Anti-lung cancer properties of cyanobacterial bioactive compounds.

Lung cancer, the most prevalent gender-independent tumor entity in both men and women, is among the leading cause of cancer-related deaths worldwide. Despite decades of effort in developing improved therapeutic strategies including immunotherapies and novel chemotherapeutic agents, only modest improvements in outcome and long-term survival of lung cancer patients have been achieved. Therefore, exploring new and exceptional sources for bioactive compounds that might serve as anti-cancer agents might be the key to improving lung cancer therapy. On account of diverse forms, cyanobacteria might serve as a potential source for compounds with potential therapeutic applicability against malignant disorders, including cancer. The assorted arrays of metabolic mechanisms synthesize a plethora of bioactive compounds with immense biological potential. These compounds have been proven to be effective against various cancer cell lines and xenograft animal models. The present review provides an overview of the most promising cyanobacteria-derived bioactive compounds proven to exhibit anti-cancer properties in in-vitro and in-vivo studies and highlights their applicability as potential therapeutic agents with a focus on their anti-lung cancer properties.

NOS1-mediated macrophage and endothelial cell interaction in the progression of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease arising due to an imbalance in lipid metabolism and maladaptive immune response driven by the accumulation of cholesterol-laden macrophages in the artery wall. Interactions between monocytes/macrophages and endothelial cells play an essential role in the pathogenesis of atherosclerosis. In our current study, nitric oxide synthase 1 (NOS1)-derived nitric oxide (NO) has been identified as a regulator of macrophage and endothelial cell interaction. Oxidized LDL (OxLDL) activates NOS1, which results in the expression of CD40 ligand in macrophages. OxLDL-stimulated macrophages produce some soluble factors which increase the CD40 receptor expression in endothelial cells. This increases the interaction between the macrophages and endothelial cells, which leads to an increase in the inflammatory response. Inhibition of NOS1-derived NO might serve as an effective strategy to reduce foam cell formation and limit the extent of atherosclerotic plaque expansion.

Natural compounds as antiatherogenic agents.

Atherosclerosis (AS) is a widespread pathological coronary heart disease (CHD), which, along with other cardiovascular diseases (CVDs), is the primary cause of global mortality. It is initiated by the accumulation of cholesterol-laden macrophages in the artery wall, thereby forming the foam-cells, the hallmark of AS. Increased influx of oxidized LDL and decreased efflux of free cholesterol from macrophages constitute major factors that mediate the progression of AS. Natural compounds treatment and prevention of AS being an effective approach for a long time. Currently, as interests in medicinally important natural products increased that including medicinal herbs, numerous studies on natural compounds effective forAS have been reported. In the current review, we shed light on the available plant-based natural compounds as AS modulators with underlying mechanisms that may lead to potential therapeutic implications.

Inhibition of mitochondria ATP synthase suppresses prostate cancer growth through reduced insulin-like growth factor-1 secretion by prostate stromal cells.

Modulation of prostate stromal cells (PrSCs) within tumor tissues is gaining attention for the treatment of solid tumors. Using our original in vitro coculture system, we previously reported that leucinostatin (LCS)-A, a peptide mycotoxin, inhibited prostate cancer DU-145 cell growth through reduction of insulin-like growth factor 1 (IGF-I) expression in PrSCs. To further obtain additional bioactive compounds from LCS-A, we designed and synthesized a series of LCS-A derivatives as compounds that target PrSCs. Among the synthesized LCS-A derivatives, LCS-7 reduced IGF-I expression in PrSCs with lower toxicity to PrSCs and mice than LCS-A. As LCS-A has been suggested to interact with mitochondrial adenosine triphosphate (ATP) synthase, a docking study was performed to elucidate the mechanism of reduced IGF-I expression in the PrSCs. As expected, LCS-A and LCS-7 directly interacted with mitochondrial ATP synthase, and like LCS-A and LCS-7, other mitochondrial ATP synthase inhibitors also reduced the expression of IGF-I by PrSCs. Furthermore, LCS-A and LCS-7 significantly decreased the growth of mouse xenograft tumors. Based on these data, we propose that the mitochondrial ATP synthases-IGF-I axis of PrSCs plays a critical role on cancer cell growth and inhibition could be a potential anticancer target for prostate cancer.

