treated with 15 compounds for 16 h (D), inflammatory factors for 32 h (50 ng/mL TNF-α, IL-4, IL-6, and IFN-γ) following eight compounds for 16 h (E), or 10 nM control siRNA (siCon) and ACE2 siRNA (siACE2) for 8 h following six compounds for 16 h (F) (n = 3). The selected compounds based on the scoring system were highlighted in red and applied for the following screening. (G) The low (L), medium (M), and high (H) concentrations of 15 compounds applied in the above three experiments. See also Figure S3 .

Article Snippet: The SPR assays were performed to analyze the interactions between the compounds and ACE2 protein (Sino Biological, China) by using a Biacore T100 machine with Sensor Chip CM7 (GE Healthcare, USA) at 25°C.

Techniques: In Silico, In Vitro, Real-time Polymerase Chain Reaction, Expressing

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib, harpagoside, and methazolamide directly bind to and activate ACE2 (A–E) The direct binding was illustrated by surface plasmon resonance (SPR) assay of purified ACE2 protein with imatinib (Ima) (A), harpagoside (Har) (B), methazolamide (Met) (C), diminazene aceturate (DIZE) (D), or xanthone (Xan) (E). Xanthone was used as a negative control. The Kd values (equilibrium dissociation constant) of compounds binding to ACE2 protein were calculated based on the fitted curves. (F and G) The detailed binding between ACE2 protein (gray) and imatinib (F, blue) and methazolamide (G, blue) was simulated with molecular dynamics simulation by GROMACS. Residues involved in binding were marked in green. (H) The HUVECs were treated with 25 μM imatinib, 100 μM harpagoside, 100 μM methazolamide, or 1 μM DX600 for 16 h, then subjected to ACE2 enzymatic activity assay. Data shown as the area under the kinetic activity curves. The ACE2 enzymatic inhibitor DX600 was used as a negative control (n = 3). (I–L) The HUVEC lysates were prepared and treated with 1.5 × 10 −6 M imatinib, 1.5 × 10 −6 M harpagoside, 1.5 × 10 −6 M methazolamide, or 1.0 × 10 −6 M DX600 for 30 min, then subjected to ACE2 enzymatic activity assay in a kinetics model. The kinetic activity curves were shown (I) (n = 3). The effects of imatinib (J) at concentrations of 1.5 × 10 −7 M (Ima-1) and 1.5 × 10 −6 M (Ima-2), harpagoside (K) at concentrations of 0.5 × 10 −7 M (Har-1) and 1.5 × 10 −6 M (Har-2), or methazolamide (L) at concentrations of 1.5 × 10 −7 M (Met-1) and 1.5 × 10 −6 M (Met-2) on the enzymatic activity of ACE2 in HUVEC lysates were shown as the area under the kinetic activity curves (n = 3). Error bars represent SEM, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figure S4 .

Article Snippet: The SPR assays were performed to analyze the interactions between the compounds and ACE2 protein (Sino Biological, China) by using a Biacore T100 machine with Sensor Chip CM7 (GE Healthcare, USA) at 25°C.

Techniques: Binding Assay, SPR Assay, Purification, Negative Control, Enzyme Activity Assay, Activity Assay

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: Imatinib and methazolamide ameliorate metabolic defects in insulin-resistant mice via ACE2 (A–I) Twenty-eight-week-old male mice with 23-week high-fat-diet treatment (DIO) and controlled lean mice (Lean) were treated with vehicle, 250 mg/kg imatinib (DIO + Ima), or 100 mg/kg methazolamide (DIO + Met) through gavage once each day for 4 weeks. (A and B) Glucose tolerance testing (GTT) was performed at 30 weeks (A); insulin tolerance testing (ITT) was performed at 31 weeks (B) (n = 6). The significance of lean versus DIO was shown as ∗ , DIO versus DIO + Ima as #, and DIO versus DIO + Met as $. ∗,#,$ p < 0.05, ∗∗,##,$$ p < 0.01, and ∗∗∗,###,$$$ p < 0.001. At 32 weeks, all mice were fasted for 6 h and sacrificed. (C–F) Fasting blood glucose (C), plasma insulin (D), homeostatic model assessment of insulin resistance (HOMA-IR) (E), and plasma TNF-α (F) (n = 6). (G and H) Livers were subjected to oil red O (ORO) staining (G, images shown on the left, quantification on the right) and real-time PCR (H) (n = 6). (I) Aortas were subjected to real-time PCR (n = 6). (J–P) For kidney conditional knockdown of ACE2 (ACE2 C-kd), 26-week-old male mice with 21-week high-fat-diet treatment (DIO) and controlled lean mice (Lean) were treated with transparenchymal renal pelvis injection of AAV9-CAG-mACE2shRNA-EGFP or control virus. After 2-week recovery, mice were given vehicle, 250 mg/kg imatinib (DIO + Ima and DIO + Ima + ACE2 C-kd), or 100 mg/kg methazolamide (DIO + Met and DIO + Met + ACE2 C-kd) through gavage once each day for 4 weeks. At 32 weeks, all mice were fasted for 6 h and sacrificed. (J and K) Plasma KIM-1 (J) and CREA (K) (n = 6). (L and M) Kidneys were subjected to periodic acid-Schiff (PAS) staining (L, images and quantification were shown; n = 5) and real-time PCR (M) (n = 6). (N) Quantification of plasma ratio of Ang II against Ang-(1–7) (n = 5). (O and P) The lysates of kidneys (O) and aortas (P) were subjected to ACE2 enzymatic activity assay, and data were shown as the area under the kinetic activity curves (n = 3). (Q–U) For A779 (MasR inhibitor) intervention, 28-week-old male mice with 23-week high-fat-diet treatment were given vehicle (DIO and DIO + A779), 250 mg/kg imatinib (DIO + Ima and DIO + Ima + A779), or 100 mg/kg methazolamide (DIO + Met and DIO + Met + A779) with or without 3 mg/kg A779 once each day for 4 weeks. (Q and R) Glucose tolerance testing (GTT) was performed at 30 weeks (Q), and insulin tolerance testing (ITT) was performed at 31 weeks (R) (n = 6). The significance of DIO versus DIO + Ima was shown as ∗ , DIO versus DIO + Met as #, DIO + Ima versus DIO + Ima + A779 as $ and DIO + Met versus DIO + Met + A779 as &. ∗,#,$,& p < 0.05, ∗∗,##,$$,&& p < 0.01, and ∗∗∗,###,$$$,&&& p < 0.001. At 32 weeks, all mice were fasted for 6 h and sacrificed. (S–U) Livers (S), kidneys (T), and aortas (U) were subjected to real-time PCR. Error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CREA, creatinine. See also Figure S5 .

