Metadichol®, a Novel Nano Lipid Formulation that

Inhibits SARS-COV-2 and a Multitude of Pathological

Viruses in vitro
Palayakotai Raghavan (  raghavan@nanorxinc.com )
NANORX Inc.

Research

Keywords: Coronavirus, SARS-COV-2, COVID-19, ACE, ACE2, TMPRSS2, VDR, Metadichol

Posted Date: March 26th, 2021

DOI: https://doi.org/10.21203/rs.3.rs-34021/v8

License:   This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License

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Abstract
New pathogenic virus outbreaks, occurring with increasing regularity, are leading us to explore novel
approaches, which will reduce the reliance on time-consuming vaccine modes to halt the outbreaks. The
requirement is to find a universal approach to disarm any new and as yet unknown viruses as they
appear. A promising approach could be targeting lipid membranes, which are common to all viruses and
bacteria.

The ongoing pandemic of severe acute respiratory syndrome-coronavirus 2 (SARS-COV-2) has reaffirmed
the importance of interactions between components of the host cell plasma membrane and the virus
envelope as a critical mechanism of infection. Metadichol®, a nano lipid emulsion, has been examined
and shown to be a strong candidate to help stop the proliferation of SARS-COV-2.

Naturally derived substances, such as long-chain saturated lipid alcohols, reduce the infectivity of various
types of viruses, including coronaviruses such as SARS-COV-2, by modifying lipid-dependent attachment
to human host cells. SARS-COV-2 uses the receptor ACE2 for entry and the serine protease TMPRSS2 for
S protein priming.

Metadichol®, a nano lipid formulation of long-chain alcohols, has been shown to inhibit TMPRSS2 (EC50
96 ng/ml). Compared to the inhibitor Camostat mesylate (CM) which has a EC50 of 26000 ng/ml, it is
270 times more potent. Additionally, Metadichol® is an inhibitor of ACE with an EC 50 of 71 ng/ml but
extremely weak inhibitor of ACE2 at 31 µg/ml. Further a live virus assay in Caco2 cells, Metadichol®
inhibited SARS-CoV-2 replication with an EC90 of 0.16 µg/ml.

Introduction
There is currently an increasing need for a broad-spectrum antimicrobial agent that could inactivate
human pathogens, such as bacteria and viruses. Rapid resistance by microorganisms has propelled this
approach to the development of focused drugs. The most recent trigger is the fear of a future pandemic
caused by new, poorly studied virulent strains, such as the present SARS-COV-2.

Background information on SARS-COV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) (causative agent of COVID-19) is causing
a pandemic1 that has produced global havoc within a few months. Medically controlling a rapidly
spreading viral pandemic utilizing specific antivirals and vaccines will prove expensive and time
consuming and is accompanied by compromises on safety and efficacy. An alternative approach is to
test molecules that are already proven safe for effectiveness against SARS-COV-2. Among the candidates
being tested are camostat mesylate (a 35-year-old Japanese drug), Avigan (another Japanese drug) and
Gilead Science Inc.'s remdesivir2.

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To enter a host cell, SARS-COV-2 requires transmembrane protease serine 2 (TMPRSS2)3, a serine
protease, and angiotensin-converting enzyme 2 (ACE2)4 to bind and thus facilitate its entry. Blocking
TMPRSS2 is the key to blocking the virus from binding to ACE2 and stops the cell entry mechanism used
by the virus.

TMPRSS2 is a protease that primes the spike protein of SARS-COV and the Middle East respiratory
syndrome-related coronavirus (MERS-COV). Camostat mesylate (CM), an inhibitor of TMPRSS2, inhibited
SARS-COV in a mouse model5,6. Hoffmann et al.7 determined that SARS-COV-2 requires TMPRSS2. They
showed that CM blocks virus entry into the lungs. To date, there are no clinical data on the use of CM in
patients.

The other receptor used by viruses to enter the host cell is ACE2. SARS-COV-2 has a spike (S) protein on
its viral envelope (exterior) that binds to the transmembrane protein ACE2, which is present on human
cells. ACE2 is essential for viral entry. However, ACE2 also regulates blood pressure and blood volume;
blocking ACE2 would be detrimental to health. An approach that inhibits TMPRSS2 would thus be an
ideal solution.

