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WO2021228570A1 - Use of sulconazole compounds for the treatment of coronavirus infections - Google Patents

Use of sulconazole compounds for the treatment of coronavirus infections Download PDF

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Publication number
WO2021228570A1
WO2021228570A1 PCT/EP2021/061299 EP2021061299W WO2021228570A1 WO 2021228570 A1 WO2021228570 A1 WO 2021228570A1 EP 2021061299 W EP2021061299 W EP 2021061299W WO 2021228570 A1 WO2021228570 A1 WO 2021228570A1
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Prior art keywords
furin
sulconazole
cov
doi
protein
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French (fr)
Inventor
Abdel-Majid Khatib
Bruno Villoutreix
Serge EVRARD
Géraldine SIEGFRIED
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Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Bergonie
Universite de Bordeaux
Institut Pasteur
Universite de Lille
Original Assignee
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Bergonie
Universite de Bordeaux
Institut Pasteur
Universite de Lille
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Publication of WO2021228570A1 publication Critical patent/WO2021228570A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4174—Arylalkylimidazoles, e.g. oxymetazolin, naphazoline, miconazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone

Definitions

  • the present invention is in the field of medicine, in particular virology.
  • SRAS-CoV-2 1 a new virus belonging to the human coronavirus family was identified in the Hubei province of central china and named SRAS-CoV-2 1 . Although the identity of the source of zoonotic infection is not yet confirmed, it is likely that this emerging virus result from a recombination between coronavirus from bat and pangolins. SRAS-CoV-2 mediates severe human respiratory disease with high death rate 2 .
  • the coronaviruses are a group of enveloped viruses with positive-sense RNA. These viruses belong to the family of Coronavirinae, order Nidovirales 3 comprising of four genera namely alpha, beta, delta, and gamma 2 . These viruses are responsible for a wide range of neurological systems, liver, hepatic and respiratory acute and chronic diseases. Prior to present crisis, only six human coronaviruses (HCoVs) have been known to mediate infection in human and induce respiratory diseases 2 ⁇ 3 . Of these, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are the highly pathogenic coronaviruses able to infect the lower respiratory tract. The other four Coronavirus namely HCoV-229E, OC43, NL63, and HKU1 are associated to upper respiratory infections and common cold 2 ⁇ 3 .
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome
  • viruses encode for viral envelope glycoproteins, synthetized in an immature polyprotein precursor. These proteins require proteolytic cleavage before they can mediate viral entry into host cells. In many aspects, viruses take advantage of cellular proteases for this key function 4 . Indeed, during viral infection, the reliance on particular proteases is a determinant factor for viral infection and spread.
  • furin-like enzymes that are ubiquitously expressed to cleave influenza A virus hemagglutinin (HA) leading to high viral spread and in turn cause higher rates of mortality 5 .
  • HA hemagglutinin
  • the ability of viruses to exploit furin-like enzymes affects the cell tropism and the virus pathogenicity.
  • furin-like proteases or pro-protein convertases (PCs) were reported to be involved in the conversion to their bioactive forms of a large majority of secretory proteins synthesized as inactive protein precursors.
  • PCs pro-protein convertases
  • Proteolytic cleavage of viral envelope glycoprotein by furin-like enzymes into a functional binding virus receptor and a fusogenic transmembrane protein is central for the mediation of virus cell entry and infectivity of the dengue virus 8 , respiratory syncytial virus (RSV) 9 , HIV 10 , human papilloma virus 11 and Chikungunya 12 .
  • RSV respiratory syncytial virus
  • HIV 10 human papilloma virus
  • Chikungunya 12 Chikungunya 12
  • the viral glycoproteins are processed at specific cleavage site, the subcellular localization of the cleavage by furin-like enzymes and the time course of the cleavage vary between viruses.
  • proteolytic activation of viral glycoproteins can occur at different steps of the viral replication cycle due to the ability of these glycoproteins to transit thought the Golgi network during virus production where converting enzyme like furin are enriched.
  • Some viral envelope proteins can also meet several furin-like enzymes in the extracellular space or during the virus entry into the endosome where the envelope protein can be processed.
  • the viral glycoprotein responsible for cell entry is the spike (S) protein 13 17 . It is processed at two different cleavages sites 13 by different proteases that drive the viral tropism.
  • the S protein is synthetized as a protein precursor transiting through the endoplasmic reticulum-Golgi apparatus intermediate compartment (ERGIC).
  • the protein can be cleaved into SI and S2 in the Trans Golgi Network (TGN) in cells expressing high level of furin.
  • TGN Trans Golgi Network
  • This priming process can also involve cell surface proteases belonging to the transmembrane protease/serine subfamily member (TMPRSS) family, which is highly, expressed in the lungs 18 .
  • TMPRSS transmembrane protease/serine subfamily member
  • the two viral subunits resulting from priming have distinct functions.
  • the SARS-CoV (1 & 2) SI subunit contains the angiotensin-converting enzyme 2 (ACE2) receptor binding domain.
