Introduction
SARS-CoV-2, the severe acquired respiratory syndrome agent coronavirus 2, has taken many lives in the past year and is continuing to create an unsafe environment. Along with numerous mutations emphasizing the quasispecies nature of the virus [
1,
2], fast transmission and a wide range of symptoms, lack of a definite therapeutic intervention has made this virus all the more deadly. Due to the ongoing mutations new variants such as Delta variants emerge with higher or different COVID-19 manifestations [
3]. Many studies have been conducted in order to recognize small compound therapeutics effective against SARS-CoV-2. Interestingly, some estrogen receptor modulators and protein synthesis inhibitors having potential antiviral against SARS-CoV2 have also been identified [
4]. It has been shown that
ESR1 as a drug target can modulate certain coronavirus associated genes. A group recently demonstrated the downregulation of
ACE2 (Angiotensin-converting enzyme 2) by estrogen [
5]. It has been shown that ACE-2 protein on the epithelial surface is the primary entry receptor for SARS-CoV-2 [
6].
Several herbal medicines and natural sources have been used to isolate antiviral compounds. Some of the examples of FDA approved antivirals from natural sources include Oseltamivir from star anise (Illicium verum) and Acyclovir from Carissa eduli [
7].
Cissampelos pareira L. is a commonly used hormone modulator which is used to treat reproductive disorders and fever in Ayurveda and also other healing systems. It has also been found to inhibit three serotypes of dengue [
8] and its effect on various hormones has also been evidenced [
9]. In a previous study we have observed that
Cipa can act as both, a protein synthesis inhibitor and an estrogen receptor inhibitor [
10]. Many of the drugs positively connected with
Cipa have been reported to be a potential antiviral agent. Since there were several overlaps between the therapeutics predicted to be effective against SARS-CoV-2 and our formulation, we decided explore the repurposing potential of
Cipa for this current pandemic. We found that Cipa can inhibit viral replication and Cipa compounds could directly to the SARS-CoV-2 spike protein, RNA‑dependent RNA‑polymerase (RdRp) and 3C‑like proteinase.
Discussion
SARS-COV-2 has shown a very diverse set of clinical presentations in various populations and among genders within the same population. It has been shown that estrogen can regulate the expression of ACE-2 receptors [
5]. Although a few known herbal medicines such as extracts of
Ganoderma lucidum,
Perilla frutescens, and
Mentha haplocalyx have been shown to be effective against SARS-CoV-2 infection [
33], only
Ganoderma lucidum has shown effect on estrogen modulation [
34]. Since
Cipa appears to have
ESR1 modulatory effects [
10], and has been shown to have antiviral potential [
8], we hypothesized it may have inhibitory effect on the novel coronavirus. CMAP analysis of
Cipa transcriptome signatures highlights several small compounds having been predicted to have inhibitory activity against SARS-CoV-2. Among these, emetine, homoharringtonine, and cycloheximide are known translation inhibitors. These have also been shown to inhibit Zika and Ebola [
35], SARS and MERS [
23], and Newcastle disease virus [
36]. Apcidin, an HDAC inhibitor has been predicted to inhibit SARS-CoV-2 in a recent study [
37].
In vitro experiments for viral inhibition of SARS-COV-2, reveal that all whole plant extracts of
Cipa (aqueous and alcoholic) could inhibit the virus at least up to 60%. Hydroalcoholic whole plant extract showed an inhibition of 98%. The single molecule constituents of Cipa could also inhibit the viral particles, with pareirarine showing the highest inhibition of 80%. This showed that
Cipa does have the potential to inhibit SARS-CoV-2 virus in vitro. Interestingly, the highest inhibition is shown by the whole plant hydroalcoholic extract which comprises of various small compound constituents. This suggests a synergistic effect of the constituents towards viral inhibition. Interestingly, among the tested constituent compounds, pareiraine was reported for the first time from
Cissampelos pareira in a study carried for antiplasmodial activity [
13]. Cissamine belongs to the protoberberine class of isoquinoline alkaloids and antiviral activity of cissamine was reported here for the first time. But berberine being the most prominent compound of protoberberine class exhibited antiviral activity against HCV, HPV, HIV-1 [
38] and H1N1 influenza A virus [
38,
39]. Magnoflorine belongs to the aporphine class of isoquinoline alkaloids and such types of alkaloids are reported to have antiviral activity against Herpes simplex virus [
40] and human poliovirus [
41]. However, there are no individual reports regarding the antiviral activity of magnoflorine. In one study, methanolic leaves extract of
Magnolia grandiflora containing magnoflorine was observed to have high antiviral activity against Herpes simplex virus (HSV-1) and possessed moderate antiviral activity against poliovirus type-1 (PV1) [
42].
In silico ligand binding assays clearly indicates the molecular therapeutic mechanism of Cipa bioactive compounds against the tested SARS-CoV2 drug targets. The in silico results presented herein corroborates with in vitro SARS-CoV2 inhibition properties of the tested compounds. Amongst the all tested Cipa compounds against three major SARS-CoV2 therapeutic targets, the 3CL -pro binding inhibitor, holds immense promise in context of its highly conserved binding site in contrast to the rapidly evolving ACE2 binding landscape of the RBD in different viral pathovariants. Furthermore, the observed synergistic antiviral property of Cipa hydroalcholic extracts in vitro, may result from the unique ability of the constituent bioactive molecules to simultaneously target several SARS-CoV2 therapeutic targets as observed in the in silico docking experiments presented here. Importantly, other than the three major viral therapeutic targets tested here, multiple crucial host derived proteins/protein complexes also play trivial role in SARS-CoV2 disease pathogenesis. Structural investigation of bioactive ligand binding to host derived therapeutic targets would further open up new arenas of future anti-SARS-CoV2 therapeutic inventions.
We also found that the signatures of the transcriptomic changes in
Cipa treated MCF7 cells and BALF from patients’ lungs, have interesting overlaps. Among the connected small compounds triciribine, torin-1 and VU-0365114–2, triciribine has been shown to inhibit Human Immunodeficiency virus sera types 1 and 2 [
43]. Another study has shown that VU-0365114–2, which is a muscarinic acetylcholine receptor M5 inhibitor, has repurposing potential against SARS-CoV-2 [
44]. While mTOR inhibitor torin-1 may modulate immune activity and enhance antiviral response, even against SARS-COV-2 [
45].
There are a number of factors which modulate the viral-host interactions which can be explored for better understanding the mechanism of action of promising antivirals such as interactions with the membrane components such as lipid rafts and ACE2, and other key non-structural proteins of the virus [
6,
46]. Interestingly, GPER, the G-protein coupled estrogen receptor is often found associated with lipid rafts and have been reported to modulate estrogenic effects in the immune cells [
47,
48]. It is worthwhile to note that in our previous study [
10], we found that
Cipa can down regulate this receptor as well. Since estrogen has been reported to effect covid related disease progression and fatality [
5,
49,
50], it will be prudent to study the interaction of GPER and associated lipids in the plasma membrane with viral proteins and explore their potential as drug targets.
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