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Metadichol ® a novel nano lipid that inhibits In Vitro, SARS-COV-2 and a multitude of pathological viruses


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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 (EC50 26000 ng/ml), it is 270 times more potent. Additionally, Metadichol® is also a 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.
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Metadichol ®a novel nano lipid that inhibits In Vitro,
SARS-COV-2 and a multitude of pathological viruses
Palayakotai R Raghavan ( )
Nanorx Inc
Research Article
Keywords: Covid-19, SARS-COV-2, Malaria, AHR, AR,COVID-19, ACE2, TMPRSS2, Furin, CD 147, VDR,
Inverse agonist, Protean agonist, Metadichol, Nano formulation, inverse agonist, SARS, H1N1, Ebola, Zika,
West Nile,
License: This work is licensed under a Creative Commons Attribution 4.0 International License. 
Read Full License
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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 nd 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
The ongoing pandemic of severe acute respiratory syndrome-coronavirus 2 (SARS-COV-2) has rearmed
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 (EC50 26000 ng/ml), it is 270 times more
potent. Additionally, Metadichol® is also a 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.
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 specic antivirals and vaccines will prove expensive and time
consuming and is accompanied by compromises on safety and ecacy. 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
both receptors can effectively stop 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 partially regulates ACE2 in concert with
inhibition of 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 parainuenza 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 signicant reduction in HSV titre. Testing between
pH 7 and 4.2 showed that a pH of 4.2 caused a more rapid inactivation of HSV–1 virus titre 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 and
TMPRSS2 and in an antiviral assay with SARS-COV–2.
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Results And 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
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 study22of 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 leukaemia cancer cells243
Metadichol also inhibits TMPRSS2 ( Table 6 gure 1,2) and is 270-fold more potent than CM24
Metadichol moderately inhibits ACE2 ( Table 7, gures 3 and 4) and, in combination with TMPRSS2
inhibition, likely leads to a pronounced synergistic effect in overcoming viral entry. The reported results
open the gateway to effective and safe therapies for COVID–19. Metadichol is a mild inhibitor of ACE2 (
table 7 and gures 4 and 6) but at the same time, not signicant to affect the physiological functions of
the host.
Vitamin D and SARS-COV–2 infection
An uncontrolled inammatory response to SARS-COV–2 is the major cause of disease severity and death
in patients with COVID–1925 and is associated with high levels of circulating cytokines, tumor necrosis
factor (TNF), CCl2, C-reactive protein (CRP), and Ferritin.
Metadichol14 is an inhibitor of CCl2 (also known as MCP–1), TNF, NF-kB, and CRP, which is a surrogate
marker for cytokine storms26 and is associated with vitamin D deciency.
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
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DNA-binding protein interacts with regulatory sequences near target genes that participate genetically
and epigenetically in the transcriptional output of genes needed for function27. Vitamin D reduces the
risk of infections by mechanisms that include inducing cathelicidins and defensins28, resulting in
lowered viral replication rates and reducing concentrations of pro-inammatory cytokines29.
Supplementation with 4000 IU/d vitamin D decreased dengue virus infection30. Inammatory cytokine
levels increase in viral and bacterial infections, as seen in COVID–19 patients. Vitamin D can reduce the
production of pro- inammatory Th1 cytokines, such as TNF and interferon (IFN)31.
Vitamin D is a modulator of adaptive immunity32 and suppresses responses mediated by T helper type 1
(Th1) cells primarily by repressing the production of the inammatory cytokines interleukin (IL)–2 and
IFN-gamma33. 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 types34.
1,25(OH)2D3 promotes T regulatory cell induction, thereby inhibiting inammatory processes35. It is
known that COVID–19 is associated with the increased production of pro- inammatory cytokines,
elevated CRP levels, increased risk of pneumonia, sepsis, acute respiratory distress syndrome (ARDS),
and heart failure36. Case fatality rates (CFRs) in China were 6%–10% for those with cardiovascular
disease, chronic respiratory tract disease, diabetes, and hypertension37. Metadichol is a inverse
agonist/protean agonist 14 of VDR ie 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-fold38. Viral infection
places a signicant strain on the body. CD8 T cells that mediate adaptive immunity39 to protect the body
from microbial invaders can easily reach their Hayick limit by depleting their telomeres40. 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 repair41. Signalling via AHR may dampen the immune
response against coronavirus42. It has been reported that although some signalling 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.43 suggested that AHR (the constitutive aryl hydrocarbon receptor)
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signalling constrains type I IFN-mediated antiviral innate defence and suggested a need to block
constitutive AHR activity; only an inverse agonist can dampen this activity. We have shown that
Metadichol® binds to AHR as an inverse/protean agonist44 and thus can reduce complications
attributed to uncontrolled inammation 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 defence. The pharmacological application of vitamin C enhances immune
function45. Vitamin C has antiviral properties leading to inhibition of the replication of HSV–1, poliovirus
type 1, inuenza virus type46, and rabies virus in vitro47.