Tumor-derived exosomes in the regulation of macrophage polarization.

BACKGROUND: This review focuses on exosomes derived from various cancer cells. The review discusses the possibility of differentiating macrophages in alternatively activated anti-inflammatory pro-tumorigenic M2 macrophage phenotypes and classically activated pro-inflammatory, anti-tumorigenic M1 macrophage phenotypes in the tumor microenvironment (TME). The review is divided into two main parts, as follows: (1) role of exosomes in alternatively activating M2-like macrophages-breast cancer-derived exosomes, hepatocellular carcinoma (HCC) cell-derived exosomes, lung cancer-derived exosomes, prostate cancer-derived exosomes, Oral squamous cell carcinoma (OSCC)-derived exosomes, epithelial ovarian cancer (EOC)-derived exosomes, Glioblastoma (GBM) cell-derived exosomes, and colorectal cancer-derived exosomes, (2) role of exosomes in classically activating M1-like macrophages, oral squamous cell carcinoma-derived exosomes, breast cancer-derived exosomes, Pancreatic-cancer derived modified exosomes, and colorectal cancer-derived exosomes, and (3) exosomes and antibody-dependent cellular cytotoxicity (ADCC). This review addresses the following subjects: (1) crosstalk between cancer-derived exosomes and recipient macrophages, (2) the role of cancer-derived exosome payload(s) in modulating macrophage fate of differentiation, and (3) intracellular signaling mechanisms in macrophages regarding the exosome's payload(s) upon its uptake and regulation of the TME. EVIDENCE: Under the electron microscope, nanoscale exosomes appear as specialized membranous vesicles that emerge from the endocytic cellular compartments. Exosomes harbor proteins, growth factors, cytokines, lipids, miRNA, mRNA, and DNAs. Exosomes are released by many cell types, including reticulocytes, dendritic cells, B-lymphocytes, platelets, mast cells, and tumor cells. It is becoming clear that exosomes can impinge upon signal transduction pathways, serve as a mediator of signaling crosstalk, thereby regulating cell-to-cell wireless communications. CONCLUSION: Based on the vesicular cargo, the molecular constituents, the exosomes have the potential to change the fate of macrophage phenotypes, either M1, classically activated macrophages, or M2, alternatively activated macrophages. In this review, we discuss and describe the ability of tumor-derived exosomes in the mechanism of macrophage activation and polarization.

Scaffolding role of TcpB in disrupting TLR4-Mal interactions: Three to tango.

Toll/interleukin-1 like receptors (TLRs) are membrane-spanning proteins crucially involved in innate immunity. On activation, the cytoplasmic toll/interleukin-1 receptor (TIR) domains of these receptors undergo homo- or heterodimerization. Brucella sp. are bacterial pathogens that affect the immune system by suppressing the TLR signaling pathway. They enact this by encoding a TIR domain-containing protein, TcpB, which suppresses NF-κB activation and proinflammatory cytokine secretion mediated by TLR4 receptors. TcpB has been shown to target the Mal-mediated pathway to suppress TLR signaling. The recent identification of its mechanism of interference with TLR4 signaling involving Mal prompted us to further study the structural aspects of TcpB binding with TLR4 and Mal. Our triprotein model displays the overall scaffolding role of TcpB in anchoring TLR4 and Mal thereby inhibiting their interaction leading to the attenuation of the TLR4 pathway.

Drug repositioning as an effective therapy for protease-activated receptor 2 inhibition.

Proteinase-activated receptor 2 (PAR-2) is a G protein-coupled receptor activated by both trypsin and a specific agonist peptide, SLIGKV-NH2. It has been linked to various pathologies, including pain and inflammation. Several peptide and peptidomimetic agonizts for PAR-2 have been developed exhibiting high potency and efficacy. However, the number of PAR-2 antagonists is smaller. We screened the Food and Drug Administration library of approved compounds to retrieve novel antagonists for repositioning in the PAR-2 structure. The most efficacious compound bicalutamide bound to the PAR-2 binding groove near the extracellular domain as observed in the in silico studies. Further, it showed reduced Ca2+ release in trypsin activated cells in a dose-dependent manner. Hence, bicalutamide is a novel and potent PAR-2 antagonist which could be therapeutically useful in blocking multiple pathways diverging from PAR-2 signaling. Further, the novel scaffold of bicalutamide represents a new molecular structure for PAR-2 antagonism and can serve as a basis for further drug development.