  • ACE2 angiotensin-converting enzyme 2
  • the S2 subunit ensures membrane fusion after a second proteolytic cleavage at the S2’ cleavages site, upstream of the fusion peptide.
  • the fusion which releases the nucleocapsid inside the infected cells depends on a conformational change of the S2 protein subunit and occur either at the plasma membrane or in the endosome depending of the protease availability.
  • Sequence analysis of the spike protein of the coronaviruses, MERS-CoV 19 , HCoV-OC43 20 and HCoV-HKUl 21 reveal the presence of a canonical furin-like cleavage site between S1/S2 and at the S2’ cleavage site 22 ( Figure la) .
  • the present invention relates to use of Sulconazole compounds for the treatment of coronavirus infections.
  • SARS-CoV-2 The spike protein (S) of SARS-CoV-2 contains two furin-like cleavage sites found only in MERS-CoV viruses that were linked to the 2003 SARS pandemic.
  • the viral infection requires the priming or cleavage of the S protein and such processing seems essential for virus entry into the host cells. Furin is highly expressed in the lung tissue and the expression is further increased in lung cancer, suggesting the exploitation of this mechanism by the virus to mediate enhanced virulence as shown by the higher risk of COVID-19 in these patients.
  • the inventors used structure-based virtual screening and a collection of about 8,000 unique approved and investigational drugs suitable for docking to search for molecules that could inhibits furin activity.
  • Sulconazole a broad-spectrum anti fungal agent, was found to be of potential interest.
  • Western blot analysis Sulconazole was found to inhibit the cleavage of the cell surface furin substrate MT1-MMP that contains two furin cleavage sites similar to those of the SARS-CoV-2 spike protein.
  • Sulconazole and analogs could be interesting for repurposing studies and to probe the yet not fully understood molecular mechanisms involved in cell entry.
  • the first object of the present invention relates to a method of treating a coronavirus infection in a subject in need thereof comprising administrating to the subject a therapeutically effective amount of a Sulconazole compound.
  • coronavirus has its general meaning in the art and refers to any member of members of the Coronaviridae family.
  • Coronavirus is a virus whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular virus.
  • the virion RNA has a cap at the 5’ end and a poly A tail at the 3’ end. The length of the RNA makes coronaviruses the largest of the RNA virus genomes.
  • coronavirus RNAs encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; plus (4) three non-structural proteins. These coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions.
  • Coronaviruses are exemplified by, but not limited to, human enteric coV (ATCC accession # VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCC accession # VR-920), and SARS-coronavirus (Center for Disease Control), in particular SARS-Covl and SARS-Cov2.
  • human enteric coV ATCC accession # VR-1475
  • human coV 229E ATCC accession # VR-740
  • human coV OC43 ATCC accession # VR-920
  • SARS-coronavirus Center for Disease Control
  • the Sulconazole compound is particularly suitable for inhibiting the replication of the coronavirus.
  • any assay well known in the art as such described in the EXAMPLE
  • for assaying a Sulconazole compound for its ability to inhibit the replication of the virus may be carried out.
  • the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS). More particularly, the method of the present invention is suitable for the treatment of COVID-19.
  • SARS Severe Acute Respiratory Syndrome
  • the subject can be human or any other animal (e.g., birds and mammals) susceptible to coronavirus infection (e.g. domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.).
  • said subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human).
  • the subject is a non-human animal.
  • the subject is a farm animal or pet.
  • the subject is a human.
  • the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant. In some embodiments, the subject suffers from a cancer.
  • cancer has its general meaning in the art and includes, but is not limited to, solid tumors and blood-bome tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term “cancer” further encompasses both primary and metastatic cancers.
  • cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.
  • the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acid
  • the subject suffers from a lung cancer.
  • treatment refers to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse.
  • the treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.
  • therapeutic regimen is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy.
  • a therapeutic regimen may include an induction regimen and a maintenance regimen.
  • the phrase “induction regimen” or “induction period” refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease.
  • the general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen.
  • An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both.
  • maintenance regimen refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years).
  • a maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
  • Sulconazole compound refers to Sulconazole itself or one of its analog.
  • Sulconazole has its general meaning in the art and refers the compound having the IUPAC name: l-[2-[(4-chlorophenyl)methylsulfanyl]-2-(2,4- dichlorophenyl)ethyl] imidazole. The compound is disclosed in U. S. Patent No. 4,055,652.
  • the Sulconazole analog is selected from the group consisting of Butoconazole, Miconazole, Oxiconazole, Tioconazole, Dapaconazole, Econazole, Isoconazole Luliconazole, Fluconazole, DB06914 and Efmaconazole.
  • the Sulconazole compound is administered to the patient in a therapeutically effective amount.
  • a therapeutically effective amount is meant a sufficient amount of the active ingredient for treating or reducing the symptoms of the coronavirus infection at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts.
  • the daily dosage of the products may be varied overawide range from 0.01 to 1,000 mg per adult per day.
