key: cord-0879620-0sdjy80s
authors: Hazra, Suvojit; Chaudhuri, Alok Ghosh; Tiwary, Basant K.; Chakrabarti, Nilkanta
title: Matrix metallopeptidase 9 as a host protein target of chloroquine and melatonin for immunoregulation in COVID-19: A network-based meta-analysis
date: 2020-07-15
journal: Life Sci
DOI: 10.1016/j.lfs.2020.118096
sha: c38cfc1d755381afc0c6361298c55ac3a2db2010
doc_id: 879620
cord_uid: 0sdjy80s

AIMS: The molecular pathogenesis of COVID-19 is similar to other coronavirus (CoV) infections viz. severe acute respiratory syndrome (SARS) in human. Due to scarcity of the suitable treatment strategy, the present study was undertaken to explore host protein(s) targeted by potent repurposed drug(s) in COVID-19. MATERIALS AND METHODS: The differentially expressed genes (DEGs) were identified from microarray data repository of SARS-CoV patient blood. The repurposed drugs for COVID-19 were selected from available literature. Using DEGs and drugs, the protein-protein interaction (PPI) and chemo-protein interaction (CPI) networks were constructed and combined to develop an interactome model of PPI-CPI network. The top-ranked sub-network with its hub-bottleneck nodes were evaluated with their functional annotations. KEY FINDINGS: A total of 120 DEGs and 65 drugs were identified. The PPI-CPI network (118 nodes and 293 edges) exhibited a top-ranked sub-network (35 nodes and 174 connectivities) with 12 hub-bottleneck nodes having two drugs chloroquine and melatonin in association with 10 proteins corresponding to six upregulated and four downregulated genes. Two drugs interacted directly with the hub-bottleneck node i.e. matrix metallopeptidase 9 (MMP9), a host protein corresponding to its upregulated gene. MMP9 showed functional annotations associated with neutrophil mediated immunoinflammation. Moreover, literature survey revealed that angiotensin converting enzyme 2, a membrane receptor of SARS-CoV-2 virus, might have functional cooperativity with MMP9 and a possible interaction with both drugs. SIGNIFICANCE: The present study reveals that between chloroquine and melatonin, melatonin appears to be more promising repurposed drug against MMP9 for better immunocompromisation in COVID-19.

J o u r n a l P r e -p r o o f (inclusion of loops i.e. self-edges in neighborhood density calculation) and 'degree cut-off 2' (number of connections necessary for a node to be scored). The cluster finding cut-off parameters were 'node score cut-off 0.2' (cluster size for selection of new members), hairfall included (deletion of all single connected nodes from clusters), fluff included (allowance of cluster expansion with one neighbor shells), node density cut-off 0.1 (controls the neighbor inclusion criteria during 'fluffing'), K-core cut-off 2 (filters out clusters by eliminating small inter-connected sub-cluster) and maximum depth of network cut-off 100 (limiting the distance from the seed node within which the cluster member can be searched).

The chemo-protein components of overall PPI-CPI interactome were analyzed in CentiScaPe 2.2 [34] plugin of Cytoscape using key network centrality parameters like node degree (the number of neighboring nodes to which the node of interest is directly connected), shortest path betweenness (the number of information streams passing through a given node) and stress (the extent to which a node can hold network communications) to identify the most potential nodes in J o u r n a l P r e -p r o o f

The list of genes of interest with their identifiers (ID) found in the top sub-network of PPI-CPI interactome was submitted to Enrichr web-tool platform (http://amp.pharm.mssm.edu/Enrichr) [35, 36] for the enrichment (i.e. over representation of common annotated biological features) analysis of functional annotations implemented across a number of resources including 'gene ontology' ('biological process', 'cellular component', 'molecular function'), 'KEGG biological pathways', 'Jensen Disease'. The Enrichr analysis was performed using statistical parameters viz. p-value (Fisher exact test), q-value (adjusted p-value for false discovery rate), old p-value, adjusted old p-value, odd ratio, z-score and combined score (log(p-value)×z-value) The Enriched results (functional annotations) were ranked based on the levels of significance with p-values < 0.05 and corresponding combined scores followed by selection of terms with certain cut-off (top 10 terms). (log(p-value)×z-value) J o u r n a l P r e -p r o o f

The results found in the present mechanistic systems biology analysis are systematically documented in the flow diagram with the findings of potential drugs against the targeted biomolecule(s) in COVID-19 ( Fig. 1 ).

