Discovery of potential anti-SARS-CoV-2 drugs based on large-scale screening in vitro and effect evaluation in vivo

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a global crisis. Clinical candidates with high efficacy, ready availability, and that do not develop resistance are in urgent need. Despite that screening to repurpose clinically approved drugs has provided a variety of hits shown to be effective against SARS-CoV-2 infection in cell culture, there are few confirmed antiviral candidates in vivo. In this study, 94 compounds showing high antiviral activity against SARS-CoV-2 in Vero E6 cells were identified from 2,580 FDA-approved small-molecule drugs. Among them, 24 compounds with low cytotoxicity were selected, and of these, 17 compounds also effectively suppressed SARS-CoV-2 infection in HeLa cells transduced with human ACE2. Six compounds disturb multiple processes of the SARS-CoV-2 life cycle. Their prophylactic efficacies were determined in vivo using Syrian hamsters challenged with SARS-CoV-2 infection. Seven compounds reduced weight loss and promoted weight regain of hamsters infected not only with the original strain but also the D614G variant. Except for cisatracurium, six compounds reduced hamster pulmonary viral load, and IL-6 and TNF-α mRNA when assayed at 4 d postinfection. In particular, sertraline, salinomycin, and gilteritinib showed similar protective effects as remdesivir in vivo and did not induce antiviral drug resistance after 10 serial passages of SARS-CoV-2 in vitro, suggesting promising application for COVID-19 treatment.

     

Phillyrin (KD-1) exerts anti-viral and anti-inflammatory activities against novel coronavirus (SARS-CoV-2) and human coronavirus 229E (HCoV-229E) by suppressing the nuclear factor kappa B (NF-κB) signaling pathway

BackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has extensively and rapidly spread in the world, causing an outbreak of acute infectious pneumonia. However, no specific antiviral drugs or vaccines can be used. Phillyrin (KD-1), a representative ingredient of Forsythia suspensa, possesses anti-inflammatory, anti-oxidant, and antiviral activities. However, little is known about the antiviral abilities and mechanism of KD-1 against SARS-CoV-2 and human coronavirus 229E (HCoV-229E).PurposeThe study was designed to investigate the antiviral and anti-inflammatory activities of KD-1 against the novel SARS-CoV-2 and HCoV-229E and its potential effect in regulating host immune response in vitro.MethodsThe antiviral activities of KD-1 against SARS-CoV-2 and HCoV-229E were assessed in Vero E6 cells using cytopathic effect and plaque-reduction assay. Proinflammatory cytokine expression levels upon infection with SARS-CoV-2 and HCoV-229E infection in Huh-7 cells were measured by real-time quantitative PCR assays. Western blot assay was used to determine the protein expression of nuclear factor kappa B (NF-κB) p65, p-NF-κB p65, IκBα, and p-IκBα in Huh-7 cells, which are the key targets of the NF-κB pathway.ResultsKD-1 could significantly inhibit SARS-CoV-2 and HCoV-229E replication in vitro. KD-1 could also markedly reduce the production of proinflammatory cytokines (TNF-α, IL-6, IL-1β, MCP-1, and IP-10) at the mRNA levels. Moreover, KD-1 could significantly reduce the protein expression of p-NF-κB p65, NF-κB p65, and p-IκBα, while increasing the expression of IκBα in Huh-7 cells.ConclusionsKD-1 could significantly inhibit virus proliferation in vitro, the up-regulated expression of proinflammatory cytokines induced by SARS-CoV-2 and HCoV-229E by regulating the activity of the NF-кB signaling pathway. Our findings indicated that KD-1 protected against virus attack and can thus be used as a novel strategy for controlling the coronavirus disease 2019.     

