Integrin/TGF-β1 Inhibitor GLPG-0187 Blocks SARS-CoV-2 Delta and Omicron Pseudovirus Infection of Airway Epithelial Cells In Vitro, Which Could Attenuate Disease Severity

Abstract

As COVID-19 continues to pose major risk for vulnerable populations, including the elderly, immunocompromised, patients with cancer, and those with contraindications to vaccination, novel treatment strategies are urgently needed. SARS-CoV-2 infects target cells via RGD-binding integrins, either independently or as a co-receptor with surface receptor angiotensin-converting enzyme 2 (ACE2). We used pan-integrin inhibitor GLPG-0187 to demonstrate the blockade of SARS-CoV-2 pseudovirus infection of target cells. Omicron pseudovirus infected normal human small airway epithelial (HSAE) cells significantly less than D614G or Delta variant pseudovirus, and GLPG-0187 effectively blocked SARS-CoV-2 pseudovirus infection in a dose-dependent manner across multiple viral variants. GLPG-0187 inhibited Omicron and Delta pseudovirus infection of HSAE cells more significantly than other variants. Pre-treatment of HSAE cells with MEK inhibitor (MEKi) VS-6766 enhanced the inhibition of pseudovirus infection by GLPG-0187. Because integrins activate transforming growth factor beta (TGF-β) signaling, we compared the plasma levels of active and total TGF-β in COVID-19+ patients. The plasma TGF-β1 levels correlated with age, race, and number of medications upon presentation with COVID-19, but not with sex. Total plasma TGF-β1 levels correlated with activated TGF-β1 levels. Moreover, the inhibition of integrin signaling prevents SARS-CoV-2 Delta and Omicron pseudovirus infectivity, and it may mitigate COVID-19 severity through decreased TGF-β1 activation. This therapeutic strategy may be further explored through clinical testing in vulnerable and unvaccinated populations.

Keywords: ACE2; COVID-19; Delta; GLPG-0187; HSAE; MEKi; Omicron; SARS-CoV-2; TGF-β1; integrin.

Figure 1

GLPG-0187 inhibits infection of SARS-CoV-2 pseudovirus variants D614, D614G, N501Y, E484K, N + E, NEK, R685A, Beta, Delta, and Omicron in small airway epithelial cells. (A) Treatment with 20 nM, 100 nM, 200 nM, or 1 µM GLPG-0187 for 2 h inhibits infection by the D614G pseudovirus variant (24 h infection time) in small airway epithelial cells compared to the VsVg positive control in a dose-dependent manner. DMSO was used as a vehicle control. (B) Treatment with 1 µM GLPG-0187 for 3 h inhibits infection by the D614, D614G, N501Y, E484K, N + E, NEK, R685A pseudovirus variants (20 h infection time). (C) Treatment with 1 µM or 2 µM GLPG-0187 for 2 h inhibits infection by the D614G, Beta, and Delta pseudovirus variants (20 h infection time). (D) Differential rates of infectivity across D614G, Delta, and Omicron variants observed after cells were spin-infected with the same amount of pseudovirus particles (1.0 × 10⁶ transduction units (TU) per 1 × 10⁵ cells/well) using the same experimental conditions described in panel C. (E) Treatment with 1 µM GLPG-0187 for 2 h inhibits infection by the Omicron pseudovirus variant (26 h infection time).

Figure 1

GLPG-0187 inhibits infection of SARS-CoV-2 pseudovirus variants D614, D614G, N501Y, E484K, N + E, NEK, R685A, Beta, Delta, and Omicron in small airway epithelial cells. (A) Treatment with 20 nM, 100 nM, 200 nM, or 1 µM GLPG-0187 for 2 h inhibits infection by the D614G pseudovirus variant (24 h infection time) in small airway epithelial cells compared to the VsVg positive control in a dose-dependent manner. DMSO was used as a vehicle control. (B) Treatment with 1 µM GLPG-0187 for 3 h inhibits infection by the D614, D614G, N501Y, E484K, N + E, NEK, R685A pseudovirus variants (20 h infection time). (C) Treatment with 1 µM or 2 µM GLPG-0187 for 2 h inhibits infection by the D614G, Beta, and Delta pseudovirus variants (20 h infection time). (D) Differential rates of infectivity across D614G, Delta, and Omicron variants observed after cells were spin-infected with the same amount of pseudovirus particles (1.0 × 10⁶ transduction units (TU) per 1 × 10⁵ cells/well) using the same experimental conditions described in panel C. (E) Treatment with 1 µM GLPG-0187 for 2 h inhibits infection by the Omicron pseudovirus variant (26 h infection time).

Figure 2

MEK inhibitor VS-6766 enhances the inhibition of SARS-CoV-2 pseudovirus infection by integrin inhibitor GLPG-0187 in small airway epithelial cells. Treatment with 5 µM VS-6766 for 24 h or with 1 µM GLPG-0187 for 3 h inhibits infection by the D614G pseudovirus variant (20 h infection time) in small airway epithelial cells compared to the VsVg positive control. DMSO was used as a vehicle control. Combination treatment involved 24 h pre-treatment with VS-6766 followed by an additional 3 h of treatment with GLPG-0187. The y axis shows side scatter, and the x axis shows FITC-pseudovirus expression.

Figure 3

Plasma TGF-β1 levels correlate with age, race, and number of medications administered upon presentation with COVID-19 to the ED, but not with sex. Total TGF-β1 levels were detected in activated plasma samples. TGF-β1 plasma concentration correlation with (A) age, (B) race, (C) number of medications administered upon presentation with COVID-19 to the emergency department (ED), (D) number of symptoms reported upon presentation to the ED, (E) sex, or (G) COVID-19 severity score. (G) COVID-19 severity score (CSS) legend. Sample values are reported in pg/mL (n = 81 samples). Statistical significance was calculated using: (A) Spearman’s correlation, (B,D,F) One-way Anova followed by a post hoc Tukey’s multiple comparisons test, and (C,E) two-tailed, unpaired Student’s t-test. The minimal level of significance was p < 0.05. Bar graphs represent the mean of the population, and error bars indicate standard deviation. * represents p < 0.05.

Figure 4

Active plasma TGF-β1 levels correlate with total TGF-β1 levels. Active TGF-β1 levels were detected in non-activated plasma samples. TGF-β1 plasma concentration correlation with (A) age, (B) race, (C) sex, or (D) COVID-19 severity score (CSS). Sample values are reported in pg/mL (n = 81 samples). Statistical significance was calculated using: (A) Spearman’s correlation, (B,D) One-way Anova followed by a post hoc Tukey’s multiple comparisons test, and (C) two-tailed, unpaired Student’s t-test. Bar graphs represent the mean of the population, and error bars indicate standard deviation. The minimal level of significance was p < 0.05.