Article Snippet: The SPR assays were performed to analyze the interactions between the compounds and ACE2 protein (Sino Biological, China) by using a Biacore T100 machine with Sensor Chip CM7 (GE Healthcare, USA) at 25°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Injection, Enzyme Activity Assay, Activity Assay

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet: ACE2 enzymatic activators improve metabolic defects and inhibit virus entry upon SARS-CoV-2 infection (A–Q) Twelve-week-old human ACE2 transgenic mice were given vehicle (Mock, CoV-2), 250 mg/kg imatinib (CoV-2 + Ima), and 100 mg/kg methazolamide (CoV-2 + Met) through gavage once each day for 4 weeks after 6-week high-fat-diet treatment and were intranasally challenged with 4 × 10 4 FFU SARS-CoV-2. After 7 days of infection, all mice were fasted for 6 h and sacrificed. (A–H) Quantification of the ratio of Ang II against Ang-(1–7) in plasma (A), fasting blood glucose (B), plasma insulin (C), homeostatic model assessment of insulin resistance (HOMA-IR) (D), plasma triglyceride (E), plasma total cholesterol (F), plasma TNF-α (G), and plasma IL-6 (H) (n = 4). (I–K) Livers were subjected to lipid assay (I and J) and real-time PCR (K) (n = 3–4). (L) Plasma KIM-1 (n = 4–5). (M and N) Kidneys were subjected to periodic acid-Schiff (PAS) staining (M, images and quantification were shown; n = 4) and real-time PCR (N) (n = 4). (O–Q) Lungs were subjected to H&E staining (O, images and quantification were shown; n = 4), real-time PCR (P) (n = 4), and immunoblotting of viral nucleocapsid protein (NP) (Q, blot shown on the left, quantification on the right; n = 4). (R) Vero E6 cells were pretreated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 6 h, followed by SARS-CoV-2 (MOI = 0.005) infection for 42 h; the supernatant was subjected to real-time PCR (n = 4). (S) HEK293T cells expressing hACE2 were pretreated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 6 h, followed by pseudovirion treatment for 66 h, and were observed with fluorescence microscopy (images shown on the left, quantification on the right; n = 5). (T) The HEK293T cells overexpressing spike-FLAG and ACE2-HA were treated with 25 μM imatinib, 100 μM harpagoside, or 100 μM methazolamide for 48 h. Precipitated proteins were subjected to immunoblotting (blot shown on the left, quantification on the right; n = 4). Error bars represent SEM. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. See also Figure S6 .

Article Snippet: The SPR assays were performed to analyze the interactions between the compounds and ACE2 protein (Sino Biological, China) by using a Biacore T100 machine with Sensor Chip CM7 (GE Healthcare, USA) at 25°C.

Techniques: Infection, Transgenic Assay, Real-time Polymerase Chain Reaction, Staining, Western Blot, Expressing, Fluorescence, Microscopy

Journal: Cell Metabolism

Article Title: Imatinib and methazolamide ameliorate COVID-19-induced metabolic complications via elevating ACE2 enzymatic activity and inhibiting viral entry

doi: 10.1016/j.cmet.2022.01.008

Figure Lengend Snippet:

Article Snippet: The SPR assays were performed to analyze the interactions between the compounds and ACE2 protein (Sino Biological, China) by using a Biacore T100 machine with Sensor Chip CM7 (GE Healthcare, USA) at 25°C.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, RNA Extraction, SYBR Green Assay, Activity Assay, Cholesterol Assay, Software

