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gene exp gpr4 hs00270999 s1  (Thermo Fisher)
86
Thermo Fisher gene exp gpr4 hs00270999 s1
Treatment with <t>GPR4</t> antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.
Gene Exp Gpr4 Hs00270999 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gpr4  (Sino Biological)
93
Sino Biological gpr4
Treatment with <t>GPR4</t> antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.
Gpr4, supplied by Sino Biological, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gpr4/product/Sino Biological
Average 93 stars, based on 1 article reviews
gpr4 - by Bioz Stars, 2026-04
93/100 stars
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gene exp gpr4 mm01322176 s1  (Thermo Fisher)
94
Thermo Fisher gene exp gpr4 mm01322176 s1
Treatment with <t>GPR4</t> antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.
Gene Exp Gpr4 Mm01322176 S1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 94 stars, based on 1 article reviews
gene exp gpr4 mm01322176 s1 - by Bioz Stars, 2026-04
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sirnas targeting human gpr4 (sigpr4)  (Thermo Fisher)
90
Thermo Fisher sirnas targeting human gpr4 (sigpr4)
Treatment with <t>GPR4</t> antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.
Sirnas Targeting Human Gpr4 (Sigpr4), supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/sirnas targeting human gpr4 (sigpr4)/product/Thermo Fisher
Average 90 stars, based on 1 article reviews
sirnas targeting human gpr4 (sigpr4) - by Bioz Stars, 2026-04
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gpr4 inhibitor ne 52‐qq57  (MedKoo Inc)
90
MedKoo Inc gpr4 inhibitor ne 52‐qq57
HKSA‐activated GPR68 mediates endothelial barrier disruption. (A) HPAECs were exposed to HKSA (5 × 10 8 particles/mL) for indicated time periods followed by qRT‐PCR analysis of <t>GPR4,</t> GPR65, and GPR68 mRNA expression levels. * p < .05, vs. GPR4 and GPR65, n = 3. (B) Cells were transfected with GPR68 PRESTO‐Tango plasmids for 24 h and stimulated with HKSA (5 × 10 8 particles/mL) or 10 μM Ogerin. GPR68 activity was determined by measuring luminescence signal as described in Methods, and values are presented after normalizing to the unstimulated control group. GPR68: * p < .05, vs. control, n = 4. (C) Cells were treated with 10 μM Ogerin alone or in combination with HKSA, followed by TER measurements. The bar graph shows permeability changes at 15 h of cell stimulation. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (D) HPAECs monolayers were transfected with non‐specific control siRNA (nsRNA) or GPR68‐specific siRNA (siGPR68) for 72 h followed by stimulation with HKSA and TER monitoring. Bar graph: Pooled data at time point 15 h * p < .05, vs. corresponding controls, n = 4. (E) HPAEC were transfected with plasmids encoding wild type (WT) or E336X GPR68 mutant (Mut). After 24 h of incubation, TER measurements of control and HKSA‐stimulated groups were performed over 25 h. Bar graph: Pooled data at 15 h of cell treatment. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 5. Shown are normalized resistance values.
Gpr4 Inhibitor Ne 52‐Qq57, supplied by MedKoo Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gpr4 inhibitor ne 52‐qq57/product/MedKoo Inc
Average 90 stars, based on 1 article reviews
gpr4 inhibitor ne 52‐qq57 - by Bioz Stars, 2026-04
90/100 stars
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gpr4 inhibitor ne 52-qq57  (MedKoo Inc)
90
MedKoo Inc gpr4 inhibitor ne 52-qq57
HKSA‐activated GPR68 mediates endothelial barrier disruption. (A) HPAECs were exposed to HKSA (5 × 10 8 particles/mL) for indicated time periods followed by qRT‐PCR analysis of <t>GPR4,</t> GPR65, and GPR68 mRNA expression levels. * p < .05, vs. GPR4 and GPR65, n = 3. (B) Cells were transfected with GPR68 PRESTO‐Tango plasmids for 24 h and stimulated with HKSA (5 × 10 8 particles/mL) or 10 μM Ogerin. GPR68 activity was determined by measuring luminescence signal as described in Methods, and values are presented after normalizing to the unstimulated control group. GPR68: * p < .05, vs. control, n = 4. (C) Cells were treated with 10 μM Ogerin alone or in combination with HKSA, followed by TER measurements. The bar graph shows permeability changes at 15 h of cell stimulation. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (D) HPAECs monolayers were transfected with non‐specific control siRNA (nsRNA) or GPR68‐specific siRNA (siGPR68) for 72 h followed by stimulation with HKSA and TER monitoring. Bar graph: Pooled data at time point 15 h * p < .05, vs. corresponding controls, n = 4. (E) HPAEC were transfected with plasmids encoding wild type (WT) or E336X GPR68 mutant (Mut). After 24 h of incubation, TER measurements of control and HKSA‐stimulated groups were performed over 25 h. Bar graph: Pooled data at 15 h of cell treatment. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 5. Shown are normalized resistance values.
Gpr4 Inhibitor Ne 52 Qq57, supplied by MedKoo Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gpr4 inhibitor ne 52-qq57/product/MedKoo Inc
Average 90 stars, based on 1 article reviews
gpr4 inhibitor ne 52-qq57 - by Bioz Stars, 2026-04
90/100 stars
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gpr4 inhibitor ne52-qq57  (MedKoo Inc)
90
MedKoo Inc gpr4 inhibitor ne52-qq57
HKSA‐activated GPR68 mediates endothelial barrier disruption. (A) HPAECs were exposed to HKSA (5 × 10 8 particles/mL) for indicated time periods followed by qRT‐PCR analysis of <t>GPR4,</t> GPR65, and GPR68 mRNA expression levels. * p < .05, vs. GPR4 and GPR65, n = 3. (B) Cells were transfected with GPR68 PRESTO‐Tango plasmids for 24 h and stimulated with HKSA (5 × 10 8 particles/mL) or 10 μM Ogerin. GPR68 activity was determined by measuring luminescence signal as described in Methods, and values are presented after normalizing to the unstimulated control group. GPR68: * p < .05, vs. control, n = 4. (C) Cells were treated with 10 μM Ogerin alone or in combination with HKSA, followed by TER measurements. The bar graph shows permeability changes at 15 h of cell stimulation. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (D) HPAECs monolayers were transfected with non‐specific control siRNA (nsRNA) or GPR68‐specific siRNA (siGPR68) for 72 h followed by stimulation with HKSA and TER monitoring. Bar graph: Pooled data at time point 15 h * p < .05, vs. corresponding controls, n = 4. (E) HPAEC were transfected with plasmids encoding wild type (WT) or E336X GPR68 mutant (Mut). After 24 h of incubation, TER measurements of control and HKSA‐stimulated groups were performed over 25 h. Bar graph: Pooled data at 15 h of cell treatment. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 5. Shown are normalized resistance values.
Gpr4 Inhibitor Ne52 Qq57, supplied by MedKoo Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gpr4 inhibitor ne52-qq57/product/MedKoo Inc
Average 90 stars, based on 1 article reviews
gpr4 inhibitor ne52-qq57 - by Bioz Stars, 2026-04
90/100 stars
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gpr4 antagonist ne-52-qq57  (Novartis)
90
Novartis gpr4 antagonist ne-52-qq57
Mice were treated with <t>GPR4</t> antagonist <t>NE-52-QQ57</t> or vehicle control for up to 6 days starting from 4 dpi. ( A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N=12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, * p < 0.05. ( B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. ( C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N=6 for PBS no virus inoculation; N=12 for vehicle, N=10 for GPR4 antagonist). Error bars indicate means ± SEM. ** p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.
Gpr4 Antagonist Ne 52 Qq57, supplied by Novartis, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/gpr4 antagonist ne-52-qq57/product/Novartis
Average 90 stars, based on 1 article reviews
gpr4 antagonist ne-52-qq57 - by Bioz Stars, 2026-04
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Treatment with GPR4 antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Treatment with GPR4 antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Control, Transgenic Assay, Quantitative RT-PCR, Expressing, Two Tailed Test, MANN-WHITNEY, Virus, Staining, Histopathology