In vitro selection of Remdesivir resistance suggests evolutionary predictability of SARS-CoV-2

Remdesivir (RDV), a broadly acting nucleoside analogue, is the only FDA approved small molecule antiviral for the treatment of COVID-19 patients. To date, there are no reports identifying SARS-CoV-2 RDV resistance in patients, animal models or in vitro. Here, we selected drug-resistant viral populations by serially passaging SARS-CoV-2 in vitro in the presence of RDV. Using high throughput sequencing, we identified a single mutation in RNA-dependent RNA polymerase (NSP12) at a residue conserved among all coronaviruses in two independently evolved populations displaying decreased RDV sensitivity. Introduction of the NSP12 E802D mutation into our SARS-CoV-2 reverse genetics backbone confirmed its role in decreasing RDV sensitivity in vitro. Substitution of E802 did not affect viral replication or activity of an alternate nucleoside analogue (EIDD2801) but did affect virus fitness in a competition assay. Analysis of the globally circulating SARS-CoV-2 variants (>800,000 sequences) showed no evidence of widespread transmission of RDV-resistant mutants. Surprisingly, we observed an excess of substitutions in spike at corresponding sites identified in the emerging SARS-CoV-2 variants of concern (i.e., H69, E484, N501, H655) indicating that they can arise in vitro in the absence of immune selection. The identification and characterisation of a drug resistant signature within the SARS-CoV-2 genome has implications for clinical management and virus surveillance.     

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.     

The potential use of Drosophila as an in vivo model organism for COVID-19-related research: a review

The world urgently needs effective antiviral approaches against emerging viruses, as shown by the coronavirus disease 2019 (COVID-19) pandemic, which has become an exponentially growing health crisis. Scientists from diverse backgrounds have directed their efforts towards identifying key features of SARS-CoV-2 and clinical manifestations of COVID-19 infection. Reports of more transmissible variants of SARS-CoV-2 also raise concerns over the possibility of an explosive trajectory of the pandemic, so scientific attention should focus on developing new weapons to help win the fight against coronaviruses that may undergo further mutations in the future. Drosophila melanogaster offers a powerful and potential in vivo model that can significantly increase the efficiency of drug screening for viral and bacterial infections. Thanks to its genes with functional human homologs, Drosophila could play a significant role in such gene-editing studies geared towards designing vaccines and antiviral drugs for COVID-19. It can also help rectify current drawbacks of CRISPR-based therapeutics like off-target effects and delivery issues, representing another momentous step forward in healthcare. Here I present an overview of recent literature and the current state of knowledge, explaining how it can open up new avenues for Drosophila in our battle against infectious diseases.     

Corilagin prevents SARS-CoV-2 infection by targeting RBD-ACE2 binding

BackgroundThe outbreak of coronavirus (SARS-CoV-2) disease caused more than 100,000,000 people get infected and over 2,200,000 people being killed worldwide. However, the current developed vaccines or drugs may be not effective in preventing the pandemic of COVID-19 due to the mutations of coronavirus and the severe side effects of the newly developed vaccines. Chinese herbal medicines and their active components play important antiviral activities. Corilagin exhibited antiviral effect on human immunodeficiency virus (HIV), hepatitis C virus (HCV) and Epstein-Barr virus (EBV). However, whether it blocks the interaction between SARS-CoV-2 RBD and hACE2 has not been elucidated.PurposeTo characterize an active compound, corilagin derived from Phyllanthus urinaria as potential SARS-CoV-2 entry inhibitors for its possible preventive application in daily anti-virus hygienic products.MethodsComputational docking coupled with bio-layer interferometry, BLI were adopted to screen more than 1800 natural compounds for the identification of SARS-CoV-2 spike-RBD inhibitors. Corilagin was confirmed to have a strong binding affinity with SARS-CoV-2-RBD or human ACE2 (hACE2) protein by the BLI, ELISA and immunocytochemistry (ICC) assay. Furthermore, the inhibitory effect of viral infection of corilagin was assessed by in vitro pseudovirus system. Finally, the toxicity of corilagin was examined by using MTT assay and maximal tolerated dose (MTD) studies in C57BL/6 mice.ResultsCorilagin preferentially binds to a pocket that contains residues Cys 336 to Phe 374 of spike-RBD with a relatively low binding energy of -9.4 kcal/mol. BLI assay further confirmed that corilagin exhibits a relatively strong binding affinity to SARS-CoV-2-RBD and hACE2 protein. In addition, corilagin dose-dependently blocks SARS-CoV-2-RBD binding and abolishes the infectious property of RBD-pseudotyped lentivirus in hACE2 overexpressing HEK293 cells, which mimicked the entry of SARS-CoV-2 virus in human host cells. Finally, in vivo studies revealed that up to 300 mg/kg/day of corilagin was safe in C57BL/6 mice. Our findings suggest that corilagin could be a safe and potential antiviral agent against the COVID-19 acting through the blockade of the fusion of SARS-CoV-2 spike-RBD to hACE2 receptors.ConclusionCorilagin could be considered as a safe and environmental friendly anti-SARS-CoV-2 agent for its potential preventive application in daily anti-virus hygienic products.     

