NEW NON-PROTEIN FURIN INHIBITOR

Description

[0001] The human genome encodes more than 550 protease-like molecular scissors. These molecular scissors digest proteins in our food, degrade non-compliant or unwanted proteins, and regulate the trafficking and activity of many cellular factors. Proteolytic cleavage is one of the major post-translational modifications in homeostasis. It regulates many processes that maintain health or lead to disease, generating a large number of bioactive proteins and peptides with key roles in cell proliferation, immunity, and inflammation (Braun E et al., 2019).

[0002] Among these molecular scissors, one is ubiquitous in mammals: furin, a protease expressed ubiquitously in many cellular tissues. Among the substrates cleaved by furin in mammals, we find cytokines, hormones, growth factors, and numerous receptors. There is evidence that furin deregulation can trigger infectious diseases as well as cancer. The enzymatic activity of furin is used by many viral and bacterial pathogens to enhance their virulence and spread (Braun E et al., 2019).

[0003] Indeed, furin plays a key role in the maturation and pathogenicity of viral glycoproteins. In mammals, it cleaves and activates a large number of viral and bacterial substrates. These substrates include viral envelope glycoproteins and bacterial toxins, but also cellular factors that promote tumor development and growth if hyperactivated (Tian S et al., 2011).

[0004] Furin is an evolutionary member of the ancient proprotein convertase family. Their similarity to bacterial subtilisin and yeast kexin proteases is the origin of the abbreviation PCSK (Proprotein Convertase Subtilisin / Kexin type). Humans encode nine members of this protease family (PCSK1 to 9), with PCSK3 representing furin. PCSKs are well known for their ability to activate other cellular proteins (Braun E et al., 2019).

[0005] To date, more than 200 cellular substrates of PCSK have been described, including hormones, receptors, growth factors, and adhesion molecules (Roeabroek A et al., 1986; Van de ven WJ et al., 1990).

[0006] PCSK1 to 7, especially furin (PCSK3), cleave their substrates after a sequence of basic residues, with a typical recognition motif (Seidah NG et al., 2012). PCSK3 cleaves basic amino acid motifs, which is why it has been called PACE (basic amino acid cleaving enzyme). Furin is expressed by FUR (FES Upstream Region), a gene carried on chromosome 15.

[0007] High levels of furin are found in the salivary glands, liver, and bone marrow, while muscle cells express relatively low amounts.

[0008] During mRNA translation, furin enters the secretory pathway as an inactive proenzyme and is integrated into the ER (Endoplasmic Reticulum) membrane. away its C-terminal transmembrane domain. As with many transmembrane proteins, it harbors a short N-terminal signal peptide that is cleaved cotranslationally. Similar to other proprotein convertases, furin contains an 83-amino acid N-terminal inhibitory propeptide, whose chaperone function is required for proper folding of the catalytic domain (Shinde U et al.,1993). The inhibitory propeptide at the N-terminal position is removed in a two-step autoproteolysis process, and it is at this point that furin becomes an active enzyme (Anderson ED, 1997). At the same time, N-linked oligosaccharides are added and cleaved. Although furin accumulates in the Golgi apparatus (TGN compartment), it can be transported to the cell surface and back away the endosomal pathway (Pesu M et al., 2006; Blanchette F et al., 1997; Decroly E et al., 1994; Hipp MM et al., 2013).

[0009] Furin can also be removed and released into the extracellular space upon protein cleavage of the catalytic domain from the membrane-bound C terminus. Whether this cleavage step is mediated by furin itself or by another protease remains to be determined (Vey M et al., 1994). The presence of furin in the Golgi apparatus at the TGN and endosomal compartments, on the cell surface, and in the extracellular space may explain its ability to process a wide variety of intra- and extracellular substrates (Plaimauer B et al., 2001).

[0010] Bioinformatics analyses and functional studies have revealed more than 100 furin cleavage sites in mammals (Tian S, 2009). These include growth factors and cytokines (e.g., IGF1, IGF2, TGFb, PDGFa, PDGFb, VEGF-C, NGF, CXCL10), hormones (e.g., PTH, TRH, GHRH), adhesion molecules (e.g., integrins, vitronectin), collagens, metalloproteinases, coagulation factors, receptors, membrane channels, and albumin.

[0011] Furin is now at the heart of the fight against cancer.