GPR4 antagonist treatment reduces cytokine and chemokine levels in K18-hACE2 mice infected with SARS-CoV-2. (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N = 12 for vehicle, N = 10 for GPR4 antagonist). *p < 0.05. Gene expression in the lung of control mice (PBS) without SARS-CoV-2 infection was set as 1. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces cytokine and chemokine levels in K18-hACE2 mice infected with SARS-CoV-2. (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N = 12 for vehicle, N = 10 for GPR4 antagonist). *p < 0.05. Gene expression in the lung of control mice (PBS) without SARS-CoV-2 infection was set as 1. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Gene Expression, Quantitative RT-PCR, Control, Luminex, Multiplex Assay, One-tailed Test, MANN-WHITNEY

SARS-CoV-2 viral load in the lungs and brains from K18-hACE2 mice that received either GPR4 antagonist or vehicle. (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N = 12 for vehicle, N = 10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. *p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N = 12 for vehicle, N = 12 for GPR4 antagonist). In the vehicle group, 3 out of 12 mouse brains (3/12) are negative for SARS-CoV-2 and 9 out of 12 are positive. In comparison, 8 out of 12 are negative and 4 out of 12 mouse brains are positive for SARS-CoV-2 in the GPR4 antagonist group. Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, *p < 0.05.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: SARS-CoV-2 viral load in the lungs and brains from K18-hACE2 mice that received either GPR4 antagonist or vehicle. (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N = 12 for vehicle, N = 10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. *p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N = 12 for vehicle, N = 12 for GPR4 antagonist). In the vehicle group, 3 out of 12 mouse brains (3/12) are negative for SARS-CoV-2 and 9 out of 12 are positive. In comparison, 8 out of 12 are negative and 4 out of 12 mouse brains are positive for SARS-CoV-2 in the GPR4 antagonist group. Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, *p < 0.05.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Quantitative RT-PCR, Two Tailed Test, Infection, Virus, Staining, Microscopy, Comparison