Lipids and viruses

Viral envelope lipids play a role in both viral stability and infective capabilities. For example, substances
that affect the lipid envelope, such as phospholipases, organic solvents, and surfactants, such as soaps,
have been shown to affect viral infectivity. Causing envelope disintegration, they stop virus transmission
to a new host. Active ingredients8 in a number of cleaning agents, wipes, and tissues target the viral lipid
envelope to render the virions nonviable. Snipes and coworkers9 showed that saturated alcohols could
inactivate viruses with chain lengths from 10 to 14 carbons. Their studies established that inactivation of
enveloped viruses by lipids varies greatly, depending on both the nature of the lipid and the type of virus.
Hilmarsson et al.10-12 studied the virucidal effects of medium- and long-chain (8 to 18 carbon) fatty
alcohols and corresponding lipids against herpes simplex viruses (HSV-1 and HSV-2), respiratory
syncytial virus (RSV), human parainfluenza virus type 2 (HPIV2) and enveloped viruses at various
concentrations, times and pH levels. After a 10-minute incubation at 37 °C with a 10 mM concentration,
14 of the lipids tested caused a 100000-fold or more significant reduction in HSV titer. Testing between
pH 7 and 4.2 showed that a pH of 4.2 caused a more rapid inactivation of HSV-1 virus titer in one minute
than higher pH values. These long-chain alcohols may act by penetrating the envelope of the virus by
hydrophobic effects, making it permeable to small molecules and thus inactivating the virus; the degree
of penetration into lipid membranes is based on the chain length of a lipid compared with the thickness
of the membrane13.

Metadichol ® is a nano lipid formulation of long-chain alcohols14. Metadichol has been shown to inhibit
viruses in vitro and in vivo15-17. Metadichol was tested for its inhibitory actions against ACE2 , ACE (
Angiotensin-converting enzyme) , and TMPRSS2 and in an antiviral assay with SARS-COV-2.

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Discussion
The results in Table 1 and 2 demonstrate Metadichol's direct antiviral effect against the SARS-COV-2
virus in Caco-2 cells, with an EC90 of 0.15 µg/ml. Comparatively, this result indicates that Metadichol has
a 2000-fold higher effectiveness than Remdesivir and 4000-fold potency over hydroxychloroquine
phosphate18..

A previously published work15 of antiviral data against other viruses is shown in Tables 3 and 4. Raw
data show the cytotoxicity of Metadichol without a virus present in Vero cells was measured by neutral
red assay. When >75% "toxicity" occurred in the absence of virus, no viral CPE value was reported.

These results suggest that it is toxic to cells at concentrations above 5 ug/ml in most cases. However,
Metadichol is not toxic, as the LD50 is 5000 mg/kg19-21. It is likely that Metadichol at higher
concentrations behaves in a soap-mimicking manner by disrupting the lipid membrane, and at lower
concentrations, it neutralizes the virus by a different mechanism. Metadichol is not toxic to cell lines, but
rather, it behaves as a "detergent" in neutralizing SARS-COV-2 and other pathogenic viruses shown in
Table 5. Additionally, Metadichol® targets selectively cancer cells in this case Caco-2 cells. In a previous
study22 of Klotho gene expression in the cancer cell lines Mia-Paca, Colo 205, and Panc1, Metadichol was
seen to be toxic to cell lines above 1 µg/ml. It is also toxic at 10 µg/ml in leukemia cancer cells 23.

Metadichol also inhibits TMPRSS2 ( Table 6 figure 1,2) and is 270-fold more potent than CM 24 but for all
practical purposes does not inhibit ACE2 ( Table 7, figures 3 and 4) and with TMPRSS2 inhibition,
prevents viral entry. The reported results open the gateway to effective and safe therapies for COVID-19.
Metadichol is a inhibitor of ACE ( table 8 and figures 5 and 6 ) . The inhibition of ACE is important in
mitigating COVID-19 infections as Guan et al, 25 reported data from 1099 patients confirmed that the
single highest risk factor was hypertension in 15% of the patients.

Vitamin D and SARS-COV-2 infection

An uncontrolled inflammatory response to SARS-COV-2 is the major cause of disease severity and death
in patients with COVID-19 26 and is associated with high levels of circulating cytokines, tumor necrosis
factor (TNF), CCl2, C-reactive protein (CRP), and Ferritin. Metadichol 14 is an inhibitor of CCl2 (also known
as MCP-1), TNF, NF-kB, and CRP, which is a surrogate marker for cytokine storms 27 and is associated
with vitamin D deficiency.