Vitamin C deciency reduces cellular48–52and humoral immune responses, and treatment of healthy
subjects promoted and enhanced natural killer (NK) cell activities53, underlining the immunological
importance of vitamin C54,55 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-inammatory
properties. Moreover, the newly characterized hydroxylase enzymes, which regulate the activity of
hypoxia-inducible factor gene transcription and cell signalling of immune cells, need vitamin C as a
cofactor for optimal activity56–58. 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 biomarkers59–61.
Gene cluster network analysis
The present drug discovery paradigm is based on the idea of one gene-one target, one disease. It has
become clear that it is dicult to achieve single target specicity. 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 progression62,63. 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.org64, which has curated genes show in Table
8 relevant to COVID–19. Table 9 lists genes and diseases states that they are involved in.
We can lter the 13 genes to a set of 5 genes: TNF, CCL2, ACE2, TMPRSS2 are modulated by Metadichol
and AGT, which is part of the renin-angiotensin system (RAS) network that ACE2 is a part of (Figure 5). A
similar analysis of these network genes shows that they are closely networked in diseases, with a highly
signicant p-value. These ve genes are closely related, and the network generated, is shown in Figure 6,
using www.innatedb.org65.
This analysis integrates known interactions and pathways from major public databases.
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The highlighted ones are SIRT1, AR (androgen receptor), and FOS. Glinsky66 suggested vitamin D as a
potential mitigation agent in preventing SARS-COV–2 entry. Metadichol binds to VDR, which controls the
expression of FOS67. AR also controls the expression of FOS, as well as that of TMPRSS2. Figure 7,
generated below using PACO68, shows the gene network and regulation relationships. VDR controls FOS
expression, FOS controls AGT, AGT controls the expression of AGTR1 and ACE, and AR controls the
expression of TMPRSS2.
Wambier and Goren69 suggested that SARS-COV–2 infection is likely to be androgen mediated. The rst
step to infection is the priming of the SARS-COV–2 spike proteins by TMPRSS2, which also cleaves ACE2
for augmented viral entry. This pathway is seen in the network (Figure 8). SIRT1 plays an active role in
enhancing immunity in viral infections70
Proteases such as Furin71 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 SRC72. Adam–17 is regulated via CEPBP73,74, which is involved in the regulation of
genes involved in immune and inammatory responses. Recently, Ulrich and Pillat75 proposed that
CD147, similar to ACE2, is another receptor used for viral entry. CD147 is a known receptor76 for the
parasite that causes malaria in humans,
Plasmodium falciparum.
Metadichol (see Ref6, US patent
9,006,292) inhibits the malarial parasite.
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 Angiostensin II77. Angiostensin II acts as a
vasoconstrictor and a pro-inammatory cytokine (Figure 9) via AT1R78. The Angiostensin II-AT1R axis
leads to a pro-inammatory state79, leading to infections through activation of NF-KB and to increased
IL–6 levels in multiple inammatory and autoimmune diseases80.
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 storms81 result in ARDS.
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 inammation. There are increased levels of the pro- inammatory cytokines IL–
6, IL–18, TNF, and IL–1-beta by macrophages and of IFN-gamma by NK cells.
Figure 9 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.
Inltration 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 inammatory cytokine response caused by SARS-COV–2 and, at the
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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 6, bringing about
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 in collaboration with
government agencies 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 partial inhibition of
ACE2 and boosts the antiviral response by enhancing 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 ght 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 associated82,83 with COVID–19, such as
hypertension and diabetes84,85, 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’s86 actions on
multiple genes and proteins 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
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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
EC90: calculated concentration to reduce virus yield by 1 log (90%); SI: CC50/EC50.
