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Pandemic SARS-CoV-2 causes a mild to severe respiratory disease called coronavirus disease 2019 (COVID-19). While control of the SARS-CoV-2 spread partly depends on vaccine-induced or naturally acquired protective herd immunity, antiviral strategies are still needed to manage COVID-19. Enisamium is an inhibitor of influenza A and B viruses in cell culture and clinically approved in countries of the Commonwealth of Independent States. In vitro, enisamium acts through metabolite VR17-04 and inhibits the activity of the influenza A virus RNA polymerase. Here we show that enisamium can inhibit coronavirus infections in NHBE and Caco-2 cells, and the activity of the SARS-CoV-2 RNA polymerase in vitro. Docking and molecular dynamics simulations provide insight into the mechanism of action and indicate that enisamium metabolite VR17-04 prevents GTP and UTP incorporation. Overall, these results suggest that enisamium is an inhibitor of SARS-CoV-2 RNA synthesis in vitro.
Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.
Although vaccination campaigns are currently being rolled out to prevent coronavirus disease (COVID-19), antivirals will remain an important adjunct to vaccination. Antivirals against coronaviruses do not exist, hence global drug repurposing efforts have been carried out to identify agents that may provide clinical benefit to patients with COVID-19. Itraconazole, an antifungal agent, has been reported to have activity against animal coronaviruses. Using cell-based phenotypic assays, the in vitro antiviral activity of itraconazole and 17-OH itraconazole was assessed against clinical isolates from a German and Belgian patient infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Itraconazole demonstrated antiviral activity in human Caco-2 cells (EC50 = 2.3 µM; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay). Similarly, its primary metabolite, 17-OH itraconazole, showed inhibition of SARS-CoV-2 activity (EC50 = 3.6 µM). Remdesivir inhibited viral replication with an EC50 = 0.4 µM. Itraconazole and 17-OH itraconazole resulted in a viral yield reduction in vitro of approximately 2-log10 and approximately 1-log10, as measured in both Caco-2 cells and VeroE6-eGFP cells, respectively. The viral yield reduction brought about by remdesivir or GS-441524 (parent nucleoside of the antiviral prodrug remdesivir; positive control) was more pronounced, with an approximately 3-log10 drop and >4-log10 drop in Caco-2 cells and VeroE6-eGFP cells, respectively. Itraconazole and 17-OH itraconazole exert in vitro low micromolar activity against SARS-CoV-2. Despite the in vitro antiviral activity, itraconazole did not result in a beneficial effect in hospitalized COVID-19 patients in a clinical study (EudraCT Number: 2020-001243-15).
Pathologic data indicate that human cytomegalovirus (HCMV) infection might be associated with the pathogenesis of several human malignancies. However, no definitive evidence of a causal link between HCMV infection and cancer dissemination has been established to date. This study describes the modulation of the invasive behavior of NCAM-expressing tumor cell lines by HCMV. Neuroblastoma (NB) cells, persistently infected with the HCMV strain AD169 (UKF-NB-4AD169 and MHH-NB-11AD169), were added to endothelial cell monolayers and adhesion and penetration kinetics were measured. The 140- and 180-kDa isoforms of the adhesion receptor NCAM were evaluated by flow cytometry, Western blot, and reverse transcriptionpolymerase chain reaction (RT-PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides or NCAM transfection. HCMV infection profoundly increased the number of adherent and penetrated NB, compared to controls. Surface expression of NCAM was significantly lower on UKF-NB-4AD169 and MHH-NB-11AD169, compared to mock-infected cells. Western-blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoform. An inverse correlation between NCAM expression and adhesion capacity of NB has been shown by antisense and transfection experiments. We conclude that HCMV infection leads to downregulation of NCAM receptors, which is associated with enhanced tumor cell invasiveness.