From in silico analysis of SARS microarray dataset we identified 120 differentially expressed genes among which 45 genes were upregulated and 75 genes were downregulated. Details of the differentially expressed genes including gene identifiers (ID), "false discovery rate" (FDR) adjusted p-values (<0.05) and log 2 (fold change) values (>1) are summarized in Figure 2 .

The interactome model of PPI network was constructed using proteins corresponding to the respective DEGs of SARS-CoV patients in STRING webtool. A total of 72 protein nodes and 212 connections had been found in PPI network (data not shown). Literature survey provided a total of 65 potential drugs proposed for treatment of COVID-19 (Table 1 ) and these drugs were included in further analysis for selection of most potent drug(s) against putative protein target(s).

The interactome model of CPI network was constructed using COVID-19 drug candidates and J o u r n a l P r e -p r o o f DEGs of SARS-CoV patients in STITCH webtool. Total 88 nodes (proteins and drugs) and 120 connections had been found in the CPI network (data not shown).

The characterization of protein-drug interactions in COVID-19 had been executed through development of interactome model of PPI-CPI network in Cytoscape by merging the already identified PPI and CPI network and that was found to be composed of 118 nodes (proteins and drugs) interconnected by 293 interactions (Fig. 3A) . The identification of nodes of gene products/proteins and drugs has been designated by the corresponding gene IDs and name of the drugs respectively in the present study. Accordingly, the respective gene IDs have been used as the node (protein) identifiers in the further descriptions.

From the interactome model of PPI-CPI network, the MCODE module in Cytoscape identified only one top-ranked sub-network (cluster having MCODE score 9.33) that comprised 35 nodes J o u r n a l P r e -p r o o f

Gene enrichment analysis study (Fig. 5 ) indicated that the gene products of the top-ranked subnetwork were associated with biological processes including neutrophil related processes (neutrophil activation, neutrophil degranulation, neutrophil mediated immunity and immune responses), innate immune responses in mucosa, humoral immune responses mediated by antimicrobial peptides, antibacterial humoral responses, defence responses to bacteria with high statistical significances (Enrichr p-value < 0.05 and combined score). The association of the gene products of this sub-network were found to be statistically significant (Enrichr p-value < 0.05 and combined score) for the cellular components involving subcellular and granular secretary functions. Statistically significant (Enrichr p-value < 0.05 and combined score) molecular functions like nuclear hormone receptor bindings, iron and transition metal ion bindings, serinetype endopeptidase activities, endonuclease and ribonuclease activities and RNA polymerase-II core promoter proximal region sequence-specific DNA bindings were also associated with the gene products of this sub-network. In addition, KEGG pathway enrichment study included 

The present study developed a chemo-protein interactome network (Fig. 3A ) on basis of differentially expressed genes in SARS-CoV infection (Fig. 2) and repurposed drugs for COVID-19 (Table 1) . A sub-network (Fig. 3B ) was identified that appeared to be involved in neutrophil activation and degranulation pathways ( ARDS is the main cause of morbidities in COVID-19 and infections of other coronaviruses [5] [6] [7] 37, 38] . Cytokine storm is a key mechanism of ARDS materialization leading to multiorgan failure and death [5, 6, 39, 40] . Till date, the molecular pathogenesis of COVID-19 is unclear.

Therefore, the similar mechanism of SARS-CoV/MERS-CoV infection can confer a lot to the molecular level of understanding in pathogenesis of COVID-19 [6] . The present study identified a sub-network (Fig. 3B ) that includes the statistically significant pathways of both neutrophil activation and degranulation (Fig. 5) . The sub-network comprised 29.66% nodes (proteins) and provided 59.38% connectivities of the overall network, which indicated the colossal importance of this sub-network in the whole network model (Fig. 3A-B) . The global and local topological analyses provided ten potential protein molecules consisting of six proteins corresponding to These ten nodes appeared to be the hub nodes of the PPI-CPI interactome network and were chosen for further study (Fig. 4C ). All these molecules had been found to be highly enriched in blood after antimicrobial-induced neutrophil-mediated humoral and innate immunity responses in COVID-19 (Fig. 5 ).