GPR4 antagonist treatment reduces CD4 + and CD8 + immune cell clusters in the brains of SARS-CoV-2-infected K18-hACE2 mice. (A) A representative image of a CD4 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, **p < 0.01. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, *p < 0.05. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces CD4 + and CD8 + immune cell clusters in the brains of SARS-CoV-2-infected K18-hACE2 mice. (A) A representative image of a CD4 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, **p < 0.01. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, *p < 0.05. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Microscopy, Two Tailed Test

Anti-SARS-CoV-2 effects of GPR4 antagonist in vitro . (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N = 3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT-qPCR targeting the nucleocapsid gene. N = 3 samples. *p < 0.05, **p < 0.01, ****p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N = 3 samples. **p < 0.01, ***p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Anti-SARS-CoV-2 effects of GPR4 antagonist in vitro . (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N = 3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT-qPCR targeting the nucleocapsid gene. N = 3 samples. *p < 0.05, **p < 0.01, ****p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N = 3 samples. **p < 0.01, ***p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: In Vitro, Incubation, Infection, Isolation, Quantitative RT-PCR, Plaque Assay

Treatment with GPR4 antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Treatment with GPR4 antagonist NE-52-QQ57 improves survival of SARS-CoV-2-infected K18-hACE2 mice. Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. (A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N = 12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, *p < 0.05. (B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. (C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N = 6 for PBS no virus inoculation; N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate means ± SEM. **p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Control, Transgenic Assay, Quantitative RT-PCR, Expressing, Two Tailed Test, MANN-WHITNEY, Virus, Staining, Histopathology

GPR4 antagonist treatment reduces cytokine and chemokine levels in K18-hACE2 mice infected with SARS-CoV-2. (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N = 12 for vehicle, N = 10 for GPR4 antagonist). *p < 0.05. Gene expression in the lung of control mice (PBS) without SARS-CoV-2 infection was set as 1. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces cytokine and chemokine levels in K18-hACE2 mice infected with SARS-CoV-2. (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N = 12 for vehicle, N = 10 for GPR4 antagonist). *p < 0.05. Gene expression in the lung of control mice (PBS) without SARS-CoV-2 infection was set as 1. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N = 12 for vehicle, N = 10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Gene Expression, Quantitative RT-PCR, Control, Luminex, Multiplex Assay, One-tailed Test, MANN-WHITNEY

SARS-CoV-2 viral load in the lungs and brains from K18-hACE2 mice that received either GPR4 antagonist or vehicle. (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N = 12 for vehicle, N = 10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. *p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N = 12 for vehicle, N = 12 for GPR4 antagonist). In the vehicle group, 3 out of 12 mouse brains (3/12) are negative for SARS-CoV-2 and 9 out of 12 are positive. In comparison, 8 out of 12 are negative and 4 out of 12 mouse brains are positive for SARS-CoV-2 in the GPR4 antagonist group. Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, *p < 0.05.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: SARS-CoV-2 viral load in the lungs and brains from K18-hACE2 mice that received either GPR4 antagonist or vehicle. (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N = 12 for vehicle, N = 10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. *p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N = 12 for vehicle, N = 12 for GPR4 antagonist). In the vehicle group, 3 out of 12 mouse brains (3/12) are negative for SARS-CoV-2 and 9 out of 12 are positive. In comparison, 8 out of 12 are negative and 4 out of 12 mouse brains are positive for SARS-CoV-2 in the GPR4 antagonist group. Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, *p < 0.05.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Quantitative RT-PCR, Two Tailed Test, Infection, Virus, Staining, Microscopy, Comparison

GPR4 antagonist treatment reduces CD4 + and CD8 + immune cell clusters in the brains of SARS-CoV-2-infected K18-hACE2 mice. (A) A representative image of a CD4 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, **p < 0.01. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, *p < 0.05. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: GPR4 antagonist treatment reduces CD4 + and CD8 + immune cell clusters in the brains of SARS-CoV-2-infected K18-hACE2 mice. (A) A representative image of a CD4 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, **p < 0.01. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster (indicated by the arrow) in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, *p < 0.05. Error bars represent mean ± SEM. N = 12 for the vehicle group, and N = 12 for the GPR4 antagonist group.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: Infection, Microscopy, Two Tailed Test