  • the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated.
  • a medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient.
  • An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
  • the active ingredient of the present invention e.g. Sulconazole compound
  • pharmaceutically acceptable excipients e.g. Sulconazole compound
  • sustained-release matrices such as biodegradable polymers
  • pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports.
  • Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
  • FIGURES are a diagrammatic representation of FIGURES.
  • FIG. 1 Schematic representation of the coronavirus spike (S) envelope glycoprotein.
  • the S protein is composed of two subunits, the SI receptor-binding subunit, and the S2 fusion subunit that contains furin cleavage sites, S1/S2 and S2’ (arrows)
  • Figure 2 Effect of Sulconazole on furin substrate cleavage
  • (a) Schematic representation of the structural aspects of the human furin cell surface substrate pro-MTl-MMP (63 kDa) and its furin-processing sites at the RRPR922 (1) and RRKR1112 (2).
  • the mature protein (63 kDa) can be further auto-catalytically cleaved into a 45-kd C-terminal form
  • Salts and duplicates were also removed. Manual inspection took place, further guided by the DataWarrior Drug-likeness scores and several others computed physicochemical properties. Further, only molecules that could be docked with a reasonable chance of success were kept (e.g., docking accuracy decreases when molecules are too flexible, thus we kept molecules with less than 20 rotatable bonds and with a MW below 900 Da). We obtained a final collection of about 8,000 molecules acting in different therapeutic areas in 2D that were generated in 3D and protonated using the Surflex tools 31 .
  • Sulconazole a broad-spectrum anti-fungal agent
  • Sulconazole is thought to inhibit the fungal cytochrome P-450 isoenzyme C-14-alpha demethylase.
  • concentration often around 100 microM and above
  • Sulconazole was found to aggregate in some assays while several closely related antifungal analogues such as Fluconazole were not 37 .
  • the molecule was indeed found to inhibit specifically a protein-protein interaction involving the WW domains of cellular ubiquitin ligases of the Nedd4 family and the PPxY motif of the adenoviral capsid protein VI 38 . Further, the molecule could also interact with other protein targets as it has been shown to impede rhodopsin (GPCR) dimerization in a dose-dependent manner 39 and to moderately inhibit the activity of heme oxygenases 40 . A potential binding pose for Sulconazole in the catalytic site of furin was determined.
  • Rivaroxaban displays at this position a chlorothiophene moiety that interacts strongly with a Tyr residue (Y228), and as such a highly basic PI group such as ami dine (arginine-Pl mimetics) is not required, enabling high potency and good oral bioavailability in contrast to molecules having a positively charged PI group.
  • the 4-chlorophenyl-Pl moiety of Sulconazole could bind to the SI pocket and replace the positively charged benzamidine group seen in the X-ray structure of furin complexed with a peptide-like inhibitors by making favourable interactions with the aromatic residue W291 of furin. This could mimic similar interactions seen between FXa and Rivaroxaban (not shown).
  • the imidazole P2 moiety of Sulconazole could also have electrostatic interactions with the conserved D154, somewhat like the arginine P2 residue of the peptide co-crystallized with furin.
  • the peptide hydrophobic valine P3 residue of the inhibitor co-crystallized with furin would be here replaced by the hydrophobic 2,4 dichlorophenyl P3 moiety of Sulconazole.
  • a binding score between Sulconazole and furin was re-computed after energy minization with different tools including the MolDock package 42 and found to be around -143 kcal/mol (dominated by favourable steric interactions with a small contribution from electrostatic interactions) and about -200 kcal/mol between furin and the modified peptide (the predicted score is better as the peptide is much larger than Sulconazole and makes many more interactions) while the score between FXa and Rivaroxaban using the same protocol was found to be around -152 kcal/mol (again Rivaroxaban is bigger than Sulconazole and makes more contacts). Scores between targets cannot be directly compared, but by taking into account these values and the structural analysis mentioned above, we expected that Sulconazole could inhibit furin.
  • Sulconazole To evaluate the ability of Sulconazole to inhibit cellular furin substrate maturation, we directly analysed in cells the cleavage of a well-established furin substrate MT1-MMP 7 that contains two cleavage sites for furin using Western blot analysis. As illustrated in Figure 2a and 2b, Sulconazole inhibits the cleavage of MT1-MMP, as assessed by the accumulation of its unprocessed form (63 KDa) and reduction of the mature form (60 KDa).
  • furin in addition to the implication of furin in the activation of viral glycoproteins required for various viral infections, elevated expression of furin was reported for a range of human cancers including lung cancer and suggested to constitute a significant prognostic factor independent of other conventional clinicopathological ones 7 .
  • patients with cancer showed higher risk of COVID-19 than individuals without cancer with poorer outcomes from COVID-19 54 , suggesting that the enhanced expression of furin in cancer patients may strongly contribute to the massive activation of S proteins leading to rapid patient’s health deterioration.
