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Cava et al. BMC Pharmacology and Toxicology (2020) 21:65 https://doi.org/10.1186/s40360-020-00444-z RESEARCH ARTICLE Open Access A protein interaction map identifies existing drugs targeting SARS-CoV-2 Claudia Cava1* , Gloria Bertoli1 and Isabella Castiglioni2 Abstract Background: Severe acute respiratory syndrome coronavirus (SARS-CoV-2), an emerging Betacoronavirus, is the causative agent of COVID-19. Angiotensin converting enzyme 2 (ACE2), being the main cell receptor of SARS-CoV-2, plays a role in the entry of the virus into the cell. Currently, there are neither specific antiviral drugs for the treatment or preventive drugs such as vaccines. Methods: We proposed a bioinformatics analysis to test in silico existing drugs as a fast way to identify an efficient therapy. We performed a differential expression analysis in order to identify differentially expressed genes in COVID- 19 patients correlated with ACE-2 and we explored their direct relations with a network approach integrating also drug-gene interactions. The drugs with a central role in the network were also investigated with a molecular docking analysis. Results: We found 825 differentially expressed genes correlated with ACE2. The protein-protein interactions among differentially expressed genes identified a network of 474 genes and 1130 interactions. Conclusions: The integration of drug-gene interactions in the network and molecular docking analysis allows us to obtain several drugs with antiviral activity that, alone or in combination with other treatment options, could be considered as therapeutic approaches against COVID-19. Keywords: COVID-19, SARS-CoV-2, Drug, Network, In silico analysis, Molecular docking Background CoV) in 2002–2003, MERS-CoV in 2012 and SARS- Coronaviruses are a group of single-stranded RNA vi- CoV-2 in 2019. Compared to SARS-CoV or MERS-CoV, ruses with a wide range of vertebrate hosts. SARS-CoV-2 spreads more rapidly making it difficult to Coronavirus infection in humans is characterized by a control [1]. wide spectrum of clinical manifestations ranging from Recently, a first model of infection has been proposed mild influenza symptoms to acute respiratory syndrome [2] in which, after an initial phase of viral exposure, the with damages in lung function, pneumonia, arrhythmia, local innate immune response generates natural IgA and or death [1]. IgM antibodies. In this first stage (5–6 days after infec- Betacoronavirus (β-CoV) is the second of four genera tion) patients show their first symptoms limited to upper of coronaviruses: alpha, beta, gamma and delta. Over the airways (cough, sore throat) with fever, fatigue and past two decades, three highly pathogenic human beta- muscle ache. If SARS-CoV-2 (the virus of COVID-19) coronaviruses have spread from animals to humans: Se- blocks the innate immunity, it spreads initially from the vere Acute Respiratory Syndrome Coronavirus (SARS- upper airways to the alveoli, causing pneumonia and dyspnea, and releasing high amounts of viral particles. * Correspondence: [email protected] While many subjects remain asymptomatic or with mild 1Institute of Molecular Bioimaging and Physiology, National Research Council (IBFM-CNR), Via F. Cervi 93, 20090 Segrate-Milan, Milan, Italy symptoms, in other individuals the response of adaptive Full list of author information is available at the end of the article immunity, releasing IgM and IgG antibodies, causes © The Author(s). 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Cava et al. BMC Pharmacology and Toxicology (2020) 21:65 Page 2 of 11 severe symptoms such as acute respiratory distress syn- Furthermore, from the Genotype-Tissue Expression drome (ARDS), due to spreading of inflammation, often (GTEx) project we collected gene expression levels of leading to complications (dyspnea, respiratory failure, lung tissues from healthy subjects (320 healthy fulminant myocarditis) that require the use of intensive volunteers). therapy. In the last phase, COVID-19 could cause death due to ARDS, acute cardiac and kidney injury, sepsis, Differential expression, pathway and network analysis and secondary infections. High mortality has been ob- We normalized and filtered RNA-seq raw counts using served in patients with older age or comorbidities the reference of hg19, following the pipeline of the R/ (hypertension, diabetes, cardiovascular disease, chronic Bioconductor package TCGAbiolinks [6]. In particular, lung disease and cancer). we used the function TCGAanalyze_Normalization Several studies reported that the first sites of infection implementing the EDASeq protocol [7], to perform seem to hit pneumocytes and enterocytes of small intes- within-lane and between-lane normalization. We ad- tine [3]. The molecular mechanisms of SARS-CoV-2 justed the data for differences in gene length and distri- entry, as suggested also for SARS-CoV [4], is mediated butional differences between samples [7]. by the binding of the viral spike glycoprotein to the Differential expression analysis between lung biopsies angiotensin-converting-enzyme-2 (ACE2) receptor, in from postmortem COVID-19 positive male patients and collaboration with a serine protease, TGRBSS2. This al- negative male controls was performed on GSE147507 lows the viral capsid fusion with the cell membrane [4]. gene expression levels with the R-package TCGABiolinks SARS-CoV-2 is a new virus for our immune system. [6]. Specifically, we used a generalized linear model which The IgG antibodies produced by other β-CoV do not is similar to linear model, but it assumes the negative bi- recognise this SARS-CoV-2, making this virus currently nomial distribution of RNA-Seq counts. P-values, gener- invincible. A possibility would be the use of immuno- ated from the differential expression analysis, were globulin collected from immunized COVID-19 patients corrected using the Benjamini–Hochberg procedure for to be injected in patients suffering from COVID-19 [5]. multiple-testing correction [6]. We defined the differen- In silico approaches on the study of the binding of exist- tially expressed genes if FDR < 0.01 and |log. FC| > 1. ing antiviral drugs on cellular receptors to block viral Pathway enrichment analysis was performed with the protein entrance could be a fast and low-cost method to R-package clusterProfiler [8]. In particular, we per- test a large amount of possible therapies. formed an enrichment test for KEGG pathways based on Furthermore, immunotherapy approach combined hypergeometric distribution [9]. We identified biological with antiviral drugs detected by in silico analyses may be functions that are over-represented in the list of differ- a stronger treatment until definitive options such as vac- entially expressed genes [10]. cines are available. We generated a protein-protein interaction network In this work we propose in silico study to identify considering the direct connections among differentially genes deregulated in COVID-19 positive patients corre- expressed genes in COVID-19 using SpidermiR [11] and lated with ACE2. Then, we performed a network analysis we selected the network with the most connected nodes. based on protein-protein interactions with the aim of We calculated for each node the degree centrality (d.c.) identifying existing drugs targeting SARS-CoV-2 deregu- that indicates how many neighbours a node has. This lated genes, to be proposed as candidate drugs for index is usually used to identify the nodes that have a COVID-19, alone or in combination with other thera- central role in the network [11]. peutic strategies. The protein interactions that emerge Drug-gene interactions were derived by the package from our study could help to decipher their mechanisms rDGIdb [12]. of action with respect complex biological processes at the basis of COVID-19 infection and progression. The Correlation drugs obtained were also investigated through docking A correlation analysis was performed between ACE2 simulations. and the other genes in GTEx data (lung tissues from normal volunteers) to obtain the genes co-expressed Methods with ACE2. The corresponding p-values of the Pearson’s Gene expression datasets correlation were considered and only ACE2 and genes We collected the gene expression levels of lung tissues significantly correlated (p-value < 0.001) were used for infected by SARS-CoV-2 from the Gene Expression the subsequent analysis. Omnibus (GEO) database. Specifically, we selected from GSE147507 2 lung biopsies from postmortem COVID- Community detection 19 positive male patients and 2

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