Identification of diphenylurea derivatives as novel endocytosis inhibitors that demonstrate broad-spectrum activity against SARS-CoV-2 and influenza A virus both in vitro and in vivo

Rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) poses enormous challenge in the development of broad-spectrum antivirals that are effective against the existing and emerging viral strains. Virus entry through endocytosis represents an attractive target for drug development, as inhibition of this early infection step should block downstream infection processes, and potentially inhibit viruses sharing the same entry route. In this study, we report the identification of 1,3-diphenylurea (DPU) derivatives (DPUDs) as a new class of endocytosis inhibitors, which broadly restricted entry and replication of several SARS-CoV-2 and IAV strains. Importantly, the DPUDs did not induce any significant cytotoxicity at concentrations effective against the viral infections. Examining the uptake of cargoes specific to different endocytic pathways, we found that DPUDs majorly affected clathrin-mediated endocytosis, which both SARS-CoV-2 and IAV utilize for cellular entry. In the DPUD-treated cells, although virus binding on the cell surface was unaffected, internalization of both the viruses was drastically reduced. Since compounds similar to the DPUDs were previously reported to transport anions including chloride (Cl-) across lipid membrane and since intracellular Cl- concentration plays a critical role in regulating vesicular trafficking, we hypothesized that the observed defect in endocytosis by the DPUDs could be due to altered Cl- gradient across the cell membrane. Using in vitro assays we demonstrated that the DPUDs transported Cl- into the cell and led to intracellular Cl- accumulation, which possibly affected the endocytic machinery by perturbing intracellular Cl- homeostasis. Finally, we tested the DPUDs in mice challenged with IAV and mouse-adapted SARS-CoV-2 (MA 10). Treatment of the infected mice with the DPUDs led to remarkable body weight recovery, improved survival and significantly reduced lung viral load, highlighting their potential for development as broad-spectrum antivirals. Author summaryMany pathogenic viruses including SARS-CoV-2 and IAV exploit endocytic pathways to initiate internalization into the host cell, and therefore, inhibiting these pathways with small molecules should effectively restrict viruses at the initial phase of infection. However, cytotoxicity represents one of the major obstacles in the therapeutic use of endocytosis inhibitors, as disruption of endocytosis may severely impact cell health. In this study, we identified DPUDs as a novel class of endocytosis inhibitors, which robustly blocked several IAV strains and SARS-CoV-2 variants of concern (VOC) in tissue culture cells. Remarkably, the DPUDs did not display any significant cytotoxicity at concentrations effective against the viruses. Our data indicate, among different endocytic pathways, clathrin-mediated endocytosis was most sensitive to DPUD treatment. We found that the DPUDs transported chloride ions into the cell, and disrupted endocytic trafficking possibly by perturbing intracellular chloride homeostasis. Confirming the antiviral activity of DPUDs in tissue culture cells, we evaluated their therapeutic potential in animal models of SARS-CoV-2 and influenza infections. SARS-CoV-2- or IAV-infected mice that received DPUD treatment displayed remarkable improvement in survival and clinical features as compared to the control animals, indicating their potential to be developed as highly-effective, broad-spectrum antivirals.