Elimination of Aicardi–Goutières syndrome protein SAMHD1 activates cellular innate immunity and suppresses SARS-CoV-2 replication

The lack of antiviral innate immune responses during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is characterized by limited production of interferons (IFNs). One protein associated with Aicardi–Goutières syndrome, SAMHD1, has been shown to negatively regulate the IFN-1 signaling pathway. However, it is unclear whether elevated IFN signaling associated with genetic loss of SAMHD1 would affect SARS-CoV-2 replication. In this study, we established in vitro tissue culture model systems for SARS-CoV-2 and human coronavirus OC43 infections in which SAMHD1 protein expression was absent as a result of CRISPR–Cas9 gene KO or lentiviral viral protein X–mediated proteosomal degradation. We show that both SARS-CoV-2 and human coronavirus OC43 replications were suppressed in SAMHD1 KO 293T and differentiated THP-1 macrophage cell lines. Similarly, when SAMHD1 was degraded by virus-like particles in primary monocyte-derived macrophages, we observed lower levels of SARS-CoV-2 RNA. The loss of SAMHD1 in 293T and differentiated THP-1 cells resulted in upregulated gene expression of IFNs and innate immunity signaling proteins from several pathways, with STAT1 mRNA being the most prominently elevated ones. Furthermore, SARS-CoV-2 replication was significantly increased in both SAMHD1 WT and KO cells when expression and phosphorylation of STAT1 were downregulated by JAK inhibitor baricitinib, which over-rode the activated antiviral innate immunity in the KO cells. This further validates baricitinib as a treatment of SARS-CoV-2–infected patients primarily at the postviral clearance stage. Overall, our tissue culture model systems demonstrated that the elevated innate immune response and IFN activation upon genetic loss of SAMHD1 effectively suppresses SARS-CoV-2 replication.     

Clinical drug response can be predicted using baseline gene expression levels and in vitro drug sensitivity in cell lines

We demonstrate a method for the prediction of chemotherapeutic response in patients using only before-treatment baseline tumor gene expression data. First, we fitted models for whole-genome gene expression against drug sensitivity in a large panel of cell lines, using a method that allows every gene to influence the prediction. Following data homogenization and filtering, these models were applied to baseline expression levels from primary tumor biopsies, yielding an in vivo drug sensitivity prediction. We validated this approach in three independent clinical trial datasets, and obtained predictions equally good, or better than, gene signatures derived directly from clinical data.     