Anti-SARS-CoV-2 effects of GPR4 antagonist in vitro . (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N = 3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT-qPCR targeting the nucleocapsid gene. N = 3 samples. *p < 0.05, **p < 0.01, ****p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N = 3 samples. **p < 0.01, ***p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Journal: Frontiers in Pharmacology

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.3389/fphar.2025.1549296

Figure Lengend Snippet: Anti-SARS-CoV-2 effects of GPR4 antagonist in vitro . (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N = 3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT-qPCR targeting the nucleocapsid gene. N = 3 samples. *p < 0.05, **p < 0.01, ****p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N = 3 samples. **p < 0.01, ***p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Article Snippet: Commercial primers/probe sets specific for mouse Gpr4 (Mm01322176_s1), Il-1β (Mm00434228_m1), Il-6 (Mm00446190_m1), Il-10 (Mm01288386_m1), Il-18 (Mm00434226_m1), Tnf-α (Mm00443258_m1), Atf3 (Mm00476033_m1), Cox2/Ptgs2 (Mm00478374_m1), Cxcl2 (Mm00436450_m1), E-selectin (Mm00441278_m1), Icam1 (Mm00516023_m1), Vcam1 (Mm01320970_m1), human GPR4 (Hs00270999_s1), mouse Ace2 (Mm01159006_m1), human ACE2 (Hs01085333_m1), mouse Tmprss2 (Mm00443687_m1), and human TMPRSS2 (Hs01122322_m1) were purchased from ThermoFisher and the expressions were normalized to 18S rRNA (HS99999901_s1) levels.

Techniques: In Vitro, Incubation, Infection, Isolation, Quantitative RT-PCR, Plaque Assay

HKSA‐activated GPR68 mediates endothelial barrier disruption. (A) HPAECs were exposed to HKSA (5 × 10 8 particles/mL) for indicated time periods followed by qRT‐PCR analysis of GPR4, GPR65, and GPR68 mRNA expression levels. * p < .05, vs. GPR4 and GPR65, n = 3. (B) Cells were transfected with GPR68 PRESTO‐Tango plasmids for 24 h and stimulated with HKSA (5 × 10 8 particles/mL) or 10 μM Ogerin. GPR68 activity was determined by measuring luminescence signal as described in Methods, and values are presented after normalizing to the unstimulated control group. GPR68: * p < .05, vs. control, n = 4. (C) Cells were treated with 10 μM Ogerin alone or in combination with HKSA, followed by TER measurements. The bar graph shows permeability changes at 15 h of cell stimulation. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (D) HPAECs monolayers were transfected with non‐specific control siRNA (nsRNA) or GPR68‐specific siRNA (siGPR68) for 72 h followed by stimulation with HKSA and TER monitoring. Bar graph: Pooled data at time point 15 h * p < .05, vs. corresponding controls, n = 4. (E) HPAEC were transfected with plasmids encoding wild type (WT) or E336X GPR68 mutant (Mut). After 24 h of incubation, TER measurements of control and HKSA‐stimulated groups were performed over 25 h. Bar graph: Pooled data at 15 h of cell treatment. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 5. Shown are normalized resistance values.

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: HKSA‐activated GPR68 mediates endothelial barrier disruption. (A) HPAECs were exposed to HKSA (5 × 10 8 particles/mL) for indicated time periods followed by qRT‐PCR analysis of GPR4, GPR65, and GPR68 mRNA expression levels. * p < .05, vs. GPR4 and GPR65, n = 3. (B) Cells were transfected with GPR68 PRESTO‐Tango plasmids for 24 h and stimulated with HKSA (5 × 10 8 particles/mL) or 10 μM Ogerin. GPR68 activity was determined by measuring luminescence signal as described in Methods, and values are presented after normalizing to the unstimulated control group. GPR68: * p < .05, vs. control, n = 4. (C) Cells were treated with 10 μM Ogerin alone or in combination with HKSA, followed by TER measurements. The bar graph shows permeability changes at 15 h of cell stimulation. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (D) HPAECs monolayers were transfected with non‐specific control siRNA (nsRNA) or GPR68‐specific siRNA (siGPR68) for 72 h followed by stimulation with HKSA and TER monitoring. Bar graph: Pooled data at time point 15 h * p < .05, vs. corresponding controls, n = 4. (E) HPAEC were transfected with plasmids encoding wild type (WT) or E336X GPR68 mutant (Mut). After 24 h of incubation, TER measurements of control and HKSA‐stimulated groups were performed over 25 h. Bar graph: Pooled data at 15 h of cell treatment. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 5. Shown are normalized resistance values.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: Disruption, Quantitative RT-PCR, Expressing, Transfection, Activity Assay, Control, Permeability, Cell Stimulation, Mutagenesis, Incubation