Vitamin D3 is generated in the skin through the action of UVB radiation, with 7-dehydrocholesterol
generated in the skin, followed by a thermal reaction. Vitamin D3 is converted to 25(OH)D in the liver and
then to 1,25(OH)2D (calcitriol) in the kidneys. Calcitriol binds to the nuclear vitamin D receptor (VDR); a
DNA-binding protein interacts with regulatory sequences near target genes that participate genetically
and epigenetically in the transcriptional output of genes needed for function 28. Vitamin D reduces the
risk of infections by mechanisms that include inducing cathelicidins and defensins 29, resulting in

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lowered viral replication rates and reducing concentrations of pro-inflammatory cytokines 30.
Supplementation with 4000 IU/d vitamin D decreased dengue virus infection31. Inflammatory cytokine
levels increase in viral and bacterial infections, as seen in COVID-19 patients. Vitamin D can reduce the
production of pro-inflammatory Th1 cytokines, such as TNF and interferon (IFN) 32.

Vitamin D is a modulator of adaptive immunity 33 and suppresses responses mediated by T helper type 1
(Th1) cells primarily by repressing the production of the inflammatory cytokines interleukin (IL)-2 and IFN-
gamma34. Additionally, 1,25(OH)2D3 promotes cytokine production by T helper type 2 (Th2) cells, which
helps enhance the indirect suppression of Th1 cells by complementing this suppression with actions
mediated by a multitude of cell types 35.

1,25(OH)2D3 promotes T regulatory cell induction, thereby inhibiting inflammatory processes 36 . It is

known that COVID-19 is associated with the increased production of pro-inflammatory cytokines, elevated
CRP levels, increased risk of pneumonia, sepsis, acute respiratory distress syndrome (ARDS), and heart
failure 37. Case fatality rates (CFRs) in China were 6%-10% for those with cardiovascular disease, chronic
respiratory tract disease, diabetes, and hypertension 38. Metadichol is an inverse agonist/protean agonist
14
of VDR i.e. it binds at the same site as calcitriol but has different properties. It is the only known inverse
agonist to VDR known in medical literature.

Telomerase and viral infections

Metadichol at one picogram increases h-TERT (telomerase) expression by 16-fold 39. Viral infection
places a significant strain on the body. CD8 T cells that mediate adaptive immunity40 to protect the body
from microbial invaders can easily reach their Hayflick limit by depleting their telomeres41. This
possibility is more likely if telomeres are already short. Infections put enormous strain on immune cells to
replicate. Naive T and B cells are particularly important when our bodies encounter new pathogens, such
as SARS-COV-2. The quantity of these cells is crucial for useful immune function.

AHR and viral infections

One of the major issues with infected COVID-19 patients has been respiratory failure. It has been
suggested that the aryl hydrocarbon receptor (AHR) is activated during coronavirus infections, impacting
antiviral immunity and lung cells associated with repair 42. Signaling via AHR may dampen the immune
response against coronavirus 43. It has been reported that although some signaling is needed for
coronavirus replication, excessive activation of this pathway may be deleterious for the virus. AHR limits
activation and interferes with multiple antiviral immune mechanisms, including IFN-I production and
intrinsic immunity. Yamada et al.44 suggested that AHR (the constitutive aryl hydrocarbon receptor)
signaling constrains type I IFN-mediated antiviral innate defense and suggested a need to block
constitutive AHR activity; only an inverse agonist can dampen this activity. We have shown that

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Metadichol® binds to AHR as an inverse/protean agonist 45 and thus can reduce complications
attributed to uncontrolled inflammation and cytokine storms.

Vitamin C and viral infections

In infectious diseases, there is also a need to boost innate and adaptive immunity. The micronutrients
with the most robust evidence for immune support are vitamins C and D. Vitamin C is essential for a
healthy and functional host defense. The pharmacological application of vitamin C enhances immune
function 46. Vitamin C has antiviral properties leading to inhibition of the replication of HSV-1, poliovirus
type 1, influenza virus type 47, and rabies virus in vitro 48.

Vitamin C deficiency reduces cellular 49-53 and humoral immune responses, and treatment of healthy
subjects promoted and enhanced natural killer (NK) cell activities 54, underlining the immunological
importance of vitamin C 55,56 and supporting its role as a crucial player in various aspects of immune cell
functions, such as immune cell proliferation and differentiation, in addition to its anti-inflammatory
properties. Moreover, the newly characterized hydroxylase enzymes, which regulate the activity of
hypoxia-inducible factor gene transcription and cell signaling of immune cells, need vitamin C as a
cofactor for optimal activity 57-59. Metadichol administration increases vitamin C levels endogenously by
recycling vitamin C and produces levels not reached by oral intake, and those reached bring about
changes in improving diverse biomarkers 60-62.