  • Silencing of furin was recently employed to treat patients with cancer using an autologous tumor-based strategy consisting of a plasmid that encodes granulocyte-macrophage colony- stimulating factor (GMCSF) and furin shRNA. This vaccine was found to be efficient during phase I and II clinical trials in patients with cancer 55 . These treatments were not associated thus far with adverse effects but are difficult to administrate and monitor. As such small drug-like molecules inhibiting furin would be very valuable.
  • Bioinformatics studies were reported to provide crucial information about the interaction between viruses and the infected cells and to assist in the choice of drug and vaccine candidates for potential antiviral treatments. These include the HIV epidemic, H1N1 influenza virus pandemic; the Zika, the Nipah and Ebola epidemic (reviewed in 56 ⁇ 57 ). Such computational procedures can for example assist the selection of virus proteins that may possibly constitute interesting targets. In the face of drastically rising drug discovery costs, strategies focusing on reducing development timelines such as drug repositioning are also relevant. It is indeed known that small molecule drugs can bind to about 3 to 6 targets on average, opening new avenues to rapidly identify potential novel treatments 58 ⁇ 59 . Molecular modelling techniques and virtual screening can definitively assist drug repositioning endeavours.
  • coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol 77, 8801-8811, doi:10.1128/jvi.77.16.8801-8811.2003 (2003).
  • Spike Glycoprotein by Proprotein Convertases Modulates Neurovirulence and Virus Spread within the Central Nervous System.

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Abstract

In December 2019, a new coronavirus was identified in the Hubei province of central china and named SRAS-CoV-2. The spike protein (S) of SARS-CoV-2 contains two furin-like cleavage sites. The viral infection requires the priming or cleavage of the S protein and such processing seems essential for virus entry into the host cells. Furin is highly expressed in the lung tissue, suggesting the exploitation of this mechanism by the virus to mediate enhanced virulence. In the present invention, the inventors used structure-based virtual screening and a collection of about 8,000 unique approved and investigational drugs suitable for docking to search for molecules that could inhibits furin activity. Sulconazole, a broad-spectrum antifungal agent, was found to be of potential interest. Using Western blot analysis, Sulconazole was found to inhibit the cleavage of the cell surface furin substrate MT1-MMP that contains two furin cleavage sites similar to those of the SARS-CoV-2 spike protein. Sulconazole and analogs could be interesting for repurposing studies and to probe the yet not fully understood molecular mechanisms involved in cell entry.

Description

USE OF SULCONAZOLE COMPOUNDS FOR THE TREATMENT OF CORONAVIRUS INFECTIONS
FIELD OF THE INVENTION:
The present invention is in the field of medicine, in particular virology.
BACKGROUND OF THE INVENTION:
In December, 2019, a new virus belonging to the human coronavirus family was identified in the Hubei province of central china and named SRAS-CoV-21. Although the identity of the source of zoonotic infection is not yet confirmed, it is likely that this emerging virus result from a recombination between coronavirus from bat and pangolins. SRAS-CoV-2 mediates severe human respiratory disease with high death rate2.
The coronaviruses are a group of enveloped viruses with positive-sense RNA. These viruses belong to the family of Coronavirinae, order Nidovirales3 comprising of four genera namely alpha, beta, delta, and gamma2. These viruses are responsible for a wide range of neurological systems, liver, hepatic and respiratory acute and chronic diseases. Prior to present crisis, only six human coronaviruses (HCoVs) have been known to mediate infection in human and induce respiratory diseases2·3. Of these, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) are the highly pathogenic coronaviruses able to infect the lower respiratory tract. The other four Coronavirus namely HCoV-229E, OC43, NL63, and HKU1 are associated to upper respiratory infections and common cold2·3.
Most enveloped viruses encode for viral envelope glycoproteins, synthetized in an immature polyprotein precursor. These proteins require proteolytic cleavage before they can mediate viral entry into host cells. In many aspects, viruses take advantage of cellular proteases for this key function4. Indeed, during viral infection, the reliance on particular proteases is a determinant factor for viral infection and spread. While several viruses mediate local infections due to the limited expression of their host proteases in a small number of cell types and/or specific tissues such as the case of the low pathogenic avian influenza A viruses, the highly pathogenic virus uses furin-like enzymes that are ubiquitously expressed to cleave influenza A virus hemagglutinin (HA) leading to high viral spread and in turn cause higher rates of mortality5. Thus, the ability of viruses to exploit furin-like enzymes affects the cell tropism and the virus pathogenicity. Previously, furin-like proteases or pro-protein convertases (PCs) were reported to be involved in the conversion to their bioactive forms of a large majority of secretory proteins synthesized as inactive protein precursors. These include growth factors, receptors, adhesion molecules, matrix metalloproteinases and viral envelope glycoproteins6·7. Precursors are usually cleaved at the general motif (K/R)-(X)n-(K/R) . where n= 0, 2, 4 or 6. To date, one or more of the seven known pro-protein convertases (PCs) family has been implicated in these processes, namely, furin, PCI, PC2, PC4, PACE4, PC5 (and its isoform PC5-B), and PC77 9. Previous studies however showed that viral glycoproteins activation including those of several coronaviruses is mediated by secreted furin-like enzymes that proteolytically process monobasic or multi-basic cleavage sites4.
