Anti-SARS-CoV-2 Activity of Extracellular Vesicle Inhibitors: Screening, Validation, and Combination with Remdesivir

Abstract

The coronavirus disease 2019 (COVID-19) pandemic severely impacts health, economy, and society worldwide. Antiviral drugs against SARS-CoV-2 are urgently needed to cope with this global crisis. It has been found that the biogenesis and release mechanisms of viruses share a common pathway with extracellular vesicles (EVs). We hypothesized that small molecule inhibitors of EV biogenesis/release could exert an anti-SARS-CoV-2 effect. Here, we screened 17 existing EV inhibitors and found that calpeptin, a cysteine proteinase inhibitor, exhibited the most potent anti-SARS-CoV-2 activity with no apparent cytotoxicity. Calpeptin demonstrated the dose-dependent inhibition against SARS-CoV-2 viral nucleoprotein expression in the infected cells with a half-maximal inhibitory concentration (IC50) of 1.44 µM in Vero-E6 and 26.92 µM in Calu-3 cells, respectively. Moreover, calpeptin inhibited the production of infectious virions with the lower IC50 of 0.6 µM in Vero E6 cells and 10.12 µM in Calu-3 cells. Interestingly, a combination of calpeptin and remdesivir, the FDA-approved antiviral drug against SARS-CoV-2 viral replication, significantly enhanced the anti-SARS-CoV-2 effects compared to monotherapy. This study discovered calpeptin as a promising candidate for anti-SARS-CoV-2 drug development. Further preclinical and clinical studies are warranted to elucidate the therapeutic efficacy of calpeptin and remdesivir combination in COVID-19.

Anti‐SARS‐CoV‐2 effect of extracellular vesicles released from mesenchymal stem cells

“…It is known that MSCs are highly resistant to viral infections (Wu et al, 2018), including SARS-CoV-2 (Avanzini et al, 2021). We, therefore, hypothesized that the EVs released from MSCs could inhibit SARS-CoV-2 infection.Accordingly, we applied in vitro anti-SARS-CoV-2 assays, which were previously developed by our group (Kanjanasirirat et al, 2020;Kongsomros et al, 2021;Sa-Ngiamsuntorn et al, 2021), to determine the dose dependent anti-SARS-CoV-2 effect of MSC-EVs. In this study, MSC-EV was referred to as the exosome (or small EV) subpopulation of extracellular vesicles.…”

“…Accordingly, we applied in vitro anti‐SARS‐CoV‐2 assays, which were previously developed by our group (Kanjanasirirat et al., 2020 ; Kongsomros et al., 2021 ; Sa‐Ngiamsuntorn et al., 2021 ), to determine the dose dependent anti‐SARS‐CoV‐2 effect of MSC‐EVs. In this study, MSC‐EV was referred to as the exosome (or small EV) subpopulation of extracellular vesicles.…”

Human Milk Oligosaccharides: Potential Applications in COVID-19

“…While hMOS have been recognized for potential application in COVID-19 treatment, direct evidence of anti-SARS-CoV-2 activity by hMOS is not evident in the literature. To obtain such data, one strategy is to utilize cell-based high-throughput antiviral screening, which was recently adapted for small molecules and natural compounds [ 113 , 114 , 115 , 116 ]. Positive results from these designs would support further development of hMOS as the antiviral agent for COVID-19.…”

Combination Therapies against COVID-19

“…IMU-838, a developmental dihydroorotate dehydrogenase (DHODH) inhibitor in phase II for autoimmune disease, showed enhanced in vitro anti-SARS-CoV-2 activity when combined with remdesivir [70] (Table 2, Ref. [2,40,[70][71][72][73][74][75][76][77][78][79][80][81][82][83][84][85][86][87][88]). Biering et al [71] identified compound B02, a human RAD51 inhibitor, exhibiting antiviral synergy with remdesivir after screening a library of FDA-approved and well-studied preclinical and clinical chemicals (Table 2).…”

“…Calpeptin exhibited high antiviral activity against SARS-CoV-2 without apparent cytotoxicity via blocking extracellular vesicles (EVs) biogenesis/release as a cysteine proteinase inhibitor. Interestingly, a cocktail of calpeptin and remdesivir signifi-cantly enhanced anti-SARS-CoV-2 activity in comparison with monotherapy [76]. Lenvatinib, as a broad-spectrum host receptor tyrosine kinase (RTK) inhibitor, showed no inhibitory activity against Mpro in vitro, but exhibited remarkable synergistic effect with remdesivir to suppress SARS-CoV-2 replication, albeit selectively in Vero-CCL81 cells.…”