OGM‐8345 attenuates HKSA‐induced GPR68 activation and endothelial barrier disruption. (A) HPAECs were transfected with GPR68 PRESTO‐Tango plasmids for 24 h followed by the addition of HKSA for indicated time periods with or without OGM‐8345 pretreatment (3 μM, 30 min). GPR68 activity was determined by measuring luminescence signal. The data are presented after normalizing to non‐treated controls. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (B) HPAEC were pre‐incubated with OGM‐8345 (3 μM, 30 min), and HKSA (5 × 10 8 particles/mL) was added for 3 h. qRT‐PCR was carried out to determine the mRNA expression levels of GPR68, GPR65, and GPR4. * p < .05, vs. GPR68 controls; ** p < .05, vs. GPR68 HKSA; ND p > .05 vs. GPR65 and GPR4 controls, n = 3. (C) HPAEC monolayers were treated with HKSA with or without pre‐treatment OGM‐8345 as above. Endothelial macromolecular permeability was determined by the XPerT assay. Increased FITC fluorescence reflects an increase in endothelial permeability. Bar = 20 μm. (D) HPAEC were pre‐incubated for 30 min with OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) followed by the addition of HKSA. Endothelial permeability was monitored by TER measurements. The bar graph represents normalized pooled data at 15 h of EC stimulation. * p < .05, vs. HKSA, n = 6.

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 attenuates HKSA‐induced GPR68 activation and endothelial barrier disruption. (A) HPAECs were transfected with GPR68 PRESTO‐Tango plasmids for 24 h followed by the addition of HKSA for indicated time periods with or without OGM‐8345 pretreatment (3 μM, 30 min). GPR68 activity was determined by measuring luminescence signal. The data are presented after normalizing to non‐treated controls. * p < .05, vs. control, ** p < .05, vs. HKSA, n = 4. (B) HPAEC were pre‐incubated with OGM‐8345 (3 μM, 30 min), and HKSA (5 × 10 8 particles/mL) was added for 3 h. qRT‐PCR was carried out to determine the mRNA expression levels of GPR68, GPR65, and GPR4. * p < .05, vs. GPR68 controls; ** p < .05, vs. GPR68 HKSA; ND p > .05 vs. GPR65 and GPR4 controls, n = 3. (C) HPAEC monolayers were treated with HKSA with or without pre‐treatment OGM‐8345 as above. Endothelial macromolecular permeability was determined by the XPerT assay. Increased FITC fluorescence reflects an increase in endothelial permeability. Bar = 20 μm. (D) HPAEC were pre‐incubated for 30 min with OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) followed by the addition of HKSA. Endothelial permeability was monitored by TER measurements. The bar graph represents normalized pooled data at 15 h of EC stimulation. * p < .05, vs. HKSA, n = 6.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: Activation Assay, Disruption, Transfection, Activity Assay, Control, Incubation, Quantitative RT-PCR, Expressing, Permeability, Fluorescence

OGM‐8345 attenuates HKSA and acidosis‐induced endothelial dysfunction. (A) HPAEC were pretreated with 3 μM OGM‐8345 at both normal and acidic pH (30 min) and stimulated with HKSA for 6 h. XPerT assay was performed to evaluate EC monolayer permeability for macromolecules. FITC fluorescence signal reflects an increase in endothelial permeability. Bar = 20 μm. (B) HPAEC were exposed to HKSA at indicated pH media with or without OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) pretreatment followed by qRT‐PCR analysis of mRNA expression of selected pro‐inflammatory marker genes. * p < .05, vs. corresponding controls, ** p < .05, vs. pH 6.5 + HKSA, n = 3. (C) Cells were challenged with HKSA at pH 6.5 or pH 7.4 in the presence or absence of OGM‐8345 for 6 h, followed by western blot analysis of ICAM‐1 protein levels; β‐tubulin was used as an internal loading control. Bar graph: Normalized densitometry data; * p < .05, vs. HKSA pH 7.4; ** p < .05, vs. pH 6.5 + HKSA, n = 3.

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 attenuates HKSA and acidosis‐induced endothelial dysfunction. (A) HPAEC were pretreated with 3 μM OGM‐8345 at both normal and acidic pH (30 min) and stimulated with HKSA for 6 h. XPerT assay was performed to evaluate EC monolayer permeability for macromolecules. FITC fluorescence signal reflects an increase in endothelial permeability. Bar = 20 μm. (B) HPAEC were exposed to HKSA at indicated pH media with or without OGM‐8345 (3 μM) or NE52‐QQ57 (1 μM) pretreatment followed by qRT‐PCR analysis of mRNA expression of selected pro‐inflammatory marker genes. * p < .05, vs. corresponding controls, ** p < .05, vs. pH 6.5 + HKSA, n = 3. (C) Cells were challenged with HKSA at pH 6.5 or pH 7.4 in the presence or absence of OGM‐8345 for 6 h, followed by western blot analysis of ICAM‐1 protein levels; β‐tubulin was used as an internal loading control. Bar graph: Normalized densitometry data; * p < .05, vs. HKSA pH 7.4; ** p < .05, vs. pH 6.5 + HKSA, n = 3.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: Permeability, Fluorescence, Quantitative RT-PCR, Expressing, Marker, Western Blot, Control