Proteolytic cleavage of viral envelope glycoprotein by furin-like enzymes into a functional binding virus receptor and a fusogenic transmembrane protein is central for the mediation of virus cell entry and infectivity of the dengue virus8, respiratory syncytial virus (RSV)9, HIV10, human papilloma virus11 and Chikungunya12. Although the viral glycoproteins are processed at specific cleavage site, the subcellular localization of the cleavage by furin-like enzymes and the time course of the cleavage vary between viruses. Furthermore, proteolytic activation of viral glycoproteins can occur at different steps of the viral replication cycle due to the ability of these glycoproteins to transit thought the Golgi network during virus production where converting enzyme like furin are enriched. Some viral envelope proteins can also meet several furin-like enzymes in the extracellular space or during the virus entry into the endosome where the envelope protein can be processed. In coronavirus, the viral glycoprotein responsible for cell entry is the spike (S) protein13 17. It is processed at two different cleavages sites13 by different proteases that drive the viral tropism. The S protein is synthetized as a protein precursor transiting through the endoplasmic reticulum-Golgi apparatus intermediate compartment (ERGIC). For some coronaviruses such as MERS-CoV and probably SARS-Cov- 2, which contain a furin-like cleavage site between SI and S2, the protein can be cleaved into SI and S2 in the Trans Golgi Network (TGN) in cells expressing high level of furin. This priming process can also involve cell surface proteases belonging to the transmembrane protease/serine subfamily member (TMPRSS) family, which is highly, expressed in the lungs18. The two viral subunits resulting from priming have distinct functions. The SARS-CoV (1 & 2) SI subunit contains the angiotensin-converting enzyme 2 (ACE2) receptor binding domain. The S2 subunit ensures membrane fusion after a second proteolytic cleavage at the S2’ cleavages site, upstream of the fusion peptide. The fusion which releases the nucleocapsid inside the infected cells, depends on a conformational change of the S2 protein subunit and occur either at the plasma membrane or in the endosome depending of the protease availability. Sequence analysis of the spike protein of the coronaviruses, MERS-CoV19, HCoV-OC4320 and HCoV-HKUl21 reveal the presence of a canonical furin-like cleavage site between S1/S2 and at the S2’ cleavage site22 (Figure la) . Similar furin-like cleavages sites were identified in the SARS-CoV-2 spike protein sequence14 (Figure lb). Thereby, the high expression of furin and other furin-like enzymes found in human lung, liver and brain tissues7·23 may be exploited by the SARS-CoV-2 for the activation of S protein leading to enhanced infection, virulence and spread of the virus. Compounds interfering with the cleavage of the SARS-CoV-2 S protein processing could be a valuable antiviral approach.
SUMMARY OF THE INVENTION:
As defined by the claims, the present invention relates to use of Sulconazole compounds for the treatment of coronavirus infections.
DETAILED DESCRIPTION OF THE INVENTION:
In December 2019, a new coronavirus was identified in the Hubei province of central china and named SRAS-CoV-2. This new virus induces COVID-19, a severe respiratory disease with high death rate. The spike protein (S) of SARS-CoV-2 contains two furin-like cleavage sites found only in MERS-CoV viruses that were linked to the 2003 SARS pandemic. The viral infection requires the priming or cleavage of the S protein and such processing seems essential for virus entry into the host cells. Furin is highly expressed in the lung tissue and the expression is further increased in lung cancer, suggesting the exploitation of this mechanism by the virus to mediate enhanced virulence as shown by the higher risk of COVID-19 in these patients. In the present invention, the inventors used structure-based virtual screening and a collection of about 8,000 unique approved and investigational drugs suitable for docking to search for molecules that could inhibits furin activity. Sulconazole, a broad-spectrum anti fungal agent, was found to be of potential interest. Using Western blot analysis, Sulconazole was found to inhibit the cleavage of the cell surface furin substrate MT1-MMP that contains two furin cleavage sites similar to those of the SARS-CoV-2 spike protein. Sulconazole and analogs could be interesting for repurposing studies and to probe the yet not fully understood molecular mechanisms involved in cell entry.