[0012] Furin has been characterized as a major initiator and key mediator in tumor growth and progression. Indeed, its excessive expression or activation can promote the formation and progression of various malignancies, including colon carcinoma, rhabdomyosarcoma, head and neck cancers, lung, skin, and brain tumors (Jaaks P et al., 2017). Often, furin levels positively correlate with cancer aggressiveness, therefore increased furin expression has been proposed as a prognostic marker for advanced cancers (Bassi DE et al., 2005; Klein Szanto AJ et al., 2017; Jaaks P et al., 2017).

[0013] The oncogenic activity of furin has been attributed to its ability to activate proteins that promote cell proliferation, angiogenesis, migration, and tissue invasion. For example, furin cleaves and activates growth factors such as IGF, PDGF, or NGF, which enhance cell proliferation and, consequently, tumor growth.

[0014] Furin expression is induced by hypoxia, as all three FUR promoters harbor binding sites for the Factor 1 hypoxia-inducible enzyme (HIF-1) (McMahon S et al., 2005).

[0015] Thus, furin-mediated vascularization may occur preferentially in growth-restricted hypoxic tumors. Compounding this, hypoxia leads to subcellular relocalization of furin to the cell surface, further promoting the processing of growth factors and other extracellular precursor protein tumorigens (Arsenault D et al., 2012).

[0016] In addition to effects on tumor growth, furin promotes the migration and extravasation of malignant cells because furin processes adhesion molecules mediating cell-cell and cell-matrix interactions. Integrin cleavage may be particularly relevant because beyond mediating cell adhesion to the extracellular matrix, they also act as signal transducers regulating cell growth, division, and survival (Stupack DG et al., 2002).

[0017] It should be noted as an aggravating factor that furin activates matrix metalloproteinases (e.g. MMP14) that facilitate the progression of metastases by degrading extracellular matrix components (Jaaks P et al., 2017). Increased furin activity promotes cancer development by suppressing protective antitumor mechanisms; for example, it is now known that increased furin-mediated activation of TGFb reduces immune surveillance by promoting the development of suppressor Treg cells and inhibiting T cell effector functions (Dahmani A et al., 2018).

[0018] Indirectly, positive feedback loops may further enhance the oncogenic potential of furin; for example, the furin substrate TGFb not only increases furin mRNA expression, but this substrate also enhances furin proteolytic activity by a yet unknown mechanism (Bourne GL et al., 2011).

[0019] Similarly, furin increases the secretion of cytokines (IFNc), which in turn activate the FUR promoter, thus promoting the transcription of the gene coding for furin (Pesu M et al., 2006; Hipp MM, 2013).

[0020] This mutual enhancement appears particularly critical given the key role of IFNc in tumor development and progression (Mojic M et al., 2017).

[0021] On the one hand, furin-driven IFNc release may have beneficial effects as it stimulates the anti-tumor activity of natural killer cells and cytotoxic T lymphocytes. Furthermore, IFNc may act as an anti-angiogenic factor and directly inhibit tumor cell proliferation by inducing the expression of tumor suppressors such as P21 or P27. On the other hand, however, recent evidence suggests that IFNc may be tumor-promoting, for example, by selecting for immune-evasive phenotypes and promoting an immunosuppressive tumor microenvironment (Mojic M et al., 2017).

[0022] Interestingly, a study in laryngeal cancer patients suggests that the IFNc-furin feedback loop may be further strengthened iatrogenically since radiotherapy increased furin expression in some patients (Lee M et al., 2016).

[0023] In summary, excessive furin activation promotes several steps of cancer development, including cell proliferation, vascularization, metastasis, and antitumor immunity.

[0024] An inhibitor of furin activity can therefore play a key role in halting the development of cancer, in particular to prevent cell proliferation, prevent metastases, restrict tumor vascularization, and improve anti-tumor immunity.

[0025] A furin activity inhibitor can also limit the side effects of radiotherapy and increase the benefit / risk ratio of this therapeutic method.

[0026] Furin is capable of proteolytically activating a wide variety of pathogen-derived proteins.

[0027] Particularly in bacteria, the AB toxin group includes several well-described furin substrates. These exotoxins are secreted by bacteria and exert their effect in the cytoplasm of the target cell. They generally consist of an A subunit that carries the enzymatic or toxic activity and a B subunit that mediates binding and translocation to the membrane. To exert its toxic effect, the A subunit must be separated from the membrane-associated B subunit by proteolytic cleavage (Gordon VM et al., 1994).