Structural insights of resveratrol with its binding partners in the toll-like receptor 4 pathway.

The benefits associated with resveratrol (Resv; 3,4',5-trihydroxy-trans-stilbene) are known for a long time. The therapeutic properties of Resv are observed in diseases like cancer, neurological disorders, atherosclerosis, aging, inflammation, etc. Multiple studies suggest that the beneficial properties of Resv are due to its binding to targets in multiple pathways. The same has been reflected in inflammation, where Resv has been shown to inhibit nuclear factor κ light-chain enhancer of activated B cells in the toll-like receptor 4 (TLR4) pathway. There are multiple cellular targets which bind to Resv, however the mode and the key interactions involved remain elusive for many of them. In the current work, we have investigated the structural insights of Resv with three of its binding partners involved in the inflammatory TLR4 signaling pathway. Through a structure-based modelling and molecular dynamics study, we have unraveled the molecular and atomic interactions involved in the Resv-binary complexes of inhibitor of κB kinase, cyclooxygeanse-2, and tank-binding kinase I, all three of which are key players in TLR4 inflammatory signaling. This study is the latest addition to the investigations of the structural partners of Resv and its molecular interactions.

NOS1-derived nitric oxide facilitates macrophage uptake of low-density lipoprotein.

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by a scavenger receptor in macrophages initiates necrosis core formation that characterizes atherosclerosis. We report that NOS1-derived nitric oxide (NO) facilitates low-density lipoprotein (LDL) uptake by macrophages independent of the inflammatory response. LDL uptake could be dramatically suppressed by NOS1 specific inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM). Importantly, the notion that NOS1 can mediate uptake of lipoproteins suggests that the foam cell formation is regulated by NOS1-derived NO-mediated mechanism. This is a novel study involving NOS1 as a critical player of foam cell formation and reveals much about the key molecular proteins involved in atherosclerosis. Targeting NOS1 would be a useful strategy in reducing LDL uptake by macrophages and hence dampening the atherosclerosis progression.

The TLR4-NOS1-AP1 signaling axis regulates macrophage polarization.

OBJECTIVE: Macrophages polarize to proinflammatory M1 or anti-inflammatory M2 states with distinct physiological functions. This transition within the M1-M2 phenotypes decides the nature, duration and severity of an inflammatory response. Although there is a substantial understanding of the fate of these phenotypes, the underlying molecular mechanism of transition within the M1-M2 phenotypes is not well understood. We have investigated the role of neuronal nitric oxide synthase (NOS1)-mediated regulation of activator protein 1 (AP-1) transcription factor in macrophages as a critical effector of macrophage phenotypic change. MATERIALS AND METHODS: Raw 264.7 and THP1 macrophages were stimulated with LPS (250 ng/ml) to activate the inflammatory signaling pathway. We analyzed the effect of pharmacological NOS1 inhibitor: TRIM (1-(2- Trifluoromethylphenyl) imidazole) on LPS-induced inflammatory response in macrophages. RESULTS: We determined that NOS1-derived nitric oxide (NO) facilitate Fos and Jun interaction which induces IL-12 & IL-23 expression. Pharmacological inhibition of NOS1 inhibits ATF2 and Jun dimer. Switching of Fos and Jun dimer to ATF2 and Jun dimerization controls phenotype transition from IL-12high IL-23high IL-10low to IL-12low IL-23lowIL-10high phenotype, respectively. CONCLUSION: These findings highlight a key role of the TLR4-NOS1-AP1 signaling axis in regulating macrophage polarization.

Deciphering the binding of caveolin-1 to client protein endothelial nitric-oxide synthase (eNOS): scaffolding subdomain identification, interaction modeling, and biological significance.