Accordingly, the first object of the present invention relates to a method of treating a coronavirus infection in a subject in need thereof comprising administrating to the subject a therapeutically effective amount of a Sulconazole compound. As used herein, the term “coronavirus” has its general meaning in the art and refers to any member of members of the Coronaviridae family. Coronavirus is a virus whose genome is plus-stranded RNA of about 27 kb to about 33 kb in length depending on the particular virus. The virion RNA has a cap at the 5’ end and a poly A tail at the 3’ end. The length of the RNA makes coronaviruses the largest of the RNA virus genomes. In particular, coronavirus RNAs encode: (1) an RNA-dependent RNA polymerase; (2) N-protein; (3) three envelope glycoproteins; plus (4) three non-structural proteins. These coronaviruses infect a variety of mammals and birds. They cause respiratory infections (common), enteric infections (mostly in infants >12 mo.), and possibly neurological syndromes. Coronaviruses are transmitted by aerosols of respiratory secretions. Coronaviruses are exemplified by, but not limited to, human enteric coV (ATCC accession # VR-1475), human coV 229E (ATCC accession # VR-740), human coV OC43 (ATCC accession # VR-920), and SARS-coronavirus (Center for Disease Control), in particular SARS-Covl and SARS-Cov2.
According to the present invention, the Sulconazole compound is particularly suitable for inhibiting the replication of the coronavirus. Typically any assay well known in the art (as such described in the EXAMPLE) for assaying a Sulconazole compound for its ability to inhibit the replication of the virus may be carried out.
In particular, the method of the present invention is suitable for the treatment of Severe Acute Respiratory Syndrome (SARS). More particularly, the method of the present invention is suitable for the treatment of COVID-19.
In some embodiments, the subject can be human or any other animal (e.g., birds and mammals) susceptible to coronavirus infection (e.g. domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.). Typically said subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant. In some embodiments, the subject suffers from a cancer. As used herein, the term "cancer" has its general meaning in the art and includes, but is not limited to, solid tumors and blood-bome tumors. The term cancer includes diseases of the skin, tissues, organs, bone, cartilage, blood and vessels. The term "cancer" further encompasses both primary and metastatic cancers. Examples of cancers that may be treated by methods and compositions of the invention include, but are not limited to, cancer cells from the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, the cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous; adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; and roblastoma, malignant; Sertoli cell carcinoma; Leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.
More particularly, the subject suffers from a lung cancer.
As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
As used herein, the term “Sulconazole compound” refers to Sulconazole itself or one of its analog.
As used herein, the term “Sulconazole” has its general meaning in the art and refers the compound having the IUPAC name: l-[2-[(4-chlorophenyl)methylsulfanyl]-2-(2,4- dichlorophenyl)ethyl] imidazole. The compound is disclosed in U. S. Patent No. 4,055,652.
In some embodiments, the Sulconazole analog is selected from the group consisting of Butoconazole, Miconazole, Oxiconazole, Tioconazole, Dapaconazole, Econazole, Isoconazole Luliconazole, Fluconazole, DB06914 and Efmaconazole.
According to the invention, the Sulconazole compound is administered to the patient in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms of the coronavirus infection at reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied overawide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.
Typically the active ingredient of the present invention (e.g. Sulconazole compound) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutical" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.
FIGURES:
Figure 1. (a) Schematic representation of the coronavirus spike (S) envelope glycoprotein. The S protein is composed of two subunits, the SI receptor-binding subunit, and the S2 fusion subunit that contains furin cleavage sites, S1/S2 and S2’ (arrows) (b) The furin cleavage sites of the human coronaviruses known to mediate infection in human and their furin cleavage site compared to the one of SARS-CoV-2. Note that only SARS-CoV-2 and MERS- CoV show optimal two furin cleavage sites.
Figure 2. Effect of Sulconazole on furin substrate cleavage, (a) Schematic representation of the structural aspects of the human furin cell surface substrate pro-MTl-MMP (63 kDa) and its furin-processing sites at the RRPR922 (1) and RRKR1112 (2). The mature protein (63 kDa) can be further auto-catalytically cleaved into a 45-kd C-terminal form (b) Inhibition of the furin protease activity in the cells in the presence of Sulconazole (10 mM), as demonstrated by the repression of MT1-MMP maturation and the accumulation of its unprocessed form (ProMTl-MMP) and shown by western blot using specific antibody.
Figure 3. Sulconazole analogs. Molecules similar to Sulconazole may also inhibit furin. Molecule DB06914 corresponds to an experimental compound reported in DrugBank. “Exp” means experimental compound and “Invest” indicates investigational compound. EXAMPLE:
Methods:
Structure-based virtual screening
In order identify approved drugs or advanced molecules acting against furin, virtual screening computations were carried out. We first generated a hand-curated database of approved and investigational drugs (small molecules). Over 20,000 non-unique approved and investigational compounds (experimental molecules were not included initially as we were interested in molecules that are approved or have entered clinical trials) were first downloaded from the last released of DrugBank25, DrugCentral26, and SWEETLEAD27. 17449 additional molecules were extracted from Wikipedia Chemical Structures using utilities implemented in the DataWarrior package28. Molecules were flagged using our FAF-Drug server29 to remove compounds with inorganic atoms and molecules with unwanted toxicophores as reported in a previous study but keeping molecules even if they could not be found in commercial vendor catalogs30. Salts and duplicates were also removed. Manual inspection took place, further guided by the DataWarrior Drug-likeness scores and several others computed physicochemical properties. Further, only molecules that could be docked with a reasonable chance of success were kept (e.g., docking accuracy decreases when molecules are too flexible, thus we kept molecules with less than 20 rotatable bonds and with a MW below 900 Da). We obtained a final collection of about 8,000 molecules acting in different therapeutic areas in 2D that were generated in 3D and protonated using the Surflex tools31. All compounds were docked with the 2019 version of Surflex-Dock32 (pgeom option to explore in depth the catalytic site) into the furin X-ray structure co-crystallized with a peptide-like inhibitor33 (PDB entry 5jxh) or co- crystallized with a small chemical compound34 (PDB entry 5mim). The protein structures were prepared with Chimera35 (water molecules, unwanted heteratoms and inhibitory molecules were removed) while exploration of the protonation states of the titratable residues was performed with our server PCE36. A short energy minimization of the 3D protein structures was then carried out priori to virtual screening.
