key: cord-0892423-qgqt76qz
authors: Gupta, R.; Nwachuku, E.; Zusman, B.; Jha, R.; Puccio, A.
title: Drug Repurposing of potential drug targets for treatment of COVID-19
date: 2021-06-08
journal: nan
DOI: 10.1101/2021.06.02.21258223
sha: 4a4347647053ea573efb2739723eed9e796556bb
doc_id: 892423
cord_uid: qgqt76qz

Drug repurposing has the potential to bring existing de-risked drugs for effective intervention in an ongoing pandemic-COVID-19 that has infected over 131 million, with 2.8 million people succumbing to the illness globally (as of April 04, 2021). We have used a novel `gene signature'-based drug repositioning strategy by applying widely accepted gene ranking algorithms to prioritize the FDA approved or under trial drugs. We mined publically available RNA sequencing (RNA-Seq) data using CLC Genomics Workbench 20 (QIAGEN) and identified 283 differentially expressed genes (FDR<0.05, log2FC>1) after a meta-analysis of three independent studies which were based on severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infection in primary human airway epithelial cells. Ingenuity Pathway Analysis (IPA) revealed that SARS-CoV-2 activated key canonical pathways and gene networks that intricately regulate general anti-viral as well as specific inflammatory pathways. Drug database, extracted from the Metacore and IPA, identified 15 drug targets (with information on COVID-19 pathogenesis) with 46 existing drugs as potential-novel candidates for repurposing for COVID-19 treatment. We found 35 novel drugs that inhibit targets (ALPL, CXCL8, and IL6) already in clinical trials for COVID-19. Also, we found 6 existing drugs against 4 potential anti-COVID-19 targets (CCL20, CSF3, CXCL1, CXCL10) that might have novel anti-COVID-19 indications. Finally, these drug targets were computationally prioritized based on gene ranking algorithms, which revealed CXCL10 as the common and strongest candidate with 2 existing drugs. Furthermore, the list of 283 SARS-CoV-2-associated proteins could be valuable not only as anti-COVID-19 targets but also useful for COVID-19 biomarker development.

A novel coronavirus (CoV) began at the end of 2019 in Wuhan, China: it infected over seventy thousand individuals within the first fifty days of the epidemic with 1800 individuals succumbing to the disease [1] . The World Health Organization (WHO) declared a public health emergency of international concern on January 30 that escalated to a pandemic on March 11, 2020 [2] . Amino acid and nucleotide sequencing studies confirmed this new CoV belonged to the same species as severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1) [3] . The

International Committee on Taxonomy of Viruses (ICTV) named this new CoV as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the disease as CoV disease 2019 [4] . SARS-CoV-2 consists of four structural proteins including spike (S) glycoprotein, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. Host cell binding and entry are mediated by the S protein. The S1 subunit of the S protein can mediate entry into human respiratory epithelial cells by interacting with cell surface receptors like angiotensin-converting enzyme 2 (ACE2). Entry also requires S protein priming by cellular proteases like Type II transmembrane serine protease (TMPRSS2), resulting in S protein cleavage and fusion of viral and airway cellular membranes [5] . Patients with infected airway epithelium can present with a range of symptoms including fever, cough, and shortness of breath, often culminating in acute lung injury, acute respiratory distress syndrome, pulmonary failure, and even death [6] . As of April 04, 2021, more than 30 million cases of COVID-19 have been reported with over 1.6% mortality rate in the U.S. alone [7] . According to WHO's International Clinical Trials Registry Platform (ICTRP), 8,936 projects including 5,126 interventional studies are registered related to COVID-19 across the globe (searched on April 04, 2021) [8] . With the possible exception of dexamethasone and anti-inflammatory therapy, the currently available medications have questionable and limited efficacy against improving outcomes after COVID- 19 . The WHO SOLIDARITY trial (an international clinical trial to seek an effective treatment for COVID- 19) results have shown that remdesivir (FDA approved drug for COVID-19 treatment) has little or no efficacy on mortality rate in hospitalized patients with COVID-19 [9] . Therefore, there is an urgent need to further identify novel medical therapies both for preventive and therapeutic use. Drug repurposing could significantly curtail the time and cost of development compared to de novo drug discovery, as toxicity and safety data are often available All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.02.21258223 doi: medRxiv preprint from former clinical trial phases [10] . In silico approaches based on functional annotations proffer novel testable hypotheses for systematic drug repurposing [11] .

