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The coronavirus SARS-CoV-2 is the cause of the ongoing COVID-19 pandemic. Most SARS-CoV-2 infections are mild or even asymptomatic. However, a small fraction of infected individuals develops severe, life-threatening disease, which is caused by an uncontrolled immune response resulting in hyperinflammation. Antiviral interventions are only effective prior to the onset of hyperinflammation. Hence, biomarkers are needed for the early identification and treatment of high-risk patients. Here, we show in a range of model systems and data from post mortem samples that SARS-CoV-2 infection results in increased levels of CD47, which is known to mediate immune escape in cancer and virus-infected cells. Systematic literature searches also indicated that known risk factors such as older age and diabetes are associated with increased CD47 levels. High CD47 levels contribute to vascular disease, vasoconstriction, and hypertension, conditions which may predispose SARS-CoV-2-infected individuals to COVID-19-related complications such as pulmonary hypertension, lung fibrosis, myocardial injury, stroke, and acute kidney injury. Hence, CD47 is a candidate biomarker for severe COVID-19. Further research will have to show whether CD47 is a reliable diagnostic marker for the early identification of COVID-19 patients requiring antiviral therapy.
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 PLpro domain that cleaves not only the viral protein but also polyubiquitin and the ubiquitin-like modifier ISG15 from host cells. 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.
SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. Since the risk of severe COVID-19 disease is higher in males than in females and increases with age, we combined proteomics data from SARS-CoV-2-infected cells with human gene expression data from the Genotype-Tissue Expression (GTEx) database to identify gene products involved in coagulation that change with age, differ in their levels between females and males, and are regulated in response to SARS-CoV-2 infection. This resulted in the identification of transferrin as a candidate coagulation promoter, whose levels increases with age and are higher in males than in females and that is increased upon SARS-CoV-2 infection. A systematic investigation of gene products associated with the GO term “blood coagulation” did not reveal further high confidence candidates, which are likely to contribute to COVID-19-related coagulopathy. In conclusion, the role of transferrin should be considered in the course of COVID-19 disease and further examined in ongoing clinic-pathological investigations.
Although vaccines are currently used to control the coronavirus disease 2019 (COVID-19) pandemic, treatment options are urgently needed for those who cannot be vaccinated and for future outbreaks involving new severe acute respiratory syndrome coronavirus virus 2 (SARS-CoV-2) strains or coronaviruses not covered by current vaccines. Thus far, few existing antivirals are known to be effective against SARS-CoV-2 and clinically successful against COVID-19.
As part of an immediate response to the COVID-19 pandemic, a high-throughput, high content imaging–based SARS-CoV-2 infection assay was developed in VeroE6-eGFP cells and was used to screen a library of 5676 compounds that passed phase 1 clinical trials. Eight candidates (nelfinavir, RG-12915, itraconazole, chloroquine, hydroxychloroquine, sematilide, remdesivir, and doxorubicin) with in vitro anti–SARS-CoV-2 activity in VeroE6-eGFP and/or Caco-2 cell lines were identified. However, apart from remdesivir, toxicity and pharmacokinetic data did not support further clinical development of these compounds for COVID-19 treatment.
Doxorubicin-loaded human serum albumin nanoparticles overcome transporter-mediated drug resistance
(2019)
Resistance to systemic drug therapies is a major reason for the failure of anti-cancer therapies. Here, we tested doxorubicin-loaded human serum albumin (HSA) nanoparticles in the neuroblastoma cell line UKF-NB-3 and its ABCB1-expressing sublines adapted to vincristine (UKF-NB-3rVCR1) and doxorubicin (UKF-NB-3rDOX20). Doxorubicin-loaded nanoparticles displayed increased anti-cancer activity in UKF-NB-3rVCR1 and UKF-NB-3rDOX20 cells relative to doxorubicin solution, but not in UKF-NB-3 cells. UKF-NB-3rVCR1 cells were resensitised by nanoparticle-encapsulated doxorubicin to the level of UKF-NB-3 cells. UKF-NB-3rDOX20 cells displayed a more pronounced resistance phenotype than UKF-NB-3rVCR1 cells and were not re-sensitised by doxorubicin-loaded nanoparticles to the level of parental cells. ABCB1 inhibition using zosuquidar resulted in similar effects like nanoparticle incorporation, indicating that doxorubicin-loaded nanoparticles circumvent ABCB1-mediated drug efflux. The limited re-sensitisation of UKF-NB-3rDOX20 cells to doxorubicin by circumvention of ABCB1-mediated efflux is probably due to the presence of multiple doxorubicin resistance mechanisms. So far, ABCB1 inhibitors have failed in clinical trials, probably because systemic ABCB1 inhibition results in a modified body distribution of its many substrates including drugs, xenobiotics, and other molecules. HSA nanoparticles may provide an alternative, more specific way to overcome transporter-mediated resistance.
Objectives Omeprazole was shown to improve the anti-cancer effect of the nucleoside-analogue 5-fluorouracil. Here, we investigated the effects of omeprazole on the activities of the antiviral nucleoside analogues ribavirin and acyclovir.
Methods West Nile virus-infected Vero cells and influenza A H1N1-infected MDCK cells were treated with omeprazole and/ or ribavirin. Herpes simplex virus 1 (HSV-1)- or HSV-2-infected Vero or HaCat cells were treated with omeprazole and/ or acyclovir. Antiviral effects were determined by examination of cytopathogenic effects (CPE), immune staining, and virus yield assay. Cell viability was investigated by MTT assay.