OGM‐8345 rescues HKSA‐induced lung injury in vivo. C57BL/6 mice were exposed to HKSA (2 × 10 8 bacterial particles/mouse, intranasal) for 18 h. (A, B) Total RNA was extracted from various tissues of control or HKSA‐challenged mice, and qRT‐PCR was used to measure mRNA expression levels of GPR4 and GPR68. * p < .05, vs. control, n = 3. (C–F) C57BL/6 mice were injected with OGM‐8345 (20 mg/kg, i.p.) immediately followed by intranasal administration of HKSA. BAL fluid was collected to analyze total PMNs (C) and protein content (D). * p < .05, vs. HKSA alone, n = 8. (E) Total RNA was extracted from lung tissues, and qRT‐PCR was carried out to determine mRNA expression of indicated pro‐inflammatory genes. p < .05, vs. HKSA alone, n = 6. (F) HKSA‐induced Evans blue accumulation in the lung reflects lung vascular hyperpermeability and the protective effect of OGM‐8345. Shown are representative images of lungs from five independent experiments.

Journal: The FASEB Journal

Article Title: Inhibition of proton sensor GPR68 suppresses endothelial dysfunction and acute lung injury caused by Staphylococcus aureus bacterial particles

doi: 10.1096/fj.202401947R

Figure Lengend Snippet: OGM‐8345 rescues HKSA‐induced lung injury in vivo. C57BL/6 mice were exposed to HKSA (2 × 10 8 bacterial particles/mouse, intranasal) for 18 h. (A, B) Total RNA was extracted from various tissues of control or HKSA‐challenged mice, and qRT‐PCR was used to measure mRNA expression levels of GPR4 and GPR68. * p < .05, vs. control, n = 3. (C–F) C57BL/6 mice were injected with OGM‐8345 (20 mg/kg, i.p.) immediately followed by intranasal administration of HKSA. BAL fluid was collected to analyze total PMNs (C) and protein content (D). * p < .05, vs. HKSA alone, n = 8. (E) Total RNA was extracted from lung tissues, and qRT‐PCR was carried out to determine mRNA expression of indicated pro‐inflammatory genes. p < .05, vs. HKSA alone, n = 6. (F) HKSA‐induced Evans blue accumulation in the lung reflects lung vascular hyperpermeability and the protective effect of OGM‐8345. Shown are representative images of lungs from five independent experiments.

Article Snippet: Heat‐killed Staph. aureus bacterial particles (HKSA) were purchased from InvivoGen (San Diego, CA, Catalog code: tlrl‐hksa), and GPR4 inhibitor NE 52‐QQ57 was from MedKoo Biosciences Inc. (Morrisville, NC).

Techniques: In Vivo, Control, Quantitative RT-PCR, Expressing, Injection

Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. ( A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N=12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, * p < 0.05. ( B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. ( C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N=6 for PBS no virus inoculation; N=12 for vehicle, N=10 for GPR4 antagonist). Error bars indicate means ± SEM. ** p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: Mice were treated with GPR4 antagonist NE-52-QQ57 or vehicle control for up to 6 days starting from 4 dpi. ( A) The survival rate of SARS-CoV-2-infected K18-hACE2 mice is increased by the administration of the GPR4 antagonist. Ten-month-old male and female K18-hACE2 transgenic mice were intranasally inoculated with 1000 PFU of SARS-CoV-2 (N=12). Survival analysis was performed using the Kaplan-Meier method with a log-rank (Mantel-Cox) test, * p < 0.05. ( B) Daily body weight changes in GPR4 antagonist-treated or vehicle control mice were recorded up to 10 dpi or until the mice reached the humane endpoint. The difference in body weight change was analyzed using multiple unpaired t-tests. ( C) RT-qPCR was conducted to quantify the expression of GPR4 in non-infected (PBS) and SARS-CoV-2-infected mouse lung tissues (compared using two-tailed Mann-Whitney test) (N=6 for PBS no virus inoculation; N=12 for vehicle, N=10 for GPR4 antagonist). Error bars indicate means ± SEM. ** p < 0.01. (D) Representative pictures of mouse lung histology (H&E staining) with mild or severe histopathology in vehicle or GPR4 antagonist-treated mice. Scale bar = 20 µm. (E) Mouse lung histopathological score. Two-tailed Student’s t-test did not indicate significance.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Control, Infection, Transgenic Assay, Quantitative RT-PCR, Expressing, Two Tailed Test, MANN-WHITNEY, Virus, Staining, Histopathology