Gene cluster network analysis in Covid-19 infections.

The present drug discovery paradigm is based on the idea of one gene-one target, one disease. It has
become clear that it is difficult to achieve single target specificity. Thus, the need to transition from
targeting a single gene to targeting multiple genes is likely to become more attractive, leading to blocking
multiple paths of disease progression 63,64. Gene network analysis can provide a minimum set of genes
that can form the basis for targeting diseases. This clustering network of genes can modulate gene
pathways and biological networks. We used www.ctdbase.org65, which has curated genes show in Table
9 relevant to COVID-19. Table 10 lists genes and diseases states that they are involved in.

We can filter the 13 genes to a set of 5 genes: TNF, CCL2, ACE2, TMPRSS2 and AGT , which is part of the
renin-angiotensin system (RAS) network that ACE2 is a part of (Figure 7 ). All these are modulated by
Metadichol via its binding to VDR. A similar analysis of these network genes shows that they are closely
networked in diseases, with a highly significant p-value. These five genes are closely related, and the
network generated, is shown in Figure 8, using www.innatedb.org66. This analysis integrates known
interactions and pathways from major public databases. The highlighted ones in yellow are SIRT1, AR
(androgen receptor) and FOS.

Glinsky 67 suggested vitamin D as a potential mitigation agent in preventing SARS-COV-2 entry.

Metadichol binds to VDR, which controls the expression of FOS 68. VDR regulates Sirt1 69 which in turn

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regulates AR 70 which controls the expression of TMPRSS2. Figure 9, generated using PACO 71, shows the
gene network and regulation relationships. VDR controls FOS expression, FOS controls AGT and AGT
controls the expression of AGTR1 and ACE, Wambier and Goren 72 have suggested that SARS-COV-2
infection is likely to be androgen mediated. The first step to infection is the priming of the SARS-COV-2
spike proteins by TMPRSS2, which also cleaves ACE2 for augmented viral entry. This key step of priming
is completely blocked by Metadichol. SIRT1 also can plays an active role in enhancing immunity in viral
infections73.

Proteases such as Furin74 and Adam-17 have been described to activate the spike protein in vitro for viral
spread and pathogenesis in infected hosts. VDR controls Furin expression, mediated through its
interaction with SRC75. Adam-17 is regulated via CEPBP76,77, which is involved in the regulation of genes
involved in immune and inflammatory responses. Recently, Ulrich and Pillat 78 proposed that CD147,
similar to ACE2, is another receptor used for viral entry. CD147 is a known receptor79 for the parasite that
causes malaria in humans, Plasmodium falciparum. Metadichol (see Ref 6) inhibits the malarial parasite.

Controlling cytokine storms

A cytokine storm develops after an initial immune response by the induction of cytokines. The response
to SARS-COV-2 leads to inflammation. There are increased levels of the pro-inflammatory cytokines IL-6,
IL-18, TNF, and IL-1-beta by macrophages and of IFN-gamma by NK cells.

Figure 10 was generated, using of PACO shows the cytokine relationship network. The cytokines can
activate T cells, which lead to tissue damage and infection in the lungs. Infiltration of T cells can also
result from the upregulation of adhesion molecules, such as ICAM1, by lung endothelial cells. Metadichol
is an inhibitor (see Ref14, US patent 8,722,093) of TNF alpha in vivo, and ICAM1 and CCl2 depress the
hyper inflammatory cytokine response caused by SARS-COV-2 and, at the same time, enhance innate and
adaptive immunity through the VDR pathways and increased vitamin C levels. Metadichol, by its binding
to VDR, leads to a network of gene control of the cytokine storms illustrated in Figure 7, bringing about
homeostasis.

The key to entry into cells by SARS-COV-2 is ACE2, which, when endocytosed with SARS-COV-2, results in
a reduction in ACE2 on cells and an increase in serum Angiotensin II 80. Angiotensin II acts as a
vasoconstrictor and a pro-inflammatory cytokine (Figure 11) via AT1R81 . The Angiotensin II-AT1R axis
leads to a pro-inflammatory state 82, leading to infections through activation of NF-KB and to increased
IL-6 levels in multiple inflammatory and autoimmune diseases 83.

