key: cord-0840304-dbmt2ydl
authors: Haider, Madiha; Anand, Vivek; Dholakia, Dhwani; Enayathullah, M. Ghalib; Parekh, Yash; Ram, Sushma; Kumari, Surekha; Anmol,; Bokara, Kiran Kumar; Sharma, Upendra; Prasher, Bhavana; Mukerji, Mitali
title: Anti-SARS-CoV-2 potential of Cissampelos pareira L. identified by Connectivity map-based analysis and in vitro studies
date: 2021-06-13
journal: bioRxiv
DOI: 10.1101/2021.06.11.448155
sha: cd675a82e50a89ceb15b3df5dcf7c3bd56597c82
doc_id: 840304
cord_uid: dbmt2ydl

Background Viral infections have a history of abrupt and severe eruptions through the years in the form of pandemics. And yet, definitive therapies or preventive measures are not present. Purpose Herbal medicines have been a source of various antiviral compounds. An accelerated repurposing potential of antiviral herbs can provide usable drugs and identify druggable targets. In this study, we dissect the anti-coronavirus activity of Cissampelos pareira L (Cipa). using an integrative approach. Methods We analysed the signature similarities between predicted antiviral agents and Cipa using the connectivity map (https://clue.io/). Next, we tested the anti-SARS-COV-2 activity of Cipa in vitro. A three-way comparative analysis of Cipa transcriptome, COVID-19 BALF transcriptome and CMAP signatures of small compounds was also performed. Results Several predicted antivirals showed a high positive connectivity score with Cipa such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its prominent pure constituents e.g pareirarine, cissamine, magnoflorine exhibited 40-80% inhibition. Comparison of genes between BALF and Cipa showed an enrichment of biological processes like transcription regulation and response to lipids, to be downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that Triciribine, torin-1 and VU-0365114-2 had positive connectivity with BALF 1 and 2, and negative connectivity with Cipa.

Background: Viral infections have a history of abrupt and severe eruptions through the 33 years in the form of pandemics. And yet, definitive therapies or preventive measures are 34 not present. 35

Purpose: Herbal medicines have been a source of various antiviral compounds. An 36 accelerated repurposing potential of antiviral herbs can provide usable drugs and identify 37 druggable targets. In this study, we dissect the anti-coronavirus activity of Cissampelos 38 pareira L (Cipa). using an integrative approach. 39

Methods: We analysed the signature similarities between predicted antiviral agents and 40

Cipa using the connectivity map (https://clue.io/). Next, we tested the anti-SARS-COV-2 41 activity of Cipa in vitro. A three-way comparative analysis of Cipa transcriptome, COVID-19 42 BALF transcriptome and CMAP signatures of small compounds was also performed. 43

Results: Several predicted antivirals showed a high positive connectivity score with Cipa 44 such as apcidin, emetine, homoharringtonine etc. We also observed 98% inhibition of 45 SARS-COV-2 replication in infected Vero cell cultures with the whole extract. Some of its 46 prominent pure constituents e.g pareirarine, cissamine, magnoflorine exhibited 40-80% 47

inhibition. Comparison of genes between BALF and Cipa showed an enrichment of 48 biological processes like transcription regulation and response to lipids, to be 49 downregulated in Cipa while being upregulated in COVID-19. CMAP also showed that 50

Triciribine, torin-1 and VU-0365114-2 had positive connectivity with BALF 1 and 2, and 51

negative connectivity with Cipa. 52 53

Keywords: Cissampelos pareira L., SARS-CoV-2, BALF, antivirus, whole plant extract, 54 Cissampelos pareira L. is a commonly used hormone modulator which is used to treat 78

reproductive disorders and fever. It has also been found to inhibit three serotypes of 79 dengue (3) and its effect on various hormones has also been evidenced (4). In a previous 80 study we have observed that Cipa can act as both, a protein synthesis inhibitor and an 81 estrogen receptor inhibitor (5). Many of the drugs positively connected with Cipa have 82 been reported to be a potential antiviral agent. Since there were several overlaps between 83 the therapeutics predicted to be effective against SARS-CoV-2 and our formulation, we 84 decided explore the repurposing potential of Cipa for this current pandemic.