Representative images of H&E staining of mouse brains and their associated hemorrhage rates. Black arrows indicate hemorrhagic areas in the brain of a SARS-CoV-2-infected mouse treated with vehicle. Note no hemorrhagic areas in the brains of mice treated with GPR4 antagonist. Scale bar = 20 μm.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: Representative images of H&E staining of mouse brains and their associated hemorrhage rates. Black arrows indicate hemorrhagic areas in the brain of a SARS-CoV-2-infected mouse treated with vehicle. Note no hemorrhagic areas in the brains of mice treated with GPR4 antagonist. Scale bar = 20 μm.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Staining, Infection

(A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N=12 for vehicle, N=10 for GPR4 antagonist). * p < 0.05. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N=12 for vehicle, N=10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: (A) Fold change in gene expression levels of specified cytokines, chemokines, and other inflammatory genes assessed via RT-qPCR and normalized to 18S rRNA, compared with vehicle controls in mouse lung homogenates (N=12 for vehicle, N=10 for GPR4 antagonist). * p < 0.05. (B) Cytokine/chemokine protein levels in mouse lung tissues measured by the Luminex multiplex platform. (C) Cytokine/chemokine protein levels in mouse serum measured by the Luminex multiplex platform. Statistical differences in cytokine/chemokine levels were analyzed using the one-tailed Mann-Whitney test (N=12 for vehicle, N=10 for GPR4 antagonist). Error bars indicate mean ± SEM.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Gene Expression, Quantitative RT-PCR, Luminex, Multiplex Assay, One-tailed Test, MANN-WHITNEY

Other cytokines and chemokines in the lung tissues of SARS-CoV-2-infected mice treated with GPR4 antagonist or vehicle. Cytokine/chemokine protein levels in mouse lung tissues were measured by the Luminex multiplex platform.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: Other cytokines and chemokines in the lung tissues of SARS-CoV-2-infected mice treated with GPR4 antagonist or vehicle. Cytokine/chemokine protein levels in mouse lung tissues were measured by the Luminex multiplex platform.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Infection, Luminex, Multiplex Assay

Other cytokines and chemokines in the serum of SARS-CoV-2-infected mice treated with GPR4 antagonist or vehicle. Cytokine/chemokine protein levels in mouse serum were measured by the Luminex multiplex platform.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: Other cytokines and chemokines in the serum of SARS-CoV-2-infected mice treated with GPR4 antagonist or vehicle. Cytokine/chemokine protein levels in mouse serum were measured by the Luminex multiplex platform.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Infection, Luminex, Multiplex Assay

(A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N=12 for vehicle, N=10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. * p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N=12 for vehicle, N=12 for GPR4 antagonist). Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, * p < 0.05.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: (A) RT-qPCR to quantify viral RNA levels in mouse lung tissues (RNA copies/μg lung RNA). The data were analyzed using the two-tailed unpaired t-test and shown in mean ± SEM (N=12 for vehicle, N=10 for GPR4 antagonist). (B) Plaque assays were analyzed to determine the infectious viral titers (PFU/mg lung) in the lungs of vehicle- and GPR4 antagonist-treated mice infected with SARS-CoV-2. The limit of detection (LOD = 5 PFU/mg lung) is indicated by the dotted horizontal line. * p < 0.05. (C) Analysis of SARS-CoV-2 virus nucleocapsid distribution in mouse brain through IHC. The percentage of SARS-CoV-2 positive viral staining in the mouse brain was assessed using a microscope (N=12 for vehicle, N=12 for GPR4 antagonist). Scale bar = 20 μm. Error bars indicate mean ± SEM. (D) SARS-CoV-2 positive ratio in the brains of mice treated with GPR4 antagonist or vehicle. Analyzed using the Chi-square test, * p < 0.05.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Quantitative RT-PCR, Two Tailed Test, Infection, Virus, Staining, Microscopy

(A) A representative image of a CD4 + immune cell cluster in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, ** p < 0.01. Error bars represent mean ± SEM. N=12 for the vehicle group, and N=12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, * p < 0.05. Error bars represent mean ± SEM. N=12 for the vehicle group, and N=12 for the GPR4 antagonist group.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: (A) A representative image of a CD4 + immune cell cluster in the mouse brain, visualized using IHC with an antibody detecting CD4. Scale bar = 20 μm. (B) Quantification of the number of CD4 + immune cell clusters in the brain using microscopy. Analyzed using the two-tailed Student’s t-test, ** p < 0.01. Error bars represent mean ± SEM. N=12 for the vehicle group, and N=12 for the GPR4 antagonist group. (C) A representative image of a CD8 + immune cell cluster in the mouse brain, visualized using IHC with an antibody detecting CD8. Scale bar = 20 μm. (D) Quantification of the number of CD8 + immune cell clusters in the brain using microscopy. Analyzed by the two-tailed Student’s t-test, * p < 0.05. Error bars represent mean ± SEM. N=12 for the vehicle group, and N=12 for the GPR4 antagonist group.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Microscopy, Two Tailed Test