The dysregulation of angiotensin 2 downstream of ACE2 leads to the cytokine release that is seen in
COVID-19 patients, resulting in increased TNF levels that lead to elevated IL-6, CCl2, and CRP levels.
Cytokine storms84 result in ARDS. Metadichol is an ACE inhibitor and blocks the Ang 1 to Ang 2 pathway
and driving the pathway towards an anti-inflammatory state,

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Clinical

A pilot study done by a third party Kasturaba Hospital in Mumbai India, on thirty COVID-19 patients with
minor symptoms showed the absence of a virus in 75% of patients after 4 days of Metadichol treatment
@ 20 mg per day. To validate this further, we have been initiated a larger study where we will have
Metadichol treatment group and control groups, with only Standard Care. We hope to communicate these
results in the near future.

Summary And Conclusions

Metadichol inhibits SARS-COV-2 entry into host cells by inhibiting TMPRSS2 and this allows ACE2 to
carry out its critical role in Renin Angiotensin pathway . Also the antiviral response is enhanced by
increased innate and adaptive immunity through the vitamin D pathway and antiviral activity by
endogenously increasing vitamin C levels. In addition, telomerase activity can also play a key role in
maintaining the levels of naive T and B cells needed to fight infections. Metadichol modulates cytokine
storms, as it is an inhibitor of TNF, ICAM1 and CCL2, which, as shown, play a key role with other
cytokines. Co morbidities associated85,86 with COVID-19, such as hypertension and diabetes87,88, are also
controlled by Metadichol, which could certainly improve the long-term prognosis for the affected patient
population. These actions on multiple genes and via multiple pathways bring about homeostasis and
prevent SARS-COV-2 infections. Metadichol’s 89 actions on multiple genes lead to over 2000 unique
interactions with other genes and result in a network that helps bring about homeostasis.

Metadichol is a safe, nontoxic product made from renewable sources and have been commercially
available for the last six years, with no reported side effects. This unique property allows for the use of
Metadichol as an immune modulator to prevent future occurrence of SARS-COV-2 and possibly other
infections being predicted, facilitating a rapid return to normal human social and economic activity
worldwide.

Glossary Of Gene Descriptions

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 Gene Description

VDR vitamin D receptor

AHR aryl hydrocarbon receptor

TERT telomerase reverse transcriptase

KL klotho

PAI1 (SERPINE1) serpin family E member 1

HIF 1 alpha hypoxia-inducible factor 1-alpha

CCL2 C-C motif chemokine ligand 2

ICAM1 intercellular adhesion molecule 1

TNF tumour necrosis factor

ACE angiotensin I-converting enzyme

ACE2 angiotensin I-converting enzyme 2

AGTR1 (ANG1) angiotensin II receptor type 1

AGTR2 (ANG2) angiotensin II receptor type 2

TMPRSS2 transmembrane serine protease 2

SIRT1 sirtuin 1

TNF tumour necrosis factor

FURIN furin, paired basic amino acid cleaving enzyme

CD 147 (BSG) Basigin (BSG), also known as extracellular matrix metalloproteinase inducer

IL6 interleukin 6

IL10 interleukin 10

CCL3 C-C motif chemokine ligand 3

IL2 interleukin 2

IL7 interleukin 7

CSF3 Colony-stimulating factor 3

IL2RA interleukin 2 receptor subunit alpha

CXCL8 C-X-C motif chemokine ligand 8

Declarations
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Competing interest: Author has no competing interests

Correspondence and requests for materials should be addressed to raghavan@nanorxinc.com

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Methods
All assays were on a fee-for-service contract basis and outsourced to bioanalytical testing companies
worldwide. SARS-COV-2 antiviral assays were performed by a bio-safety level 3 (BSL3) facility at the Anti-
viral research Institute at Utah State University in the USA. The other assays were performed at Southern
Research Infectious Disease Research Facility in Frederick, Maryland, USA and IBT Bio services in
Rockville, Maryland, USA. ACE2, ACE and TMPRSS2 Assays were carried out by by Skanda Life Sciences
Pvt Ltd Bangalore , India.

Antiviral assay

Metadichol was serially diluted using eight half-log dilutions in test medium (MEM supplemented with 2%
FBS and 50 µg/mL gentamicin) so that the starting (high) test concentration was 100 µg/ml. Each
dilution was added to 5 wells of a 96-well plate with 80-100% confluent Caco-2 cells.