Results Omeprazole concentrations up to 80μg/mL did not affect the antiviral effects of ribavirin. In contrast, omeprazole increased the acyclovir-mediated effects on HSV-1- and HSV-2-induced CPE formation in a dose-dependent manner in Vero and HaCat cells. Addition of omeprazole 80μg/mL resulted in a 10.8-fold reduction of the acyclovir concentration that reduces CPE formation by 50% (IC50) in HSV-1-infected Vero cells and in a 47.7-fold acyclovir IC50 reduction in HSV-1-infected HaCat cells. In HSV-2-infected cells, omeprazole reduced the acyclovir IC50 by 7.3-fold (Vero cells) or by 12.9-fold (HaCat cells). Omeprazole also enhanced the acyclovir-mediated effects on viral antigen expression and virus replication in HSV-1- and HSV-2-infected cells. In HSV-1-infected HaCat cells, omeprazole 80μg/mL reduced the virus titre in the presence of acyclovir 1μg/mL by 1.6×105-fold. In HSV-2-infected HaCat cells omeprazole 80μg/mL reduced the virus titre in the presence of acyclovir 2μg/mL by 9.2×103-fold. The investigated drug concentrations did not affect cell viability, neither alone nor in combination.
Conclusions Omeprazole increases the anti-HSV activity of acyclovir. As clinically well-established and tolerated drug, it is a candidate drug for antiviral therapies in combination with acyclovir.
Survivin is a drug target and the survivin suppressant YM155 a drug candidate for high-risk neuroblastoma. Findings from one YM155-adapted subline of the neuroblastoma cell line UKF-NB-3 had suggested that increased ABCB1 (mediates YM155 efflux) levels, decreased SLC35F2 (mediates YM155 uptake) levels, decreased survivin levels, and TP53 mutations indicate YM155 resistance. Here, the investigation of ten additional YM155-adapted UKF-NB-3 sublines only confirmed the roles of ABCB1 and SLC35F2. However, cellular ABCB1 and SLC35F2 levels did not indicate YM155 sensitivity in YM155-naïve cells, as indicated by drug response data derived from the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Moreover, the resistant sublines were characterised by a remarkable heterogeneity. Only seven sublines developed on-target resistance as indicated by resistance to RNAi-mediated survivin depletion. The sublines also varied in their response to other anti-cancer drugs. In conclusion, cancer cell populations of limited intrinsic heterogeneity can develop various resistance phenotypes in response to treatment. Therefore, individualised therapies will require monitoring of cancer cell evolution in response to treatment. Moreover, biomarkers can indicate resistance formation in the acquired resistance setting, even when they are not predictive in the intrinsic resistance setting.
SARS-CoV-2 infections are rapidly spreading around the globe. The rapid development of therapies is of major importance. However, our lack of understanding of the molecular processes and host cell signaling events underlying SARS-CoV-2 infection hinder therapy development. We employed a SARS-CoV-2 infection system in permissible human cells to study signaling changes by phospho-proteomics. We identified viral protein phosphorylation and defined phosphorylation-driven host cell signaling changes upon infection. Growth factor receptor (GFR) signaling and downstream pathways were activated. Drug-protein network analyses revealed GFR signaling as key pathway targetable by approved drugs. Inhibition of GFR downstream signaling by five compounds prevented SARS-CoV-2 replication in cells, assessed by cytopathic effect, viral dsRNA production, and viral RNA release into the supernatant. This study describes host cell signaling events upon SARS-CoV-2 infection and reveals GFR signaling as central pathway essential for SARS-CoV-2 replication. It provides with novel strategies for COVID-19 treatment.
Reliable, easy-to-handle phenotypic screening platforms are needed for the identification of anti-SARS-CoV-2 compounds. Here, we present caspase 3/7 activity as a read-out for monitoring the replication of SARS-CoV-2 isolates from different variants, including a remdesivir-resistant strain, and of other coronaviruses in a broad range of cell culture models, independently of cytopathogenic effect formation. Compared to other cell culture models, the Caco-2 subline Caco-2-F03 displayed superior performance, as it possesses a stable SARS-CoV-2 susceptible phenotype and does not produce false-positive hits due to drug-induced phospholipidosis. A proof-of-concept screen of 1796 kinase inhibitors identified known and novel antiviral drug candidates including inhibitors of PHGDH, CLK-1, and CSF1R. The activity of the PHGDH inhibitor NCT-503 was further increased in combination with the HK2 inhibitor 2-deoxy-D-glucose, which is in clinical development for COVID-19. In conclusion, caspase 3/7 activity detection in SARS-CoV-2-infected Caco-2F03 cells provides a simple phenotypic high-throughput screening platform for SARS-CoV-2 drug candidates that reduces false positive hits.
The COVID-19 pandemic and the associated prevention measures did not only impact on the transmission of COVID-19 but also on the spread of other infectious diseases in an unprecedented natural experiment. Here, we analysed the transmission patterns of 22 different infectious diseases during the COVID-19 pandemic in England. Our results show that the COVID-19 prevention measures generally reduced the spread of pathogens that are transmitted via the air and the faecal-oral route. Moreover, the COVID-19 prevention measures resulted in the sustained suppression of vaccine-preventable infectious diseases also after the removal of restrictions, while non-vaccine preventable diseases displayed a rapid rebound. Despite concerns that a lack of exposure to common pathogens may affect population immunity and result in large outbreaks by various pathogens post-COVID-19, only four of the 22 investigated diseases and disease groups displayed higher post-than pre-pandemic levels without an obvious causative relationship. Notably, this included chickenpox for which an effective vaccine is available but not used in the UK, which provides strong evidence supporting the inclusion of the chickenpox vaccination into the routine vaccination schedule in the UK. In conclusion, our findings provide unique, novel insights into the impact of non-pharmaceutical interventions on the spread of a broad range of infectious diseases.