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An epidemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is spreading worldwide. SARS-CoV-2 relies on its spike protein to invade host cells by interacting with the human receptor protein Angiotensin-Converting Enzymes 2 (ACE2). Therefore, designing an antibody or small-molecular entry blockers is of great significance for virus prevention and treatment. This study identified five potential small molecular anti-virus blockers via targeting SARS-CoV-2 spike protein by combining in silico technologies with in vitro experimental methods. The five molecules were natural products that binding to the RBD domain of SARS-CoV-2 was qualitatively and quantitively validated by both native Mass Spectrometry (MS) and Surface Plasmon Resonance (SPR). Anti-viral activity assays showed that the optimal molecule, H69C2, had a strong binding affinity (dissociation constant K D ) of 0.0947 µM and anti-virus IC 50 of 85.75 µM.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is still an emergent pandemic for humans. The virus infection is achieved by penetrating its spike protein to host cells via binding with ACE2. Moreover, recent studies show that SARS-CoV-2 may have multiple receptors that need to be further revealed. SARS-CoV-2 shares similar sequences of the spike protein with the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which can invade host cells by binding to either DPP4 or sialic acids. Sialic acids can be linked to the terminal of glycoproteins and gangliosides are used as one of the receptors of many types of viruses. Therefore, it is very interesting to determine whether sialic acid is a potential receptor of SARS-CoV-2. To address this question, we took N-Acetylneuraminic acid (Neu5Ac), a type of predominant sialic acid found in human cells, as the molecular probe to computationally search the surface of the spike protein to locate the potential binding sites of Neu5Ac. SPR analysis and mass spectrum analysis confirmed the interaction between Neu5Ac and spike protein. This study shows that sialic acids can moderately interact with the spike protein of SARS-CoV-2 by binding between the two RBDs of the spike protein, indicating it could be a potential secondary or auxiliary receptor of SARS-CoV-2.

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Chimeric antigen receptor (CAR) T cells are powerful in eradicating hematological malignancies, but their efficacy is limited in treating solid tumors. One of the barriers is the immunosuppressive response induced by immunomodulatory signaling pathways. Pharmacological targeting of these immunosuppressive pathways may be a simple way to improve the efficacy of CAR T cells. In this study, anti-CD133 and anti-HER2 CAR T cells were generated from healthy donors, and combination therapy using CAR T cells and small molecules targeting adenosine receptors was performed in vitro and in vivo with the goal of probing for potential synergistic antitumor activities. The adenosine A2b receptor agonist, BAY 60-6583, was found to significantly increase cytokine secretion of CD133-or HER2-specific CAR T cells when co-cultured with the respective target tumor cells. The in vitro cytotoxicity and proliferation of CAR T cells were also enhanced when supplied with BAY 60-6583. Furthermore, the combination with this small molecule facilitated the anti-HER2 CAR T cell-mediated elimination of tumor cells in a xenograft mouse model. However, the enhanced antitumor activities could not be suppressed by knockout of the adenosine A2b receptor in CAR T cells. Furthermore, mass spectrometry and computational methods were used to predict several potential alternative targets. Four potential targets (pyruvate kinase M (PKM), Talin-1, Plastin-2, and lamina-associated polypeptide 2) were captured by a photo-affinity probe, of which PKM and Talin-1 were predicted to interact with BAY 60-6583. Overall, our data suggest that BAY 60-6583 upregulates T cell functions through a mechanism independent of the adenosine A2b receptor.