Cisplatin, which induces DNA damage, is standard chemotherapy for advanced bladder cancer (BCa). However, efficacy is limited due to resistance development. Since artesunate (ART), a derivative of artemisinin originating from Traditional Chinese Medicine, has been shown to exhibit anti-tumor activity, and to inhibit DNA damage repair, the impact of artesunate on cisplatin-resistant BCa was evaluated. Cisplatin-sensitive (parental) and cisplatin-resistant BCa cells, RT4, RT112, T24, and TCCSup, were treated with ART (1–100 µM). Cell growth, proliferation, and cell cycle phases were investigated, as were apoptosis, necrosis, ferroptosis, autophagy, metabolic activity, and protein expression. Exposure to ART induced a time- and dose-dependent significant inhibition of tumor cell growth and proliferation of parental and cisplatin-resistant BCa cells. This inhibition was accompanied by a G0/G1 phase arrest and modulation of cell cycle regulating proteins. ART induced apoptos is by enhancing DNA damage, especially in the resistant cells. ART did not induce ferroptosis, but led to a disturbance of mitochondrial respiration and ATP generation. This impairment correlated with autophagy accompanied by a decrease in LC3B-I and an increase in LC3B-II. Since ART significantly inhibits proliferative and metabolic aspects of cisplatin-sensitive and cisplatin-resistant BCa cells, it may hold potential in treating advanced and therapy-resistant BCa.
Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air–liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.
Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. Patients with relapsed disease have a poor prognosis despite intense treatment. In the present study, we aimed to identify chemoresistance gene expression signatures in vincristine resistant neuroblastoma cells. We found that vincristine-resistant neuroblastoma cells formed larger clones and survived under reduced serum conditions as compared with non-resistant parental cells. To identify the possible mechanisms underlying vincristine resistance in neuroblastoma cells, we investigated the expression profiles of genes known to be involved in cancer drug resistance. This specific gene expression patterns could predict the behavior of a tumor in response to chemotherapy and for predicting the prognosis of high-risk neuroblastoma patients. Our signature could help chemoresistant neuroblastoma patients in avoiding useless and harmful chemotherapy cycles.
The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.
The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), is effective against T-cell acute lymphoblastic leukaemia (T-ALL) but not against B-cell ALL (B-ALL). The underlying mechanisms have remained elusive. Here, data from pharmacogenomics studies and a panel of ALL cell lines reveal an inverse correlation between nelarabine sensitivity and the expression of SAMHD1, which can hydrolyse and inactivate triphosphorylated nucleoside analogues. Lower SAMHD1 abundance is detected in T-ALL than in B-ALL in cell lines and patient-derived leukaemic blasts. Mechanistically, T-ALL cells display increased SAMHD1 promoter methylation without increased global DNA methylation. SAMHD1 depletion sensitises B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induces AraG resistance. SAMHD1 has a larger impact on nelarabine/AraG than on cytarabine in ALL cells. Opposite effects are observed in acute myeloid leukaemia cells, indicating entity-specific differences. In conclusion, SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine.
Objectives: Given the high need and the absence of specific antivirals for treatment of COVID-19 (the disease caused by severe acute respiratory syndrome-associated coronavirus-2 [SARS-CoV-2]), human immunodeficiency virus (HIV) protease inhibitors are being considered as therapeutic alternatives. Methods: Prezcobix/Rezolsta is a fixed-dose combination of 800 mg of the HIV protease inhibitor darunavir (DRV) and 150 mg cobicistat, a CYP3A4 inhibitor, which is indicated in combination with other antiretroviral agents for the treatment of HIV infection. There are currently no definitive data on the safety and efficacy of DRV/cobicistat for the treatment of COVID-19. The in vitro antiviral activity of darunavir against a clinical isolate from a patient infected with SARS-CoV-2 was assessed. Results: DRV showed no antiviral activity against SARS-CoV-2 at clinically relevant concentrations (EC50 > 100 μM). Remdesivir, used as a positive control, demonstrated potent antiviral activity (EC50 = 0.38 μM). Conclusions: Overall, the data do not support the use of DRV for the treatment of COVID-19.