The Effect of Statins on Blood Gene Expression in COPD

BackgroundCOPD is currently the fourth leading cause of death worldwide. Statins are lipid lowering agents with documented cardiovascular benefits. Observational studies have shown that statins may have a beneficial role in COPD. The impact of statins on blood gene expression from COPD patients is largely unknown.ObjectiveIdentify blood gene signature associated with statin use in COPD patients, and the pathways underpinning this signature that could explain any potential benefits in COPD.MethodsWhole blood gene expression was measured on 168 statin users and 451 non-users from the ECLIPSE study using the Affymetrix Human Gene 1.1 ST microarray chips. Factor Analysis for Robust Microarray Summarization (FARMS) was used to process the expression data. Differential gene expression analysis was undertaken using the Linear Models for Microarray data (Limma) package adjusting for propensity score and surrogate variables. Similarity of the expression signal with published gene expression profiles was performed in ProfileChaser.Results25 genes were differentially expressed between statin users and non-users at an FDR of 10%, including LDLR, CXCR2, SC4MOL, FAM108A1, IFI35, FRYL, ABCG1, MYLIP, and DHCR24. The 25 genes were significantly enriched in cholesterol homeostasis and metabolism pathways. The resulting gene signature showed correlation with Huntington’s disease, Parkinson’s disease and acute myeloid leukemia gene signatures.ConclusionThe blood gene signature of statins’ use in COPD patients was enriched in cholesterol homeostasis pathways. Further studies are needed to delineate the role of these pathways in lung biology.     

Cross-Study Projections of Genomic Biomarkers: An Evaluation in Cancer Genomics

Human disease studies using DNA microarrays in both clinical/observational and experimental/controlled studies are having increasing impact on our understanding of the complexity of human diseases. A fundamental concept is the use of gene expression as a “common currency” that links the results of in vitro controlled experiments to in vivo observational human studies. Many studies – in cancer and other diseases – have shown promise in using in vitro cell manipulations to improve understanding of in vivo biology, but experiments often simply fail to reflect the enormous phenotypic variation seen in human diseases. We address this with a framework and methods to dissect, enhance and extend the in vivo utility of in vitro derived gene expression signatures. From an experimentally defined gene expression signature we use statistical factor analysis to generate multiple quantitative factors in human cancer gene expression data. These factors retain their relationship to the original, one-dimensional in vitro signature but better describe the diversity of in vivo biology. In a breast cancer analysis, we show that factors can reflect fundamentally different biological processes linked to molecular and clinical features of human cancers, and that in combination they can improve prediction of clinical outcomes.     

Diltiazem inhibits SARS-CoV-2 cell attachment and internalization and decreases the viral infection in mouse lung

The continuous emergence of severe acute respiratory coronavirus 2 (SARS-CoV-2) variants and the increasing number of breakthrough infection cases among vaccinated people support the urgent need for research and development of antiviral drugs. Viral entry is an intriguing target for antiviral drug development. We found that diltiazem, a blocker of the L-type calcium channel Ca_v1.2 pore-forming subunit (Ca_v1.2 α_1c) and an FDA-approved drug, inhibits the binding and internalization of SARS-CoV-2, and decreases SARS-CoV-2 infection in cells and mouse lung. Ca_v1.2 α_1c interacts with SARS-CoV-2 spike protein and ACE2, and affects the attachment and internalization of SARS-CoV-2. Our finding suggests that diltiazem has potential as a drug against SARS-CoV-2 infection and that Ca_v1.2 α_1c is a promising target for antiviral drug development for COVID-19.     

The SARS-CoV-2 RNA polymerase is a viral RNA capping enzyme

SARS-CoV-2 is a positive-sense RNA virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic, which continues to cause significant morbidity, mortality and economic strain. SARS-CoV-2 can cause severe respiratory disease and death in humans, highlighting the need for effective antiviral therapies. The RNA synthesis machinery of SARS-CoV-2 is an ideal drug target and consists of non-structural protein 12 (nsp12), which is directly responsible for RNA synthesis, and numerous co-factors involved in RNA proofreading and 5′ capping of viral RNAs. The formation of the 5′ 7-methylguanosine (m⁷G) cap structure is known to require a guanylyltransferase (GTase) as well as a 5′ triphosphatase and methyltransferases; however, the mechanism of SARS-CoV-2 RNA capping remains poorly understood. Here we find that SARS-CoV-2 nsp12 is involved in viral RNA capping as a GTase, carrying out the addition of a GTP nucleotide to the 5′ end of viral RNA via a 5′ to 5′ triphosphate linkage. We further show that the nsp12 NiRAN (nidovirus RdRp-associated nucleotidyltransferase) domain performs this reaction, and can be inhibited by remdesivir triphosphate, the active form of the antiviral drug remdesivir. These findings improve understanding of coronavirus RNA synthesis and highlight a new target for novel or repurposed antiviral drugs against SARS-CoV-2.     