Three wells of each dilution were inoculated with virus, with two wells uninoculated (as toxicity controls);
six wells were inoculated and untreated (as virus controls); and six wells were uninoculated and untreated
(as cell controls). SARS-COV-2 was prepared to achieve the lowest possible multiplicity of infection (MOI)
that would yield >80% cytopathic effect (CPE) within five days. M128533 (protease specific for SARS-
COV) was tested in parallel as a positive control. The plates were incubated at 37±2°C and 5% CO2. On
day three post-infection, once untreated virus control wells reached maximum CPE, plates were stained
with neutral red dye for approximately 2 hours (±15 minutes). The supernatant dye was removed, and the
wells were rinsed with PBS. The incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol
for >30 minutes, and the optical density was read on a spectrophotometer at 540 nm.

Optical densities were converted to percent of that of cell controls, and the concentration of compound
that would cause 50% cell death (CC50) in the absence of virus was calculated by regression analysis.
The selective index (SI) is the CC50 divided by the EC90. The results are shown in Table 7.

For a virus yield reduction (VYR) assay, the supernatant fluid from each compound concentration was
collected on day three post-infection before neutral red staining (3 wells pooled) and tested for virus titre
using a standard endpoint dilution CCID50 assay in Vero 76 cells and titre calculations via the Reed-
Muench (1948) equation. The concentration of compound required to reduce virus yield by one log10 was
calculated by regression analysis (EC90).

As shown in Table 2 the virus reduction assay did not follow a typical dose-response relationship, with
virus reduction seen at concentrations of 0.3 µg/ml and 3.2 µg/ml but no reduction seen at a
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concentration of 1 µg/ml. It was assumed that breakthrough of the virus at 1 µg/ml was an outlier. The
calculated SI was 20 (Table 1), indicating an EC90 of 0.15 µg/ml.

The results for other viruses shown in Table 3 and 4 and 5 were carried out in a similar procedure by
various labs using Vero cells.

TMPRSS2 inhibition assay

Procedure

TMPRSS purified from LNCaP cells (ATCC) was used as an enzyme source. The reaction mixture
contained purified TMPRSS2 protease in TBS with or without a range of various concentrations from
1.56 to 100 ng/ml of test sample or inhibitor. The reaction mixture was incubated for 10 minutes at 37°C.
To the reaction mixture, 1 µl of 10 mM the fluorogenic trypsin substrate Cbz-Gly-Gly-Arg-AMC was added,
and kinetic fluorescence readings were recorded after 2 minutes of incubation at 37°C at Ex383 nm and
Em455 nm at 5-10 minutes using a SpectraMax i3X (Molecular Devices). The change in fluorescence
(delta RFU) was calculated to determine the inhibitory effects of the test sample. CM ( Cayman
Chemicals) at a two-fold range of concentrations from 1.56 to 100 nM was used as a positive control for
TMPRSS2 protease inhibition.

ACE2 Inhibition assay

An ACE2 Inhibitor Screening Assay Kit, catalogue no. 79923 (BPS Biosciences, San Diego, USA), was
used to measure the exopeptidase activity of ACE2 and inhibition by Metadichol and the control inhibitor
DX600. The inhibitory activity was measured based on the fluorescence emitted by the cleavage of a
chromogenic substrate.

Procedure

Enzyme (ACE2) stocks were prepared from the supplied kit. Twenty micro litres of enzyme solution (0.5
ng/µl) was added to all the wells designated for the assay. DX600, a potent ACE2 inhibitor, was used as a
positive control for ACE2 inhibition at various concentrations ranging from 0.0156 µg/ml to 1 µg/ml. The
test sample at concentrations ranging from 0.125 µg/ml to 40 µg/ml was used. To each well containing
the enzyme solution, 5 µl of inhibitor solution was added to the respective designated wells. The reaction
mixture was incubated at room temperature for 5 minutes. After incubation, 25 µl of ACE2 substrate was
added to the mixture and incubated for 1 hour at room temperature. The RFU due to cleavage of the
substrate were read at Ex555 nm and Em585 nm using a Spectra Max i3x (Molecular Devices). The IC50
values were calculated based on the readings obtained.

ACE inhibition assay

ACE inhibitory activity of test sample was assessed using Angiotensin I Converting Enzyme (ACE)
Fluorometric Activity Assay Kit (Sigma, Cat. No., CS0002). The procedure was followed as per the

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manufacturer’s instructions with slight modifications

Sample Preparation

1. ACE positive control is used as a main enzyme source and working stock was prepared by diluting
the ACE 250-fold in assay buffer.
2. Desired concentrations of test samples of various concentrations were prepared using the sample
stock of 5mg/ml.
3. All the samples were prepared in a final volume of 25μl (2X concentration)

Assay procedure

All the reagents were equilibrated to 37 ˚C for 5 minutes before starting the assay. To the 96 well flat-
bottom black plates, 25μl of freshly prepared ACE enzyme was added and 25μl of various 2X
concentrations of freshly prepared test samples were added. The reaction mixture was gently mixed with
the pipette and the contents were incubated for 5 mins at RT. The reaction was initiated by adding 50μl of
100-fold diluted substrate for a final reaction volume of 100μl. The reaction mixture was incubated for 5
mins at RT and the fluorescence of the reaction was measured at excitation of 320nm and emission of
405nm at the end of the incubation.