Material and Methods: 87

Transcriptome meta-analysis: 88 We obtained the raw RNA sequencing data from SARS-CoV-2 patients Broncho-alveolar 89 lavage fluid (BALF) 1 and 2 from the recent publications and analyzed them inhouse 90 (supplementary information 1.1). The gene expression data for Cissampelos pareira L. was 91 taken from (5), which can be accessed at GSE156445.

Functional enrichment and connectivity map of the differentially expressed genes.

The differentially expressed genes were analyzed for functional enrichment using enrichr 95 (6) and for similar signatures using clue.io (7). The results were then compared with Cipa 96 to find intersections between gene ontologies, enriched gene sets, and connectivity map 97

perturbations between upregulated genes of BALF 1 and 2 and downregulated genes of 98

Cipa and between downregulated genes of BALF 1 and 2 and upregulated genes of Cipa. 99

Enrichments were also done for the genes whose knockdown signatures score >90 100 connectivity with Cipa and then compared with the upregulated processes and gene sets in 101 SARS-CoV-2. 102 103

Whole plant of Cissampelos pareira L was collected from the Palampur, HP, India (alt. 1350 105 m). The identification of the plant material was done by a taxonomy expert in CSIR-IHBT, 106

Palampur and a voucher specimen (no. PLP16688) was deposited in the herbarium of 107 CSIR-IHBT, Palampur, HP-176,061, India. The plant has been obtained and isolated as per 108

CSIR, India guidelines (details for extract preparation in supplementary information 1.2). 109

The pure molecules from roots of the plant were isolated as reported recently (8) 

Since metanalysis highlighted an inhibitory potential of Cipa against SARS-COV-2, we tested 146 the effect of whole plant and root extracts of Cipa in Vero cell culture assays infected with 147 SARS-CoV-2. The relative viral RNA (%) was calculated by considering the values averaged 148 from N (Nucleoprotein), ORF1ab (19 non-structural proteins, NSP1-16), and E (Envelope) 149 viral genes. The whole plant aqueous extract showed a definite antiviral activity, evidenced 150 by decreased relative viral RNA content with a reduction by 57% at 100µg/ml where the 151 viral particle number reduced from 10 5.9 to 10 5.6 ( Figure 1A-C) . 152 153

Next, we wanted to see whether the Cipa pure constituents can inhibit SARS-COV-2. The 154

total alkaloid content in the extract was 46.4 mg/g with cissamine (18.6 mg/g) being the 155 major one, followed by magnoflorine (12.9 mg/g). The pure molecules namely hayatinin and 2 respectively in comparison with the virus control ( Figure 1D ).

Cipa transcriptome oppositely regulates several biological pathways compared to 163

We looked for the differentially expressed genes which were common between Cipa and 166 BALF samples from two studies (10, 11), hereby referred to as BALF-1 and BALF-2 167 respectively. We observed that 39 genes were common between Cipa and BALF-1 and 168

BALF-2, out of which 29 showed an opposite expression (Table S1) . Individually, 134 genes 169

were common between Cipa and BALF-1, and 174 genes common between Cipa and BALF-170 2. Upon functional enrichment analysis we observed that the genes enriched for regulation 171 of vascular endothelial growth were upregulated in both BALF-1 and 2 datasets while 172

being downregulated by Cipa. Similarly, while regulation of gene expression was 173 upregulated by Cipa it was downregulated in BALF-1 and BALF-2 data sets (Figure 2A-C) .

The connectivity map analysis of the gene signatures of BALF-1 and BALF-2 comparison 176

with Cipa results revealed a number of small compounds that had high positive scores with 177

Cipa and negative scores with either BALF transcriptomes. We observed several small 178 compounds having opposite signature similarities between Cipa and BALF. Triciribine, 179

torin-1 and VU-0365114-2 were three compounds with negative scores for Cipa and 180

positive scores for both BALF 1 and 2 ( Figure 2D ). Also, knockdown signatures of a number 181 of genes were found to exhibit opposing scores between Cipa and BALF transcriptomes.