RNA-sequencing (RNA-Seq) based transcriptional profiling of SARS-CoV-2 infected airway epithelial cells provides a remarkable opportunity for understanding the relationship between infection-triggered gene expression signature and viral pathogenesis. A meta-analysis using differentially expressed genes (DEGs) obtained from RNA-Seq studies has the potential to advance our understanding of SARS-CoV-2 pathogenesis and facilitates the process of anti-COVID-19 drug repurposing. This current study systematically and quantitatively combined analysis of multiple RNA-Seq studies using a meta-analysis approach. This helps decrease the inconsistency in individual studies by increasing the sample size and statistical power to enlist more robust SARS-CoV-2-associated genes [12] . Furthermore, existing, approved drugs with opposing effects on these SARS-CoV-2-associated genes could have the potential to reverse COVID-19 symptoms. Therefore, we employed an in silico approach to discover potential anti-COVID-19 drug targets and suggest a priority for repurposing drugs which are FDA approved or under clinical trial investigation as potential COVID-19 therapies. Finally, this work provides insights into the untested but potential gene targets in relation to COVID-19 pathogenesis.

This study utilized the CLC Genomics Workbench 20.0.3 database [13] to search for deposited RNA-Sequencing experiments related to SARS-CoV-2 infections that matched the following criteria: only whole transcriptome studies; experiments carried out in human airway epithelial cells infected with SARS-CoV-2 in vitro; and availability of the raw data (.fastq files) for each sample. We searched on Dec 03, 2020 with search terms: "SARS-CoV-2", "SARS-CoV-2 pandemic", "SARS-CoV-2 host response", "SARS-CoV-2 transcriptome", "COVID-19", "COVID-19 pandemic", "COVID-19 host response", and "COVID-19 transcriptome". We identified three independent studies shown in Table 1 and selected the reads associated with SARS-CoV-2 or mock-treated airway epithelial cells. Details of selected reads and experimental conditions are given in Table S1 . All rights reserved. No reuse allowed without permission.

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Raw reads (.fastq files) were downloaded from CLC genomics workbench 20.0.3 [13] . A quality check for raw reads was performed. Low-quality bases (Phred score<20) and adapters were excluded. Trimmed reads were mapped to the human_sequence_hg38 reference genome to verify valid reads. Reads were extracted, counted, and normalized. Genes with false discovery rate (FDR)-adjusted p-values<0.05 and log2 fold change (log2FC)>1 were considered to be statistically significant ( Figure 1 ).

The gene datasets were analyzed for disease and disorders, molecular and cellular functions, and canonical pathways using Ingenuity Pathway Analysis (IPA) version 60467501 [14] . Only experimentally observed studies associated to the human species were used for IPA analysis. Fisher's exact test was used to calculate a p-value determining the probability that All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.02.21258223 doi: medRxiv preprint overlap with each canonical pathway is due to chance alone. In contrast to canonical pathways, which are relatively immutable in IPA, gene networks were also generated de novo in IPA based on the list of genes that are imported. IPA takes Network Eligible molecules from the gene list, searches the Ingenuity Knowledge Base, and uses a network algorithm to draw connections between molecules based on biological function [14] . 

To establish a link between SARS-CoV-2-related genes to drugs, we used a commercial drug database of Metacore version 20.3 build 70200 from Clarivate Analytics [15] and IPA [14] .

To highlight the most promising drugs that might be repurposed for COVID-19 treatment, only drug targets that were either FDA approved or had been examined in clinical trials were selected.

From these drug databases, we extracted information on drug name, drug target, and drugs' mechanism of action.

The list of all genes involved in the pathogenesis of COVID-19 was extracted (as of April 04, 2021) from the IPA database. Information on specific target activity and the effect of each target on disease/function were examined. Target pathogenesis information with the drugs' mechanisms of action retrieved from the drug databases was used to rationally shortlist promising anti-SARS-CoV-2 drugs.