Caveolin-1 (Cav-1) gene inactivation interferes with caveolae formation and causes a range of cardiovascular and pulmonary complications in vivo. Recent evidence suggests that blunted Cav-1/endothelial nitric-oxide synthase (eNOS) interaction, which occurs specifically in vascular endothelial cells, is responsible for the multiple phenotypes observed in Cav-1-null animals. Under basal conditions, Cav-1 binds eNOS and inhibits nitric oxide (NO) production via the Cav-1 scaffolding domain (CAV; amino acids 82-101). Although we have recently shown that CAV residue Phe-92 is responsible for eNOS inhibition, the "inactive" F92A Cav-1 mutant unexpectedly retains its eNOS binding ability and can increase NO release, indicating the presence of a distinct eNOS binding domain within CAV. Herein, we identified and characterized a small 10-amino acid CAV subsequence (90-99) that accounted for the majority of eNOS association with Cav-1 (Kd = 49 nM), and computer modeling of CAV(90-99) docking to eNOS provides a rationale for the mechanism of eNOS inhibition by Phe-92. Finally, using gene silencing and reconstituted cell systems, we show that intracellular delivery of a F92A CAV(90-99) peptide can promote NO bioavailability in eNOS- and Cav-1-dependent fashions. To our knowledge, these data provide the first detailed analysis of Cav-1 binding to one of its most significant client proteins, eNOS.

An improved mouse model of sepsis based on intraperitoneal injections of the enriched culture of cecum slurry.

AIM: Sepsis is a life-threatening clinical syndrome comprising multiorgan dysfunctions caused by a disproportionate body immune response. There are several animal sepsis models which are based on cecum ligation, cecal puncture, and cecum slurry injection. The major limitation of all current sepsis models is the high variability owing to the variable degree of ligation, puncture and inconsistent microbial composition used for sepsis initiation. The primary objective of this work is to demonstrate the feasibility of a standardized method for sepsis development. MATERIALS AND METHODS: The cecal slurry bacterial culture was developed and preserved in glycerol stocks. Antibiotics aztreonam and vancomycin were used for generating several defined, enriched cecal slurry bacterial cultures. Mice survival was assessed until 48 hrs post injection, and the tissue samples were collected after 10 hrs from sepsis initiation. KEY FINDINGS: The results indicate that increasing polymicrobial load resulted in lower survival rates and was associated with the higher number of infiltrating immune cells and necrosis. H&E (haematoxylin & eosin) staining & serum markers revealed that septic mice exhibited increased inflammation and significant damage to the liver and kidneys. The defined Gram-negative and Gram-positive specific cecal slurry bacterial cultures were developed and their efficiency in inducing sepsis was characterized. SIGNIFICANCE: Enriched cecal slurry bacterial cultures can be stored in glycerol stocks at -80 °C. This has an ethical advantage of avoiding unnecessary animal euthanasia for each experiment and provides a standardization capability of sepsis development.

Structural and accessibility studies highlight the differential binding of clemizole to TRPC5 and TRPC6.

Transient Receptor Potential Canonical 5 (T RP C5) and T RP C6 channels play critical physiological roles in various cell types. Their involvement in numerous disease progression mechanisms has led to extensive searches for their inhibitors. Although several potent T RP C inhibitors have been developed and the structure of their binding sites were mapped using cryo electron microscopy, a comprehensive understanding of the molecular interactions within the inhibitor binding site of T RP Cs remains elusive. This study aimed to decipher the structural determinants and molecular mechanisms contributing to the differential binding of clemizole to T RP C5 and T RP C6, with a particular focus on the accessibility of binding site residues. This information can help better understand what molecular features allow for selective binding, which is a key characteristic of clinically effective pharmacological agents. Using computational methodologies, we conducted an in-depth molecular docking analysis of clemizole with T RP C5 and T RP C6 channels. The protein structures were retrieved from publicly accessible protein databases. Discovery Studio 2020 Client Visualizer and Chimera software facilitated our in-silico mutation experiments and enabled us to identify the critical structural elements influencing clemizole binding. Our study reveals key molecular determinants at the clemizole binding site, specifically outlining the role of residues' Accessible Surface Area (ASA) and Relative Accessible Surface Area (RASA) in differential binding. We found that lower accessibility of T RP C6 binding site residues, compared to those in T RP C5, could account for the lower affinity binding of clemizole to T RP C6. This work illuminates the pivotal role of binding site residue accessibility in determining the affinity of clemizole to T RP C5 and T RP C6. A nuanced understanding of the distinct binding properties between these homologous proteins may pave the way for the development of more selective inhibitors, promising improved therapeutic efficacy and fewer off-target effects. By demystifying the structural and molecular subtleties of T RP C inhibitors, this research could significantly accelerate the drug discovery process, offering hope to patients afflicted with T RP C-related diseases.