In the present study, MMP9 was found as the functionally important hub node (protein) and target of the drugs in the central hub node of the PPI-CPI interactome network ( Fig. 3B and Fig   4C) . Matrix metallopeptidases (MMPs), the zinc-containing and calcium-dependent proteolytic endopeptidases, are released from the intracellular stores and become active extracellularly [41] .

MMPs cause deterioration of a number of extracellular matrix proteins that help in the extracellular matrix remodelling in various physiological and pathological processes including inflammation [41] [42] [43] [44] . MMPs are widely distributed in tissues and their expressions are controlled by cytokines, growth factors and hormones. MMP9 (83kD), a glycoprotein with type-IV collagenase activity, is found in neutrophils, lymphocytes and dendritic cells that are involved in angiogenesis and inflammatory cytokine generation [41, 44] . Notably, the Enrichr functional analysis in the current study revealed MMP9 as to be associated with transition ion binding (molecular function under gene ontology) and diverse pathophysiological conditions viz. (Fig. 5) . MMP9 is reported to relate with tertiary lumens for release of cytokines during neutrophil activation [53] . In monocytes and macrophages, MMP9 is over-activated by plasmin and reacts with 'toll like receptor 9' (TLR9) signalling to induce formation of tumour necrosis factor (TNF) that leads to the development of pro-inflammatory cytokine storm [54, 55] . The involvement of MMP9 with serine protease activities was also noticed in Enrichr functional analysis with molecular function under gene ontology (Fig. 5) . MMP9 deficiency is found to be protective against severe H1N1 influenza virus A infection in mice model [56] . Therefore, our J o u r n a l P r e -p r o o f findings and other reports support the view that the approach to MMP9 inhibition and/or alterations of its activators (plasmin) and inhibitors (TIMP1) may prevent the deadly cytokine storm and the life-risk of COVID-19 patients.

Here, both chloroquine and melatonin were identified as the repurposed drug candidates that appeared to be the interacting partners of MMP9 (Fig. 3B and Fig. 4) . Chloroquine treatment decreases serum MMP9 level in systemic lupus erythematosus [57] and suppresses MMP9 activity and its mRNA expression in breast cancer [58] . Chloroquine can also lower the TNFmediated neutrophil apoptosis, neutrophil degranulation and cytokine burst [59] [60] [61] [62] [63] .

Chloroquine/hydroxychloroquine efficiently inhibits the human retrovirus activities in vitro [64] .

Recently, a number of studies [17, 65, 66] and a clinical trial [67] support the use of chloroquine/hydroxychloroquine for the treatment of COVID-19. Conversely, certain clinical studies strongly disagree with its uses [68] [69] [70] , as it develops cardiac failure in COVID-19 patients [68] , indicating the clinical data about chloroquine/hydroxychloroquine are not conclusive [71] . Notably, hydroxychloroquine did not appear in the sub-network in the present study (Fig-3B ) and remained unattained for further consideration.

Melatonin is responsible for normal sleep and maintenance of the 'biological clock' in human [72] . It has anti-inflammatory properties [73, 74] , exerts its potential role in anti-viral mechanism [74] [75] [76] [77] [78] and has also been used in Ebola virus infection [78] . The pathophysiological observation in COVID-19 patients supports the chronobiological uses of melatonin in its treatment [79, 80] and a therapeutic algorithm in this regard has also been proposed recently [81] . In addition, melatonin is reported as the promising adjuvant for COVID-19 treatment [21, 82] and its J o u r n a l P r e -p r o o f deficiencies may enhance susceptibility of diabetic and hypertensive elderly patients in SARS-CoV-2 infection [80] . The first clinical trial of melatonin in a small cohort indicates its involvement in abating ARDS in COVID-19 patients [83] . Interestingly, melatonin can bind to the active site of MMP9 and inhibit the latter to arrest immunoinflammation [84] . Thus, the present network based meta-analysis justified the involvement of melatonin interaction with MMP9 in immunocompromised COVID-19.