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This meta-analysis identified 283 DEGs at FDR<0.05 and log2FC>1 (Table S2) The IPA network algorithm created 25 connection network between molecules based on biological function (Table S3D ) and ranked them as per the network score. The score takes into All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.02.21258223 doi: medRxiv preprint account the number of Network Eligible molecules in the network and its size, as well as the total number of Network Eligible molecules analyzed and the total number of molecules in the Ingenuity Knowledge Base that could potentially be included in networks. The higher the score, the lower the probability of finding the observed number of Network Eligible molecules in a given network by random chance. Figure 3 shows three most relevant networks. Network-1 (score=35; Figure 3A ) was associated with 23 genes of our dataset and was associated with 'Cellular movement, hematological system development and function, immune cell trafficking'. This network was characterized by key cytokines IL6, IL16, CXCL1, CXCL3, CXCL5, CXCL6, CXCL8, CXCL10, and CCL20. Network-2 (score=25; Figure 3B All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. supports the ability of our approach to identify anti-SARS-CoV-2 drugs and suggests its potential to discover novel anti-COVID-19 indications for existing drugs. We also identified 35 drugs targeting ALPL, CXCL8, and IL6 that were not previously tested against COVID-19 symptoms and could be repurposed for anti-SARS-CoV-2 management. We additionally found 4 potential targets (CCL20, CSF3, CXCL1, CXCL10) with 6 existing drugs that could be repurposed for the treatment of COVID-19. Eight further targets (CXCL6, IFI44, IFI44L, RSAD2, S100A8, SPRR2A, SYNE1, XAF1) associated with COVID-19 pathogenesis were identified, but drugs of any therapies that are either approved for another indication or being studied in a clinical trial. All rights reserved. No reuse allowed without permission.

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We subsequently implemented publicly available online gene ranking algorithms to Table 2, Table S5 ).

Another online gene ranking algorithm, ToppNet has mapped training and test set genes to protein-protein interaction network. Scoring and ranking of test set genes based on relative location to all of the training set genes using global network-distance measures in the proteinprotein interaction network (PPIN) ( Table 2, Table S5 ). CXCL10 was the topmost prioritized target with the maximum score analyzed by ToppGene and ToppNet. CXCL10 is involved in the regulation of inflammatory and immune responses ( Figure 4 ) and may be a potential candidate target for treating COVID-19 related lung pathology.

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(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (Table 2 ). Our drug discovery pipeline suggested an additional 35 existing drugs (Table S4 ) targeting these molecules that can be repurposed. Importantly, our meta-analysis identified 4 potential novel targets (CCL20, CSF3, CXCL1, CXCL10) associated with COVID-19 pathogenesis, targeted by 6 existing drugs that have not been studied in clinical trials for treatment of COVID-19 and could be repurposed. An additional 8 targets (CXCL6, IFI44, IFI44L, RSAD2, S100A8, SPRR2A, SYNE1, XAF1) are associated with COVID-19 pathogenesis, but do not have therapies targeting them available for potential repurposing. The list of 283 SARS-CoV-2-related proteins could be useful not only as potential anti-COVID-19

targets, but could also be considered COVID-19 biomarkers of disease progression and/or response to therapy.

The interaction network among these 283 gene points to core hub genes that could be responsible for altered molecular and cellular functions 'cell movement', 'cell death & survival'

and 'cell signaling/proliferation' associated with enriched diseases 'infection' and 'inflammatory response' following SARS-CoV-2 infection in airway epithelium. Inflammatory response in viral infection is a double-edged sword: Although inflammation is necessary to orchestrate the All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.02.21258223 doi: medRxiv preprint recruitment and coordination of immune cells at the infection site, over-stimulation of the inflammatory response results in a surge in cytokines and could culminate into secondary injury or destruction of the respiratory epithelium. Our IPA analysis showed that activation of cytokines IL6, IL16, CXCL1, CXCL3, CXCL5, CXCL6, CXCL8, CXCL10, and CCL20 are a hallmark signature of cytokine response in COVID-19 patients [17, 18] . We also found increased levels of key pro-apoptotic factors like BCL2A1, LTF, SOX6, SPP1, and SYNE1. Li et al. has recently detected apoptosis in bronchial and lung epithelial cells during the initial exudative phase (day 2-4 post infection) of SARS-CoV-2 infection in humanized ACE2 transgenic mice [19] . These proapoptotic factors could represent key nodes associated with epithelial cell death and subsequent diffuse alveolar damage (a critical feature of acute lung injury). Further, canonical pathway analysis suggested an enriched 'IL17 signaling' pathway. IL17 family cytokine levels are associated with respiratory viral infections [20] . It regulates key inflammatory cytokines like CSF3, TNFα, IL6, IL-1β, CXCL10, IL-8, MIP2A, MMPs that control granulopoiesis and recruitment of neutrophils, fever, chemoattraction, and tissue damage and remodeling [21] .