Structural modelling studies and immunoprophylactic potential of Brugia malayi DEAD Box RNA helicase.

DEAD Box RNA helicases are essential enzymes that are involved in RNA metabolic processes such as transcription, pre-mRNA splicing, translation initiation and RNA decay. We have previously over-expressed and biochemically characterized an immunodominant cDNA clone encoding DEAD box RNA helicase (BmL3-Helicase) isolated by immunoscreening of the larval stage cDNA library of Brugia malayi. In the current study, the 3D structure was determined and the immunoprophylactic efficacy of BmL3-Helicase was investigated by immunizing Mastomys coucha with the recombinant protein and subsequently challenging with B. malayi infective larvae. The immunization had an adverse outcome on the establishment of challenged larvae resulting in a 67.4% reduction in adult parasite recovery, a 86.7% decrease in the microfilarial density and profound sterility of the recovered female worms. The immune response thus generated was investigated by measuring the levels of specific antibodies including IgG subclasses, reactive oxygen species and cytokines.

Transient Receptor Potential Canonical 6 (TRPC6) Channel in the Pathogenesis of Diseases: A Jack of Many Trades.

The mammalian Transient Receptor Potential Canonical (TRPC) subfamily comprises seven transmembrane proteins (TRPC1-7) forming cation channels in the plasma membrane of mammalian cells. TRPC channels mediate Ca2+ and Na+ influx into the cells. Amongst TRPCs, TRPC6 deficiency or increased activity due to gain-of-function mutations has been associated with a multitude of diseases, such as kidney disease, pulmonary disease, and neurological disease. Indeed, the TRPC6 protein is expressed in various organs and is involved in diverse signalling pathways. The last decade saw a surge in the investigative studies concerning the physiological roles of TRPC6 and describing the development of new pharmacological tools modulating TRPC6 activity. The current review summarizes the progress achieved in those investigations.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Epigallocatechin gallate (EGCG) attenuates severe acute respiratory coronavirus disease 2 (SARS-CoV-2) infection by blocking the interaction of SARS-CoV-2 spike protein receptor-binding domain to human angiotensin-converting enzyme 2.

The outbreak of the coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2 triggered a global pandemic where control is needed through therapeutic and preventive interventions. This study aims to identify natural compounds that could affect the fusion between the viral membrane (receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 spike protein) and the human cell receptor angiotensin-converting enzyme 2. Accordingly, we performed the enzyme-linked immunosorbent assay-based screening of 10 phytochemicals that already showed numerous positive effects on human health in several epidemiological studies and clinical trials. Among these phytochemicals, epigallocatechin gallate, a polyphenol and a major component of green tea, could effectively inhibit the interaction between the receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 spike protein and the human cell receptor angiotensin-converting enzyme 2. Alternately, in silico molecular docking studies of epigallocatechin gallate and angiotensin-converting enzyme 2 indicated a binding score of -7.8 kcal/mol and identified a hydrogen bond between R393 and angiotensin-converting enzyme 2, which is considered as a key interacting residue involved in binding with the severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain, suggesting the possible blocking of interaction between receptor-binding domain and angiotensin-converting enzyme 2. Furthermore, epigallocatechin gallate could attenuate severe acute respiratory syndrome coronavirus 2 infection and replication in Caco-2 cells. These results shed insight into identification and validation of severe acute respiratory syndrome coronavirus 2 entry inhibitors.