Effect of identified molecule on cell surface furin substrate cleavage
Cells were incubated with the selected molecule and the maturation of MT1-MMP, a cell surface substrate that contains two cleavage sites of furin similar to the ones of the S protein7, was analysed in cell lysates that were subjected to immunoblotting analysis as previously described6·7. Results:
Of the identified molecules with high and intermediate Surflex docking scores (e.g., an estimation of binding affinity) and favourable non-covalent interactions in the catalytic site of furin as judged by interactive structural analysis, Sulconazole, a broad-spectrum anti-fungal agent, was unexpected (as not a known protease inhibitor) and found to be of potential interest. Sulconazole is thought to inhibit the fungal cytochrome P-450 isoenzyme C-14-alpha demethylase. At high concentration (often around 100 microM and above), Sulconazole was found to aggregate in some assays while several closely related antifungal analogues such as Fluconazole were not37. This drug and several related analogs do not however seem to be promiscuous at reasonable concentration. The molecule was indeed found to inhibit specifically a protein-protein interaction involving the WW domains of cellular ubiquitin ligases of the Nedd4 family and the PPxY motif of the adenoviral capsid protein VI38. Further, the molecule could also interact with other protein targets as it has been shown to impede rhodopsin (GPCR) dimerization in a dose-dependent manner39 and to moderately inhibit the activity of heme oxygenases40. A potential binding pose for Sulconazole in the catalytic site of furin was determined. The docked compound and a co-crystallized inhibitory peptide (PDB entry 5jxh) displaying a basic residue (eg., arginine) at the PI position can be seen (not shown). As Sulconazole does not display a positively charged group at this position, we thought to investigate different proteases such as to gain additional knowledge over our docked poses. We for instance used the crystal structure of human coagulation factor Xa serine protease (SP) domain in complex with the approved anticoagulant drug Rivaroxaban41. Many proteases have substrates or inhibitors with a positively charged PI residue that makes favourable interactions with the negatively charged D189 (chymotrypsinogen numbering) at the bottom of the SI specificity pocket (not shown). Rivaroxaban displays at this position a chlorothiophene moiety that interacts strongly with a Tyr residue (Y228), and as such a highly basic PI group such as ami dine (arginine-Pl mimetics) is not required, enabling high potency and good oral bioavailability in contrast to molecules having a positively charged PI group. By comparison, the 4-chlorophenyl-Pl moiety of Sulconazole could bind to the SI pocket and replace the positively charged benzamidine group seen in the X-ray structure of furin complexed with a peptide-like inhibitors by making favourable interactions with the aromatic residue W291 of furin. This could mimic similar interactions seen between FXa and Rivaroxaban (not shown). Further, the imidazole P2 moiety of Sulconazole could also have electrostatic interactions with the conserved D154, somewhat like the arginine P2 residue of the peptide co-crystallized with furin. In addition, the peptide hydrophobic valine P3 residue of the inhibitor co-crystallized with furin would be here replaced by the hydrophobic 2,4 dichlorophenyl P3 moiety of Sulconazole. A binding score between Sulconazole and furin was re-computed after energy minization with different tools including the MolDock package42 and found to be around -143 kcal/mol (dominated by favourable steric interactions with a small contribution from electrostatic interactions) and about -200 kcal/mol between furin and the modified peptide (the predicted score is better as the peptide is much larger than Sulconazole and makes many more interactions) while the score between FXa and Rivaroxaban using the same protocol was found to be around -152 kcal/mol (again Rivaroxaban is bigger than Sulconazole and makes more contacts). Scores between targets cannot be directly compared, but by taking into account these values and the structural analysis mentioned above, we expected that Sulconazole could inhibit furin.
To evaluate the ability of Sulconazole to inhibit cellular furin substrate maturation, we directly analysed in cells the cleavage of a well-established furin substrate MT1-MMP7 that contains two cleavage sites for furin using Western blot analysis. As illustrated in Figure 2a and 2b, Sulconazole inhibits the cleavage of MT1-MMP, as assessed by the accumulation of its unprocessed form (63 KDa) and reduction of the mature form (60 KDa).