Captopril was used as a positive control at various concentrations.

Tables
Table 1. In vitro antiviral assay results

CC50 EC90 SI90

Metadichol (µg/ml) 4 0.15 20

M128533 (µg/ml) >10 0.2 >33

CC50: 50% cytotoxic concentration of compound without virus added;

EC50: 50% effective antiviral concentration;

EC90: calculated concentration to reduce virus yield by 1 log (90%);

SI: CC50/EC50.

Table 2. Cytotoxicity and virus yield data for each concentration of Metadichol tested

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 Metadichol Concentration (µg/ml) Cytotoxicity (%) Virus Titre (CCID50 per 0.1 ml)

100 100% <0.7

32 100% <0.7

10 83% <0.7

3.2 54% 0.7

1 17% 4.3

0.3 26% 1.5

0.1 26% 5.7

0.03 26% 5.3

Table 3. Raw data for cytotoxicity of Metadichol without virus present, as measured by neutral red assay

Units are µg/ml unless

noted

Metadichol (µg/ml) Adenovirus Tacaribe Rift SARS Japanese West Nile Yellow fever
valley encephalitis virus Powassan virus

500 95% 98% 96% 96% 100% 100% 100% 100%

160 92% 98% 96% 95% 100% 100% 100% 100%

50 90% 97% 97% 95% 100% 100% 100% 100%

16 85% 95% 81% 92% 88% 77% 98% 100%

5 0% 23% 26% 35% 33% 28% 35% 44%

1.6 0% 2% 10% 15% 12% 14% 19% 6%

0.5 0% 3% 9% 0% 2% 3% 2% 0%

0.16 0% 17% 3% 0% 0% 0% 4% 0%

CC50 9.90 7.30 8.40 6.70 7.20 8.50 5.00 5.1

Table 4 Antiviral assay of Metadichol against various viruses, as measured by neutral red assay

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 Metadichol Adenovirus Tacaribe Rift valley SARS Japanese West Yellow fever Powassan
(µg/ml) fever encephalitis Nile

5 100% 31% 100% 0% 56% 84% 70% 53%

1.6 100% 69% 100% 52% 87% 100% 73% 100%

0.5 100% 97% 100% 100% 100% 100% 95% 100%

0.16 100% 100% 100% 100% 100% 100% 96% 100%

EC50 >9.9 2.8 >8.4 1.7 >7.2 >8.5 >5 >5.1

Table 5. List of viruses inhibited by Metadichol in vitro

Adenovirus Rift valley

Japanese encephalitis Marburg

Tacaribe SARS

Powassan Respiratory syncytial virus

Zika Chikungunya

Ebola Influenza A (H1N1)

Yellow fever Dengue

West Nile virus HIV

Table 6. TMPRSS2 assay data

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 Sample Concentration RFU % Inhibition IC50

Control 0 43233358 0.00

Metadichol (ng/ml) 1.56 41305150 4.46 96.65 ng/ml

3.12 39329385 9.03

6.25 36713767 15.08

12.5 33778222 21.87

25 30695684 29.00

50 26087008 39.66

100 16009312 62.97

Camostat mesylate (µg/ml) 0.78 37984828 12.14 26.46 ug/ml

1.56 35235186 18.50

3.125 31685728 26.71

6.25 29234396 32.38

12.5 23276839 46.16

25 18931887 56.21

50 8797988 79.65

Table 7 ACE2 assay data

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 Sample Concentration (µg/ml) RFU % Inhibition IC50 (µg/ml)

Control 0 308315546 0.00

Metadichol 0.125 290309918 5.84 30.15

0.25 260064163 15.65

0.5 249149792 19.19

1 240301136 22.06

10 212275253 31.15

20 187702504 39.12

40 139821100 54.65

DX600 0.0156 252855648 17.99 0.1027

0.031 231028864 25.07

0.0625 193810784 37.14

0.125 145881248 52.68

0.25 127485752 58.65

0.5 111498760 63.84

Table 8 ACE assay data

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 Captopril (control) Concentration ng/ml Percent Inhibition
0.63 6.12