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Table 2. Cytotoxicity and virus yield data for each concentration of Metadichol tested
Metadichol Concentration(µg/ml) Cytotoxicity (%) Virus Titre (CCID50 per 0.1
100 100% <0.7
32 100% <0.7
10 83% <0.7
3.2 54% 0.7
117% 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
 
Metadichol (µg/ml) Adenovirus Tacaribe Rift
SARS Japanese
West Nile
Yellow fever Powassan
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
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Table 4 Antiviral assay of Metadichol against various viruses, as measured by neutral red assay
Adenovirus Tacaribe Rift valley
SARS Japanese
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 R ift 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
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
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. COVID-19 and 13 curated genes
CSF3  
Table 9. Disease network of the 13 curated genes
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Disease name Disease categories Corrected
Annotated genes
COVID-19 Respiratory tract disease, viral
3.10E-47 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
Pneumonia, viral Respiratory tract disease, viral
4.34E-46 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
Coronavirus infections Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
Nidovirales infections Viral disease 1.74E-44 13 ACE2, AGT, CCL2, CCL 3, CSF3, CXCL10,
RNA virus infections Viral disease 4.92E-27 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
Virus diseases Viral disease 1.73E-25 13 ACE2, AGT, CCL2, CCL3, CSF3, CXCL10,
diseases, viral
Viral disease 1.38E-12 7 CCL2, CCL3, IL10, IL2, IL2RA, IL6, TNF
HIV infections Immune system disease,viraldisease 1.56E-12 7 CCL2, CCL3, IL10, IL2, IL2RA, IL6, TNF
Lentivirus infections Viral disease 1.56E-12 7 CCL2, CCL3, IL10, IL2, IL2RA, IL6, TNF
Retroviridae infections Viral disease 1.56E-12 7 CCL2, CCL3, IL10, IL2, IL2RA, IL6, TNF
HIV wasting
Immune system disease,metabolic
disease, nutrition disorder, viral disease
4.00E-04 2 IL6, TNF
Viral disease 0.001 2 IL6, TNF
Enterovirus infections Viral disease 0.0044 2 IL6, TNF
Viral disease 0.00519 2 IL6, TNF
Table 10. Disease network of genes implicated in SARS-COV-2 infection
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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
All assays were on a fee-for-service contract basis and outsourced to bioanalytical testing companies
worldwide. Antiviral assays were performed by a bio-safety level 3 (BSL3) facility in the USA.
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% conuent 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 ve days. M128533 (protease specic 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
Page 20/31
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 uid 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
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
TMPRSS puried from LNCaP cells (Cayman Chemicals) was used as an enzyme source. The reaction
mixture contained puried 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 uorogenic trypsin substrate Cbz-Gly-Gly-Arg-AMC was
added, and kinetic uorescence 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
uorescence (delta RFU) was calculated to determine the inhibitory effects of the test sample. CM 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
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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 uorescence emitted by the cleavage of a
chromogenic substrate.
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 SpectraMax i3x (Molecular Devices). The IC50
values were calculated based on the readings obtained.
Competing interest: Author has no competing interests
Correspondence and requests for materials should be addressed to
Glossary Of Gene Descriptions
Page 22/31
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
Page 23/31
Figure 1
TMPRSS2 inhibition with Camostat mesylate (control)
Page 24/31
Figure 2
TMPRSS2 inhibition with Metadichol
Page 25/31
Figure 3
Ace-2 inhibition and DX 600 (control)
Page 26/31
Figure 4
ACE2 inhibition with Metadichol
Page 27/31
Figure 5
Potential key gene targets in SARS-COV-2 infection
Page 28/31
Figure 6
Network analysis of genes involved in SARS-COV-2 infections
Page 29/31
Figure 7
Cytokine relationship and network
Page 30/31
Figure 8
RAS- and VDR- gene relationships
Page 31/31
Figure 9
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Therapeutic options in response to the 2019-nCoV outbreak are urgently needed. Here, we discuss the potential for repurposing existing antiviral agents to treat 2019-nCoV infection (now known as COVID-19), some of which are already moving into clinical trials. Therapeutic options in response to the 2019-nCoV outbreak are urgently needed. Here, we discuss the potential for repurposing existing antiviral agents to treat 2019-nCoV infection (now known as COVID-19), some of which are already moving into clinical trials.