There is a growing body of evidence supporting the role of IL17 in COVID-19 pathogenesis [20, 22] We further used globally accepted ranking algorithms to prioritize the drug targets. These algorithms analyzed the targets' functional and topological similarity to established COVID-19 pathogenesis genes and both scored CXCL10 the highest priority drug target. CXCL10 has been implicated in SARS-CoV [23] and other viral infections such as rhinovirus, respiratory syncytial virus (RSV), Coxsackie virus, hepatitis virus B and C, Ebola, dengue (DENV), and equine infectious anemia virus (EIAV) [24] . Ichikawa et al. have shown that mice deficient in CXCL10 or its receptor CXCR3 have decreased lung injury severity and increased survival after viral and non-viral lung injury [25] . CXCL10 levels were positively correlated with the extent of organ damage and pathogen burden [26] . Interestingly, increased levels of CXCL10 were found in plasma samples of patients with COVID-19 who died [17] . Furthermore, Chua et al. have All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. [29] . The safety profile seems favorable, with the most commonly reported treatment-related adverse event being a mild to moderate headache (5% patients) with 10 or 20 mg/kg (intravenous infusion) doses [30] , though the optimal treatment dose and safety profile would need to be investigated specifically in the COVID-19 patient population.

Fenofibrate is another therapy that may reduce CXCL10 activity and is hypothesized to have therapeutic activity in inflammatory diseases like Crohn's disease [31] . Fenofibrate may also decrease the expression of other cytokines (IL17, CCL2, and CCL20) implicated in COVID-19 pathology [31] . As a peroxisome proliferator-activated receptor alpha (PPARα) agonist, fenofibrate may also prevents phospholipid accumulation within SARS-CoV-2 infected cells, blocking viral replication [32] . It may also suppress microvascular inflammation and apoptosis through inhibition of nuclear factor-κB and activation of adenosine monophosphate (AMP)activated protein kinase [33] , suggesting the fenofibrate may further have favorable systemic anti-inflammatory and endothelial effects.

In conclusion, systematic analyses of the SARS-CoV-2 triggered gene signature in airway epithelium revealed 15 protein targets linked to 46 existing drugs. This include 35 drugs modifying the activity of molecules already being studied as therapeutic targets for COVID-19 disease (IL6, ALPL, CXCL8 targets) and which could likely be repurposed for a similar aim.

Our study also found 4 additional targets (CCL20, CSF3, CXCL1, CXCL10) with existing All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.02.21258223 doi: medRxiv preprint therapies that have yet to be trialed in the COVID-19 patient population. CXCL10 appears to be a particular strong candidate based on high target scores and the availability of two existing drugs inhibiting the action of this cytokine. Our study has several limitations. This investigation is based on in vitro RNA-Seq data, resulting in an under-appreciation of significant inter-cellular signaling that may occur differently in the human body. Furthermore, our computational approach is limited as a tool for evaluating drugs to be repurposed because most available computational tools are used for small molecule drugs only. However, given the pressing need for effective targeted therapies for the treatment of COVID-19, further studies are crucially needed to experimentally validate these results and, if promising, rapidly transition to clinical trials. Table S1 . Details of selected reads and experimental conditions for each GEO dataset Table S2 . A complete list of differential expressed genes in airway epithelium after SARS-CoV-2 infection identified in meta-analysis. Table S3 . List of (A) Disease and disorders, (B) cellular and molecular functions, (C) canonical pathways, and gene networks that were enriched in the meta-analysis of three RNA-sequencing assays associated with SARS-CoV-2 infected (in vitro) airway epithelium All rights reserved. No reuse allowed without permission.

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The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.02.21258223 doi: medRxiv preprint Table S4 . Potential anti-SARS-CoV-2 drug targets with existing FDA approved or clinical trial drugs. Table S5 . Ranking of the potential AD drug targets using (A) ToppGene and (B) ToppNet web tools.

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The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.02.21258223 doi: medRxiv preprint

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Clinical features of patients infected with 2019 novel coronavirus in Wuhan

Attenuating the Effects of Novel COVID-19 (SARS-CoV-2) Infection-Induced Cytokine Storm and the Implications

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CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin

CXCL10-CXCR3 enhances the development of neutrophil-mediated fulminant lung injury of viral and nonviral origin

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Eldelumab [anti-interferon-γ-inducible protein-10 antibody] induction therapy for active Crohn's disease: a randomised, double-blind, placebo-controlled phase IIa study

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Fenofibrate suppresses microvascular inflammation and apoptosis through adenosine monophosphate-activated protein kinase activation

Authors thank to Molecular Biology Information Service of the Health Sciences Library System, University of Pittsburgh for support on data analysis by CLC Workbench Genomics 20and Ingenuity pathway analysis (IPA).

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