CD4 + and CD8 + T cell clusters in the mouse lung. GPR4 antagonist treatment reduced CD4 + and CD8 + immune cell clusters in the lungs of SARS-CoV-2-infected K18-hACE2 mice. Black arrows indicate immune cell clusters. Scale bar = 20 μm.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: CD4 + and CD8 + T cell clusters in the mouse lung. GPR4 antagonist treatment reduced CD4 + and CD8 + immune cell clusters in the lungs of SARS-CoV-2-infected K18-hACE2 mice. Black arrows indicate immune cell clusters. Scale bar = 20 μm.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Infection

(A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N=3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT- qPCR targeting the nucleocapsid gene. N=3 samples. * p < 0.05, ** p < 0.01, **** p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N=3 samples. ** p < 0.01, *** p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: (A) GPR4 antagonist incubated with SARS-CoV-2 (100 PFU) for 1h before infecting Vero E6 cells. GPR4 antagonist-containing medium was removed after infection. Plaque formation was measured to determine the infectious SARS-CoV-2 viral titer. N=3 samples. (B) Viral RNA in the SARS-CoV-2-infected Vero E6 cells (100 PFU inoculum) treated with various concentrations of GPR4 antagonist was determined 24 h post-infection. The GPR4 antagonist was maintained in the medium until cell assessment 24 h after treatment. Viral RNA isolated from Vero E6 cells was quantified by RT- qPCR targeting the nucleocapsid gene. N=3 samples. * p < 0.05, ** p < 0.01, **** p < 0.0001. (C) The infectious viral load by the plaque assay in SARS-CoV-2-infected Vero E6 cells (30 PFU inoculum) in response to treatment of vehicle DMSO or GPR4 antagonist at 72 h post-infection. The GPR4 antagonist was maintained in the medium for 72 h until cell assessment. N=3 samples. ** p < 0.01, *** p < 0.001. Comparisons between groups were analyzed by one-way ANOVA followed by post hoc Dunnett’s test. Error bars indicate mean ± SEM.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Incubation, Infection, Isolation, Quantitative RT-PCR, Plaque Assay

Viability of Vero E6 cells treated with the GPR4 antagonist. The viability of Vero E6 cells was approximately 100% relative to the DMSO control in the presence of 20 μM, 10 μM, 1 μM, and 0.1 μM GPR4 antagonist for 24h. (A) CellTiter-Glo (CTG) assay. (B) MTT assay.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: Viability of Vero E6 cells treated with the GPR4 antagonist. The viability of Vero E6 cells was approximately 100% relative to the DMSO control in the presence of 20 μM, 10 μM, 1 μM, and 0.1 μM GPR4 antagonist for 24h. (A) CellTiter-Glo (CTG) assay. (B) MTT assay.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: Control, CTG Assay, MTT Assay

ACE2 and TMPRSS2 RNA expressions in cells treated with the GPR4 antagonists in vitro . Quantitative RT-PCR was performed to assess ACE2 or TMPRSS2 RNA expressions in Vero E6, A549, Caco-2, and Lewis lung carcinoma cells after a 24h GPR4 antagonist treatment. Two-tailed Student’s t-tests were used to compare ACE2 and TMPRSS2 expressions between vehicle and GPR4 antagonist treatment in cell lines.

Journal: bioRxiv

Article Title: The GPR4 antagonist NE-52-QQ57 increases survival, mitigates the hyperinflammatory response and reduces viral load in SARS-CoV-2-infected K18-hACE2 transgenic mice

doi: 10.1101/2024.12.26.630404

Figure Lengend Snippet: ACE2 and TMPRSS2 RNA expressions in cells treated with the GPR4 antagonists in vitro . Quantitative RT-PCR was performed to assess ACE2 or TMPRSS2 RNA expressions in Vero E6, A549, Caco-2, and Lewis lung carcinoma cells after a 24h GPR4 antagonist treatment. Two-tailed Student’s t-tests were used to compare ACE2 and TMPRSS2 expressions between vehicle and GPR4 antagonist treatment in cell lines.

Article Snippet: At 4 dpi (days post-infection), mice showed some body weight loss with respiratory symptoms and were randomly assigned to orally receive either the GPR4 antagonist (NE-52-QQ57, provided by Novartis, 30 mg/kg, q.d.) or the vehicle control (0.5% methylcellulose, 0.5% Tween 80, and 99% water) (12 mice per group, 6 males and 6 females, from a total of 3 independent experiments).

Techniques: In Vitro, Quantitative RT-PCR, Two Tailed Test