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Importance In December 2019, novel coronavirus (2019-nCoV)–infected pneumonia (NCIP) occurred in Wuhan, China. The number of cases has increased rapidly but information on the clinical characteristics of affected patients is limited. Objective To describe the epidemiological and clinical characteristics of NCIP. Design, Setting, and Participants Retrospective, single-center case series of the 138 consecutive hospitalized patients with confirmed NCIP at Zhongnan Hospital of Wuhan University in Wuhan, China, from January 1 to January 28, 2020; final date of follow-up was February 3, 2020. Exposures Documented NCIP. Main Outcomes and Measures Epidemiological, demographic, clinical, laboratory, radiological, and treatment data were collected and analyzed. Outcomes of critically ill patients and noncritically ill patients were compared. Presumed hospital-related transmission was suspected if a cluster of health professionals or hospitalized patients in the same wards became infected and a possible source of infection could be tracked. Results Of 138 hospitalized patients with NCIP, the median age was 56 years (interquartile range, 42-68; range, 22-92 years) and 75 (54.3%) were men. Hospital-associated transmission was suspected as the presumed mechanism of infection for affected health professionals (40 [29%]) and hospitalized patients (17 [12.3%]). Common symptoms included fever (136 [98.6%]), fatigue (96 [69.6%]), and dry cough (82 [59.4%]). Lymphopenia (lymphocyte count, 0.8 × 10⁹/L [interquartile range {IQR}, 0.6-1.1]) occurred in 97 patients (70.3%), prolonged prothrombin time (13.0 seconds [IQR, 12.3-13.7]) in 80 patients (58%), and elevated lactate dehydrogenase (261 U/L [IQR, 182-403]) in 55 patients (39.9%). Chest computed tomographic scans showed bilateral patchy shadows or ground glass opacity in the lungs of all patients. Most patients received antiviral therapy (oseltamivir, 124 [89.9%]), and many received antibacterial therapy (moxifloxacin, 89 [64.4%]; ceftriaxone, 34 [24.6%]; azithromycin, 25 [18.1%]) and glucocorticoid therapy (62 [44.9%]). Thirty-six patients (26.1%) were transferred to the intensive care unit (ICU) because of complications, including acute respiratory distress syndrome (22 [61.1%]), arrhythmia (16 [44.4%]), and shock (11 [30.6%]). The median time from first symptom to dyspnea was 5.0 days, to hospital admission was 7.0 days, and to ARDS was 8.0 days. Patients treated in the ICU (n = 36), compared with patients not treated in the ICU (n = 102), were older (median age, 66 years vs 51 years), were more likely to have underlying comorbidities (26 [72.2%] vs 38 [37.3%]), and were more likely to have dyspnea (23 [63.9%] vs 20 [19.6%]), and anorexia (24 [66.7%] vs 31 [30.4%]). Of the 36 cases in the ICU, 4 (11.1%) received high-flow oxygen therapy, 15 (41.7%) received noninvasive ventilation, and 17 (47.2%) received invasive ventilation (4 were switched to extracorporeal membrane oxygenation). As of February 3, 47 patients (34.1%) were discharged and 6 died (overall mortality, 4.3%), but the remaining patients are still hospitalized. Among those discharged alive (n = 47), the median hospital stay was 10 days (IQR, 7.0-14.0). Conclusions and Relevance In this single-center case series of 138 hospitalized patients with confirmed NCIP in Wuhan, China, presumed hospital-related transmission of 2019-nCoV was suspected in 41% of patients, 26% of patients received ICU care, and mortality was 4.3%.
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Background: A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. Methods: All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. Findings: By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0-58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0-13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. Interpretation: The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. Funding: Ministry of Science and Technology, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technology Commission.
The COVID-19 pandemic caused by infection with SARS-CoV-2 has led to more than 200,000 deaths worldwide. Several studies have now established that the hyperinflammatory response induced by SARS-CoV-2 is a major cause of disease severity and death in infected patients. Macrophages are a population of innate immune cells that sense and respond to microbial threats by producing inflammatory molecules that eliminate pathogens and promote tissue repair. However, a dysregulated macrophage response can be damaging to the host, as is seen in the macrophage activation syndrome induced by severe infections, including in infections with the related virus SARS-CoV. Here we describe the potentially pathological roles of macrophages during SARS-CoV-2 infection and discuss ongoing and prospective therapeutic strategies to modulate macrophage activation in patients with COVID-19. This Progress article from Merad and Martin examines our current understanding of the excessive inflammatory responses seen in patients with severe COVID-19. The authors focus on the emerging pathological roles of monocytes and macrophages and discuss the inflammatory pathways that are currently being targeted in the clinic.
The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
What′s sauce for the goose? Little is known about the coronavirus causing the current outbreak; however, it shares strong sequence homology with its better‐studied cousin SARS‐CoV. Based on previous studies of targeting SARS‐CoV, we suggest four potential candidates that could be used to drug the viral spike protein, RNA‐dependent RNA polymerase, and coronavirus main proteinase. Abstract With the current trajectory of the 2019‐nCoV outbreak unknown, public health and medicinal measures will both be needed to contain spreading of the virus and to optimize patient outcomes. Although little is known about the virus, an examination of the genome sequence shows strong homology with its better‐studied cousin, SARS‐CoV. The spike protein used for host cell infection shows key nonsynonymous mutations that might hamper the efficacy of previously developed therapeutics but remains a viable target for the development of biologics and macrocyclic peptides. Other key drug targets, including RNA‐dependent RNA polymerase and coronavirus main proteinase (3CLpro), share a strikingly high (>95 %) homology to SARS‐CoV. Herein, we suggest four potential drug candidates (an ACE2‐based peptide, remdesivir, 3CLpro‐1 and a novel vinylsulfone protease inhibitor) that could be used to treat patients suffering with the 2019‐nCoV. We also summarize previous efforts into drugging these targets and hope to help in the development of broad‐spectrum anti‐coronaviral agents for future epidemics.