Several approved, investigational and experimental Sulconazole analogs are known and some are shown in Figure 3. These compounds could be further investigated with regard to the inhibition of furin and the cleavage of the SARS-CoV-2 S protein.
Discussion:
The recent pandemic of the SARS-CoV-2 proves the complexity of responding to infection diseases. Although considerable progresses were made in various fields of medicine and virology, the emergence of COVID-19 revealed that we were not prepared to take rapid actions so as to reduce the impact of the emerging infection. There are growing evidences that the proteolytic activation of fusion proteins used by coronaviruses for their entry into host cells participate to the virus spreading and favours the virus dissemination in different cell types and species43. The SARS-CoV-2 and the previously reported MERS-CoV viruses, that are to date the only viruses with two optimal furin cleavage sites (Figure lb) reinforce seriously this concept and highlights the implication of the proteases expressed by a cell type as a decisive factor during viral infection44. Like MERS-CoV, SARS-CoV-2 S protein contains furin cleavage sites in the S1/S2 domain and S2’ domain suggesting the S2' site in the emergence and virulence of COVID-19 and its tropism.
The potential clinical and pharmacological role of the furin-like enzymes has fostered the development of both peptide- and protein-based PC-inhibitors7·45·46. In various preclinical studies, the most promising protein-based specific inhibitors of PCs were reported to be attributed to a 1 -antitrypsin Portland also known as al-PDX47, an a 1 -antitrypsin variant, and the individual convertases-pro-segment based inhibitors48. al-PDX, was first shown to be a potent inhibitor of furin-mediated cleavage of HIV gpl6049, but subsequently demonstrated to also inhibit all the furin-like enzymes involved in processing within the constitutive secretory pathway50·51. Other studies further showed that endogenous inhibition of precursor convertases by ai-PDX reduces the maturation of the surface glycoproteins of infectious pathogens52. Interestingly, exogenous addition of al-PDX potentially inhibits the furin-dependent processing of HCMV gB, thus reducing the titer of infectious human cytomegalovirus more effectively than currently used antiherpetic agents53. The reported furin inhibition by the external application of al-PDX occurs since furin is localized to the trans-Golgi network (TGN) and cycles to the cell surface, where it could meet al-PDX, and back via endosomal compartments53. In addition to the implication of furin in the activation of viral glycoproteins required for various viral infections, elevated expression of furin was reported for a range of human cancers including lung cancer and suggested to constitute a significant prognostic factor independent of other conventional clinicopathological ones7. Interestingly, patients with cancer showed higher risk of COVID-19 than individuals without cancer with poorer outcomes from COVID-1954, suggesting that the enhanced expression of furin in cancer patients may strongly contribute to the massive activation of S proteins leading to rapid patient’s health deterioration. Silencing of furin was recently employed to treat patients with cancer using an autologous tumor-based strategy consisting of a plasmid that encodes granulocyte-macrophage colony- stimulating factor (GMCSF) and furin shRNA. This vaccine was found to be efficient during phase I and II clinical trials in patients with cancer55. These treatments were not associated thus far with adverse effects but are difficult to administrate and monitor. As such small drug-like molecules inhibiting furin would be very valuable.
Bioinformatics studies were reported to provide crucial information about the interaction between viruses and the infected cells and to assist in the choice of drug and vaccine candidates for potential antiviral treatments. These include the HIV epidemic, H1N1 influenza virus pandemic; the Zika, the Nipah and Ebola epidemic (reviewed in56·57). Such computational procedures can for example assist the selection of virus proteins that may possibly constitute interesting targets. In the face of drastically rising drug discovery costs, strategies focusing on reducing development timelines such as drug repositioning are also relevant. It is indeed known that small molecule drugs can bind to about 3 to 6 targets on average, opening new avenues to rapidly identify potential novel treatments58·59. Molecular modelling techniques and virtual screening can definitively assist drug repositioning endeavours. Thus, facing the pandemic COVID-19 situation and the lack of validated treatment or vaccine, we decided to use different bioinformatics approaches and our novel collection of about 8,000 approved and investigational compounds to search for novel potential furin inhibitors. The anti-fungal agent Sulconazole was identified after structural analysis and was further found to inhibit the maturation of a major cell surface furin substrate in human cells. Here, we suggest that the inhibition of furin by Sulconazole or one of its analogs could be of high interest in SARS-CoV-2 infection.
REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
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Claims

CLAIMS:
1. A method of treating a coronavirus infection in a subject in need thereof comprising administrating to the subject a therapeutically effective amount of a Sulconazole compound.
2. The method of claim 1 wherein the coronavirus is SARS-Cov2.
3. The method of claim 1 wherein the subject suffers from a cancer, in particular a lung cancer.
4. The method of claim 1 wherein the Sulconazole compound is selected from the group consisting of Sulconazole, Butoconazole, Miconazole, Oxiconazole, Tioconazole, Dapaconazole, Econazole, Isoconazole Luliconazole, Fluconazole, DB06914 and Efmaconazole.
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