Repurposing dyphylline as a pan-coronavirus antiviral therapy.

Background: In the last two decades, the world has witnessed the emergence of zoonotic corona viruses (CoVs), which cause mild to severe respiratory diseases in humans. Human coronaviruses (HCoVs), mainly from the alpha-CoV and beta-CoV genera, have evolved to be highly pathogenic, such as SARS-CoV-2 causing the COVID-19 pandemic. These coronaviruses carry functional enzymes necessary for the virus life cycle, which represent attractive antiviral targets. Methods & Results: We aimed to therapeutically target the main protease (Mpro) of HCoV-NL63 and HCoV-229E (from alpha-CoV genus) and HCoV-OC43 and SARS-CoV-2 (from beta-CoV genus). Through virtual screening, we identified an FDA-approved drug dyphylline, a xanthine derivate, that binds to the catalytic dyad residues; histidine and cystine of the Mpro structures. Importantly, dyphylline dose-dependently inhibited the viral replication in cell culture models infected with the viruses. Conclusion: Our findings support the repurposing of dyphylline as a pan-coronavirus antiviral agent.

TIRAP in the Mechanism of Inflammation.

The Toll-interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP) represents a key intracellular signalling molecule regulating diverse immune responses. Its capacity to function as an adaptor molecule has been widely investigated in relation to Toll-like Receptor (TLR)-mediated innate immune signalling. Since the discovery of TIRAP in 2001, initial studies were mainly focused on its role as an adaptor protein that couples Myeloid differentiation factor 88 (MyD88) with TLRs, to activate MyD88-dependent TLRs signalling. Subsequent studies delineated TIRAP's role as a transducer of signalling events through its interaction with non-TLR signalling mediators. Indeed, the ability of TIRAP to interact with an array of intracellular signalling mediators suggests its central role in various immune responses. Therefore, continued studies that elucidate the molecular basis of various TIRAP-protein interactions and how they affect the signalling magnitude, should provide key information on the inflammatory disease mechanisms. This review summarizes the TIRAP recruitment to activated receptors and discusses the mechanism of interactions in relation to the signalling that precede acute and chronic inflammatory diseases. Furthermore, we highlighted the significance of TIRAP-TIR domain containing binding sites for several intracellular inflammatory signalling molecules. Collectively, we discuss the importance of the TIR domain in TIRAP as a key interface involved in protein interactions which could hence serve as a therapeutic target to dampen the extent of acute and chronic inflammatory conditions.

Comparative assessment of favipiravir and remdesivir against human coronavirus NL63 in molecular docking and cell culture models.

Human coronavirus NL63 (HCoV-NL63) mainly affects young children and immunocompromised patients, causing morbidity and mortality in a subset of patients. Since no specific treatment is available, this study aims to explore the anti-SARS-CoV-2 agents including favipiravir and remdesivir for treating HCoV-NL63 infection. We first successfully modelled the 3D structure of HCoV-NL63 RNA-dependent RNA polymerase (RdRp) based on the experimentally solved SARS-CoV-2 RdRp structure. Molecular docking indicated that favipiravir has similar binding affinities to SARS-CoV-2 and HCoV-NL63 RdRp with LibDock scores of 75 and 74, respectively. The LibDock scores of remdesivir to SARS-CoV-2 and HCoV-NL63 were 135 and 151, suggesting that remdesivir may have a higher affinity to HCoV-NL63 compared to SARS-CoV-2 RdRp. In cell culture models infected with HCoV-NL63, both favipiravir and remdesivir significantly inhibited viral replication and production of infectious viruses. Overall, remdesivir compared to favipiravir is more potent in inhibiting HCoV-NL63 in cell culture. Importantly, there is no evidence of resistance development upon long-term exposure to remdesivir. Furthermore, combining favipiravir or remdesivir with the clinically used antiviral cytokine interferon-alpha resulted in synergistic effects. These findings provided a proof-of-concept that anti-SARS-CoV-2 drugs, in particular remdesivir, have the potential to be repurposed for treating HCoV-NL63 infection.