1.25 15.04

2.50 32.42

5.00 43.5

10.00 57.2

20 76.51

Metadichol 3.9 4.61

7.8 6.37

15.6 14.28

31.25 22.59

62.5 36.71

125 54.89

250 60.51

500 66.23

1000 78.1

Table 9. COVID-19 and 13 curated genes

CCL2 IL6 IL7

TNF TMPRSS2 ACE2

IL10 CCL3 AGT

IL2 IL8 IL2RA

CSF3

Table 10. Disease network of the 13 curated genes

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Disease name Disease categories Corrected Annotated Annotated genes
P-value gene
quantity

COVID-19 Respiratory tract disease, viral 3.10E-47 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
disease IL10, IL2, IL2RA, IL6, IL7, TMPRSS2,
TNF

Pneumonia, viral Respiratory tract disease, viral 4.34E-46 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
disease IL10, IL2, IL2RA, IL6, IL7, TMPRSS2,
TNF

Coronaviridae Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
infections IL10, IL2, IL2RA, IL6, IL7, TMPRSS2,
TNF

Coronavirus Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
infections IL10, IL2, IL2RA, IL6, IL7, TMPRSS2,
TNF

Nidovirales Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
infections IL10, IL2, IL2RA, IL6, IL7, TMPRSS2,
TNF

RNA virus Viral disease 4.92E-27 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
infections IL10, IL2, IL2RA, IL6, IL7, TMPRSS2,
TNF

Virus diseases Viral disease 1.73E-25 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
IL10, IL2, IL2RA, IL6, IL7, TMPRSS2,
TNF

Sexually Viral disease 1.38E-12 7 CCL2, CCL3, IL10, IL2, IL2RA, IL6, TNF
transmitted
diseases, viral

HIV infections Immune system disease, viral disease 1.56E-12 7 CCL2, CCL3, IL10, IL2, IL2RA, IL6, TNF

Lentivirus Viral disease 1.56E-12 7 CCL2, CCL3, IL10, IL2, IL2RA, IL6, TNF
infections

Retroviridae Viral disease 1.56E-12 7 CCL2, CCL3, IL10, IL2, IL2RA, IL6, TNF
infections

HIV wasting Immune system disease, metabolic 4.00E-04 2 IL6, TNF

syndrome disease, nutrition disorder, viral
disease

Coxsackievirus Viral disease 0.001 2 IL6, TNF

infections

Enterovirus Viral disease 0.0044 2 IL6, TNF

infections

Picornaviridae Viral disease 0.00519 2 IL6, TNF

infections

Page 23/33
Table 11. Disease network of genes implicated in SARS-COV-2 infection

Disease name P-value Corrected P-value Genes Annotated genes

COVID-19 1E-18 5.44E-16 5 ACE2, AGT, CCL2, TMPRSS2, TNF

Pneumonia, viral 1.56E-18 8.46E-16 5 ACE2, AGT, CCL2, TMPRSS2, TNF

Coronaviridae infections 3.4E-18 1.85E-15 5 ACE2, AGT, CCL2, TMPRSS2, TNF

Coronavirus infections 3.4E-18 1.85E-15 5 ACE2, AGT, CCL2, TMPRSS2, TNF

Nidovirales infections 3.4E-18 1.85E-15 5 ACE2, AGT, CCL2, TMPRSS2, TNF

Pneumonia 9.42E-15 5.11E-12 5 ACE2, AGT, CCL2, TMPRSS2, TNF

Respiratory tract infections 3.13E-13 1.7E-10 5 ACE2, AGT, CCL2, TMPRSS2, TNF

RNA virus infections 2.46E-12 1.34E-09 5 ACE2, AGT, CCL2, TMPRSS2, TNF

Virus diseases 9.48E-12 5.15E-09 5 ACE2, AGT, CCL2, TMPRSS2, TNF

Figures

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Figure 1

TMPRSS2 inhibition with Camostat mesylate (control)

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Figure 2

TMPRSS2 inhibition with Metadichol

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Figure 3

Ace-2 inhibition and DX 600 (control)

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Figure 4

ACE2 inhibition with Metadichol

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Figure 5

ACE Inhibition with Captopril (control)

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Figure 6

Ace inhibition with Metadichol

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Figure 7

Potential key gene targets in SARS-COV-2 infection

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Figure 8

Network analysis of genes involved in SARS-COV-2 infections

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Figure 10

Cytokine gene relationships

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