The cell culture study (kidney cell line Vero E6) and network analysis reveal that chloroquine [85] and melatonin [21] respectively may have potential anti-viral role in targeting human cell membrane-bound ACE2 receptor, a zinc dependent carboxypeptidase, and other associated protein partners in COVID-19. Interestingly, separate studies report that MMP9 level increases in ACE2-knockout mice model [86] and ACE inhibitors (lisinopril and imidapril) also target MMP9 along with ACE2 [87] . Therefore, both metallopeptidases i.e. ACE2 and MMP9 may have cooperativities for immunoinflammation in COVID-19 and can be potential targets of both chloroquine and melatonin therapies. [6, [88] [89] [90] [91] [92] [93] . The ectodomain of membrane-bound ACE2 is shed into the extracellular fluid as its soluble form [88, [94] [95] [96] . In vitro studies also indicate that the soluble ACE2 can bind to SARS-CoV/SARS-CoV-2 and is supposed to limit the availability of viruses to interact with the membrane-bound ACE2 receptors thus minimizing the chances of viral load [88, 92, 94] . Notably, ACE2 converts angiotensin-II to angiotensin(1-7), a peptide which causes modification of inflammatory processes [97] . Angiotensin(1-7) also has a vasodilatory effect [98] and enhances the heart rate acting on the caudal ventrolateral medulla in the brain [99] , which may contribute to the development of hypotension and tachycardia as found in SARS/COVID-19 patients [100] .

Moreover, the interaction of SARS/SARS-CoV-2 with the ACE2 receptors of the support cells in olfactory mucosa also results in inflammation-induced anosmia [101] . 

Return of the Coronavirus: 2019-nCoV

A new coronavirus associated with human respiratory disease in China

We have therefore made the assessment that COVID-19 can be characterized as a pandemic

Clinical characteristics of coronavirus disease 2019 (COVID-19) in China: A systematic review and meta-analysis

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

Molecular immune pathogenesis and diagnosis of COVID-19

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting Wuhan

SARS coronavirus pathogenesis: host innate immune responses and viral antagonism of interferon

Interferon-α2b Treatment for COVID-19

Coronaviruses-drug discovery and therapeutic options

First Case of 2019 Novel Coronavirus in the United State

IRCCS. Recommendations for COVID-19 Clinical Management

The origin, transmission and clinical therapies on coronavirus disease 2019 (COVID-19) outbreak-an update on the status

Case of the Index Patient Who Caused Tertiary Transmission of Coronavirus Disease

Pneumonia Monitored by Quantitative RT-PCR

Potential interventions for novel coronavirus in China: A systematic review

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

The FDA-approved drug ivermectin inhibits the replication of SARS-CoV-2 in vitro

COVID-19 infection and rheumatoid arthritis: Faraway, so close!

Potent binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human monoclonal antibody

Network-based drug repurposing for novel coronavirus 2019-nCoV/SARS-CoV-2

Expression profile of immune response genes in patients with severe acute respiratory syndrome

Bioconductor: open software development for computational biology and bioinformatics

R: A language and environment for statistical computing, R Foundation for Statistical Computing

limma powers differential expression analyses for RNA-sequencing and microarray studies

Linear models and empirical bayes methods for assessing differential expression in microarray experiments

Controlling the false discovery rate: a practical and powerful approach to multiple testing

STRING v11: protein-protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets

Recent Aspects on the Pathogenesis Mechanism, Animal Models and Novel Therapeutic Interventions for Middle East Respiratory Syndrome Coronavirus Infections

High-throughput screening and identification of potent broad-spectrum inhibitors of coronaviruses

STITCH: interaction networks of chemicals and proteins

Cytoscape: A Software Environment for Integrated Models of Biomolecular Interaction Networks

An automated method for finding molecular complexes in large protein interaction networks

Analyzing biological network parameters with CentiScaPe

Enrichr: interactive and collaborative HTML5 gene list enrichment analysis tool

Enrichr: a comprehensive gene set enrichment analysis web server 2016 update

Coronavirus pathogenesis

MERS, SARS and other coronaviruses as causes of pneumonia

The mercurial nature of neutrophils: still an enigma in ARDS?

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

Seesaw of matrix metalloproteinases (MMPs)

Physiology and pathophysiology of matrix metalloproteases

Overview of MMP biology and gene associations in human diseases

MMP-9 in translation: from molecule to brain physiology, pathology, and therapy

Identification of common biological pathways and drug targets across multiple respiratory viruses based on human host gene expression analysis

Tissue inhibitors of metalloproteinases: structure, regulation and biological functions

Series "matrix metalloproteinases in lung health and disease" edited by J. Müller-Quernheim and O. Eickelberg number 1 in this series: Biological role of matrix metalloproteinases: a critical balance