Journal: Molecular Medicine

Article Title: SARS-CoV-2 and interferon blockade

doi: 10.1186/s10020-020-00231-w

Figure Lengend Snippet: ACE2 is a key enzyme in the RAS, catalyzing the metabolism of Ang II to Ang(1-7) and Ang I to Ang(1-9). ACE2 also mediates degradation of ACE-catalyzed breakdown products, Des-arg9-Bk (B1R agonist) and Lys-des-arg9-Bk. The net result of ACE2 in these two systems is to counterbalance ACE/Ang II/AT1R and Bradykinin/Des-arg9-Bk/B1R pathways. Through its cellular binding and entry mechanisms, SARS-CoV-2 is proposed to result in a reduction of ACE2, leading to elevations in Ang I and II, and leading to AT1R stimulation, and Des-arg9-Bk leading to B1R stimulation thus exacerbating inflammation, vascular leakage, and pro-fibrotic events. Potential therapeutics include those targeted to angiotensin and bradykinin system related peptides, in addition to peptides targeting the ACE2-viral spike (S) protein interaction

Article Snippet: Potential therapeutics include those targeted to angiotensin and bradykinin system related peptides, in addition to peptides targeting the ACE2-viral spike (S) protein interaction Several candidate therapeutics focus on the virus, targeting viral replication (remdesivir), viral entry (Arbidol, APN01, convalescent plasma, monoclonal antibodies (REGN-COV2), camostat mesylate), or critical viral proteins (protease inhibitors).

Techniques: Binding Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis

doi: 10.1038/sigtrans.2017.22

Figure Lengend Snippet: AT2R activation increases UCP1 and CITED1 expressions in white adipocytes. ( a and b ) Mouse white adipose cells (day 4) were transfected with control siRNAs, ACE2-siRNA, AT1R-siRNA or AT2R-siRNAs for 2–3 days, before treated without (−) (control) or with (+) AngII, ZD7155 or PD123319 for another 4 days. ( c ) Mouse white adipocytes were treated without (−) (control) or with (+) 100 nM M024/C21, 100 nM CGP42112 or PD123319 for 4 days. The representative immunoblots of protein expressions (UCP1, CITED1, ACE2, AT1R, AT2R, PPARγ, PRDM16, aP2 and actin), and the statistics (mean±s.e.m., n =4; normalized to actin densities) are shown accordingly. * P <0.05, ** P <0.01, *** P <0.001 versus control; # P <0.05 and ## P <0.01 between indicated pairs.

Article Snippet: For gene silencing of ACE2 ( ACE2 ), AT1R ( AGTR1 ) or AT2R ( AGTR2 ), adipose cells (day 4 after induction of differentiation) were transfected with a mouse ACE2 siRNA (sc-41401), AT1R siRNA (sc-29751), AT2R siRNA (sc-29753) or control siRNA (sc-37007) (Santa Cruz Biotechnology) as previously described., Specifically, adipose cells were incubated with Opti-MEM containing a complex of Lipofectamine-RNAiMAX transfection reagent (0.5% (v/v), Life Technologies, ThermoFisher Scientific) with siRNAs (25 n m ) for 4 h, followed by the addition of grown medium and incubated for another 2–3 days.

Techniques: Activation Assay, Transfection, Control, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Angiotensin type 2 receptor activation promotes browning of white adipose tissue and brown adipogenesis

doi: 10.1038/sigtrans.2017.22

Figure Lengend Snippet: Signaling pathways underlying the AngII–AT2R-induced browning of white adipocytes. ( a – c ) Time course of the phosphorylation and total protein levels of ERK1/2, Akt and AMPK in mouse white adipocytes, without (−) (control) or with (+) exposure to AngII and ZD7155. Top panels, representative immunoblots; bottom panels, statistics (mean±s.e.m., n =3) of the optical density ratio between pERK and ERK, pAkt and Akt, or pAMPK and AMPK. ( d and e ) Mouse white adipose cells (day 4) were transfected with control siRNA, ERK1/2-siRNAs, Akt-siRNA or AMPK-siRNAs for 2–3 days before treated without (−) (control) or with (+) AngII and ZD7155 for 4 days. The representative immunoblots and the statistics (mean±s.e.m., n =4; normalized to actin densities) are shown accordingly. * P <0.05, ** P <0.01, *** P <0.001 versus control; # P <0.05 and ## P <0.01 between indicated pairs.

Article Snippet: For gene silencing of ACE2 ( ACE2 ), AT1R ( AGTR1 ) or AT2R ( AGTR2 ), adipose cells (day 4 after induction of differentiation) were transfected with a mouse ACE2 siRNA (sc-41401), AT1R siRNA (sc-29751), AT2R siRNA (sc-29753) or control siRNA (sc-37007) (Santa Cruz Biotechnology) as previously described., Specifically, adipose cells were incubated with Opti-MEM containing a complex of Lipofectamine-RNAiMAX transfection reagent (0.5% (v/v), Life Technologies, ThermoFisher Scientific) with siRNAs (25 n m ) for 4 h, followed by the addition of grown medium and incubated for another 2–3 days.

Techniques: Protein-Protein interactions, Phospho-proteomics, Control, Western Blot, Transfection