Some of these include, MICALL1, CRK, FKBP1A, CBX4, and FGR10P ( Figure 2E) . We also found that the signatures of the transcriptomic changes in Cipa treated MCF7 cells 207

and BALF from patients' lungs, have interesting overlaps. Among the connected small 208 compounds triciribine, torin-1 and VU-0365114-2, triciribine has been shown to inhibit 209

Human Immunodeficiency virus sera types 1 and 2 (16). Another study has shown that VU-210 0365114-2, which is a muscarinic acetylcholine receptor M5 inhibitor, has repurposing 211 potential against SARS-CoV-2 (17). While mTOR inhibitor torin-1 may modulate immune 212 activity and enhance antiviral response, even against SARS-COV-2 (18).

In summary, we report here a framework applicable for repurposing of herbal 216

formulations using an integrated multi-pronged approach using transcriptome-based 217 connectivity mapping, in vitro validation and conjoint analysis with disease signatures. We 218

demonstrate the potential repurposing of Cissampelos pareira L for sars-cov-2 using this 219 approach. 220 221 nCoV/SARS-CoV-2. Cell Discov. 6, 14 (2020 

A SARS-CoV-2 protein interaction map reveals targets for drug 238 repurposing

Estrogen regulates the expression of SARS-CoV-2 receptor ACE2 in 240 differentiated airway epithelial cells

Cissampelos pareira Linn: Natural Source of Potent Antiviral Activity 243 against All Four Dengue Virus Serotypes

Antifertility activity of the 245 methanolic leaf extract of Cissampelos pareira in female albino mice

Transcriptome analysis and connectivity mapping of 248 <em>Cissampelos pareira</em> L. provides molecular links of ESR1 249 modulation to viral inhibition

Enrichr: a comprehensive gene set enrichment analysis web 251 server 2016 update

A Next Generation Connectivity Map: L1000 Platform and the 253 First 1,000,000 Profiles

Chemical profiling and quantification of potential active constituents 255 responsible for the antiplasmodial activity of Cissampelos pareira

The FDA-approved drug 258 ivermectin inhibits the replication of SARS-CoV-2 in vitro

Transcriptomic characteristics of bronchoalveolar lavage fluid and 261 peripheral blood mononuclear cells in COVID-19 patients

Heightened Innate Immune Responses in the Respiratory Tract of 264 COVID-19 Patients

Emetine inhibits Zika and Ebola virus infections through two molecular 266 mechanisms: inhibiting viral replication and decreasing viral entry

Repurposing of clinically developed drugs for treatment of Middle East 269 respiratory syndrome coronavirus infection

The Natural Compound Homoharringtonine Presents Broad 272

Clinical HDAC Inhibitors Are Effective Drugs to Prevent the Entry of 274 SARS-CoV2

Inhibition of Human Immunodeficiency Virus Type 1 by Triciribine 276 Involves the Accessory Protein Nef

Identification of Repurposable Drugs and Adverse Drug Reactions for 279 Various Courses of COVID-19 Based on Single-Cell RNA Sequencing Data

Immunoregulation with mTOR inhibitors to prevent COVID-19 282 severity: A novel intervention strategy beyond vaccines and specific antiviral 283 medicines

Based Docking to the SARS-CoV-2 Viral Spike Protein and Viral Spike Protein-Human 286 ACE2 Interface

Figure 1: Inhibition of SARS-CoV-2 in vitro by Cipa whole extract and its constituents: 335 Relative viral RNA % and Log reduction in viral particles in vero cells upon treatment at

150 and 200μg of A) whole plant aqueous extract (PE), B) root extract (PER) and C) 337

D) Sars-cov-2 viral titers inhibition by Cipa 338 constituents CP-2 Salutaridine, CP-3 Cissamine, CP-5 pareirarine, CP-7 Magnoflorine, PE 339 aqueous extract, PE50 50% hydroalcoholic extract and PER root extract