Increased Ratio of Matrix Metalloproteinase-9 (MMP-9)/Tissue Inhibitor Metalloproteinase-1 from Alveolar Macrophages in Chronic Asthma with a Fast Decline Journal Pre-proof 5-Year Follow-up

Neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential

Neutrophilic inflammation in asthma and association with disease severity

Matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) as non-invasive biomarkers of remodelling in asthma

Transcriptomic characteristics of bronchoalveolar lavage fluid and peripheral blood mononuclear cells in COVID-19 patients

Biological roles of neutrophil-derived granule proteins and cytokines

Pharmacological targeting of plasmin prevents Journal Pre-proof activation syndrome

Plasmin regulation of acute cytokine storm

Matrix metalloproteinase-9 deficiency protects mice from severe influenza A viral infection

Effect of chloroquine phosphate treatment on serum MMP-9 and TIMP-1 levels in patients with systemic lupus erythematosus

Chloroquine has tumor inhibitory and tumor promoting effects in triple negative breast cancer

Differential effects of the autophagy inhibitors 3-methyladenine and chloroquine on spontaneous and TNF-α-induced neutrophil apoptosis

Neutrophils and malaria

Neutrophil degranulation and cell lysis is associated with clinical severity in virusinduced asthma

A role for neutrophils in viral respiratory disease

Effects of amodiaquine, chloroquine, and mefloquine on human polymorphonuclear neutrophil function in vitro

Chloroquine and hydroxychloroquine as inhibitors of human immunodeficiency virus (HIV-1) activity

A systematic review on the efficacy and safety of chloroquine for the treatment of COVID-19

A systematic review on use of aminoquinolines for the therapeutic management of COVID-19: Efficacy, safety and clinical trials

Breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated pneumonia in clinical studies

Clinical efficacy of hydroxychloroquine in patients with covid-19 pneumonia who require oxygen: observational comparative study using routine care data

A Randomized Trial of Hydroxychloroquine as Postexposure Prophylaxis for Covid-19

Lack of efficacy of hydroxychloroquine in covid-19

Hydroxychloroquine or Chloroquine for Treatment or Prophylaxis of COVID-19: A Living Systematic Review

Melatonin and sleep in aging population

Melatonin: a pleiotropic molecule regulating inflammation

Melatonin possesses an anti-influenza potential through its immune Journal Pre-proof

Melatonin: its possible role in the management of viral infections-a brief review

Beneficial actions of melatonin in the management of viral infections: a new use for this "molecular handyman"?

Regression of herpes viral infection symptoms using melatonin and SB-73: comparison with Acyclovir

Ebola virus disease: potential use of melatonin as a treatment

Can melatonin reduce the severity of COVID-19 pandemic?

Is melatonin deficiency a unifying pathomechanism of high risk patients with COVID-19?

Therapeutic Algorithm for Use of Melatonin in Patients With COVID-19

COVID-19: Melatonin as a potential adjuvant treatment

Melatonin as adjuvant treatment for coronavirus disease 2019 pneumonia patients requiring hospitalization (MAC-19 PRO): a case series

Melatonin inhibits matrix metalloproteinase-9 activity by binding to its active site

Chloroquine is a potent inhibitor of SARS coronavirus infection and spread

Angiotensin-converting enzyme 2: the first decade

ACE inhibitors to block MMP-9 activity: new functions for old inhibitors

Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus

A pneumonia outbreak associated with a new coronavirus of probable bat origin

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

SARS-CoV-2 receptor ACE2 and TMPRSS2 are primarily expressed in bronchial transient secretory cells

Soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy?

Role of the ACE2/angiotensin 1-7 axis of the renin-angiotensin system in heart failure

Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor

Advances in the renin angiotensin system: focus on angiotensin-converting enzyme 2 and angiotensin-(1-7)

Ectodomain shedding of ACE and ACE2 as regulators of their protein functions

ACE2, angiotensin-(1-7) and M as receptor axis in inflammation and fibrosis

Role of the vasodilator peptide angiotensin-(1-7) in cardiovascular drug therapy

Angiotensin-(1-7) antagonist, A-779, microinjection into the caudal ventrolateral medulla of renovascular hypertensive rats restores baroreflex bradycardia

Cardiovascular complications of severe acute respiratory syndrome

Non-neural expression of SARS-CoV-2 entry genes in the olfactory epithelium suggests mechanisms underlying anosmia in COVID-19 patients