MD-Miner: a network-based approach for personalized drug repositioning

Background

Due to advances in next generation sequencing technologies and corresponding reductions in cost, it is now attainable to investigate genome-wide gene expression and variants at a patient-level, so as to better understand and anticipate heterogeneous responses to therapy. Consequently, it is feasible to inform personalized drug treatment decisions using personal genomics data. However, these efforts are limited due to a lack of reliable computational approaches for predicting effective drugs for individual patients. The reverse gene set enrichment analysis (i.e., connectivity mapping) approach and its variants have been widely and successfully used for drug prediction. However, the performance of these methods is limited by undefined mechanism of action (MoA) of drugs and reliance on cohorts of patients rather than personalized predictions for individual patients.

Results

In this study, we have developed and evaluated a computational approach, known as Mechanism and Drug Miner (MD-Miner), using a network-based computational approach to predict effective drugs and reveal potential drug mechanisms of action at the level of signaling pathways. Specifically, the patient-specific signaling network is constructed by integrating known disease associated genes with patient-derived gene expression profiles. In parallel, a drug mechanism of action network is constructed by integrating drug targets and z-score profiles of drug-induced gene expression (pre vs. post-drug treatment). Potentially effective candidate drugs are prioritized according to the number of common genes between the patient-specific dysfunctional signaling network and drug MoA network. We evaluated the MD-Miner method on the PC-3 prostate cancer cell line, and showed that it significantly improved the success rate of discovering effective drugs compared with the random selection, and could provide insight into potential mechanisms of action.

Conclusions

This work provides a signaling network-based drug repositioning approach. Compared with the reverse gene signature based drug repositioning approaches, the proposed method can provide clues of mechanism of action in terms of signaling transduction networks.

     

Drug response prediction by ensemble learning and drug-induced gene expression signatures

Chemotherapeutic response of cancer cells to a given compound is one of the most fundamental information one requires to design anti-cancer drugs. Recently, considerable amount of drug-induced gene expression data has become publicly available, in addition to cytotoxicity databases. These large sets of data provided an opportunity to apply machine learning methods to predict drug activity. However, due to the complexity of cancer drug mechanisms, none of the existing methods is perfect. In this paper, we propose a novel ensemble learning method to predict drug response. In addition, we attempt to use the drug screen data together with two novel signatures produced from the drug-induced gene expression profiles of cancer cell lines. Finally, we evaluate predictions by in vitro experiments in addition to the tests on data sets. The predictions of the methods, the signatures and the software are available from http://mtan.etu.edu.tr/drug-response-prediction/.     

Synergy of Omeprazole and Praziquantel In Vitro Treatment against Schistosoma mansoni Adult Worms

BackgroundTreatment and morbidity control of schistosomiasis relies on a single drug, praziquantel (PZQ), and the selection of resistant worms under repeated treatment is a concern. Therefore, there is a pressing need to understand the molecular effects of PZQ on schistosomes and to investigate alternative or synergistic drugs against schistosomiasis.MethodologyWe used a custom-designed Schistosoma mansoni expression microarray to explore the effects of sublethal doses of PZQ on large-scale gene expression of adult paired males and females and unpaired mature females. We also assessed the efficacy of PZQ, omeprazole (OMP) or their combination against S. mansoni adult worms with a survival in vitro assay.ConclusionsFunctional analysis of gene interaction networks is an important approach that can point to possible novel synergistic drug candidates. We demonstrated the potential of this strategy by showing that PZQ in combination with OMP displayed increased efficiency against S. mansoni adult worms in vitro when compared with either drug alone.