[0028] With the case of diphtheria toxin (from Corynebacterium diphtheriae Or Löffler Glue bowl ) or exotoxin A of Pseudomonas aeruginosa,toxin studied in the treatment of hepatitis B (Hafkemeyer P, 2008 DOI: 10.3748 / wjg.14.2810), cleavage most likely occurs in endosomes before the A subunit moves into the nucleus where it inhibits protein synthesis by inhibiting the elongation factor EF2 (Collier RJ, 1967).

[0029] With the case of anthrax toxin (from Bacillus anthracis), a three-protein exotoxin consisting of receptor-binding protective antigen (PA) and edema factor (EF) and lethal factor (LF), the cleavage pattern differs. Indeed, upon binding to its receptor, PA is cleaved by furin on the cell surface. This cleavage step triggers oligomerization of PA into a complex that binds EF and LF. Subsequently, this toxin complex is endocytosed and PA forms a channel that allows translocation of EF and LF into the cytoplasm (Gordon VM et al., 1994). Although PA can be activated by different members of the proprotein convertase family, furin appears to be the major anthrax toxin activating the toxic protease (Molloy SS et al., 1992).

[0030] These examples illustrate that several bacterial pathogens exploit furin and related convertases for the activation of their exotoxins. Strictly speaking, however, some toxins produced by bacteria (e.g., diphtheria toxin) represent viral gene products because they are encoded by bacteriophages (Wagner PL et al., 2002). In these cases, the term “viral exotoxin” is more appropriate. This strongly suggests that furin-mediated toxin activation confers a selection advantage to both the bacterium and its phage.

[0031] For example, induction of cell death by furin-activated toxins promotes tissue invasion, increases transmission rates (e.g., by causing diarrhea), or suppresses cellular immune responses. Without the proteolytic activation of exotoxins, diseases such as dysentery or diphtheria would not occur.

[0032] A furin activity inhibitor can therefore prevent the activation of many bacterial toxins (themselves, possibly of viral origin), notably the group of AB toxins with an intracellular inhibitor that will keep the toxins passive and without virulence. A furin activity inhibitor can also stop and avoid toxins such as anthrax in the extracellular environment.

[0033] In the fight against bacterial toxins and for their neutralization, furin inhibitors are substances to be integrated in the prevention and treatment of infectious diseases involving these toxins.

[0034] Like bacterial and viral exotoxins, most viral envelope glycoproteins must be proteolytically cleaved before they can mediate viral entry into host cells. In many cases, viruses exploit cellular trypsin- or subtilisin-like endoproteases for this purpose. While subtilisin-like proteases such as furin require polybasic cleavage sites, trypsin-like proteases also recognize monobasic motifs and cleave after single arginine or lysine residues (Klenk HD et al., 1994 https: / / doi.org / 10.1016 / 0966-842X(94)90123-6).

[0035] It is noteworthy that the dependence on specific proteases is a key point in tissue tropism and viral spread in an infected organism. For example, avirulent strains of Newcastle disease virus (NDV) harbor a monobasic cleavage site in their Fusion (F) protein and result in only local infections (mainly in the respiratory tract) because the expression of the respective host proteases is limited to a few cell types. In contrast, the F proteins of virulent NDV strains can be cleaved by furin. Therefore, these viruses can spread systemically and cause high mortality rates in infected birds (Nagai Y et al., 1989). Another well-described example is the cleavage of the hemagglutinin (HA) of influenza A virus.Unlike low-pathogenic avian influenza A viruses, their highly pathogenic counterparts harbor a polybasic furin cleavage site in the HA48 protein. Thus, the ability of viruses to exploit furin may have critical effects on their pathogenicity.

[0036] To date, proteolytic cleavage of furin is known and operational on envelope glycoproteins encoded by many diverse virus families, including Herpesviruses, Coronavirus, Flavivirus, Togavirus, Bornavirus, Bunyavirus, Filovirus, Orthomyxovirus, Paramyxovirus, Pneumo and Retroviridae. The envelope proteins of some viruses are cleaved in the producing cell, others are processed in the extracellular space or upon entry into their target cells.

[0037] In the case of retroviral glycoprotein trimers, such as those of human immunodeficiency, Rous sarcoma, or murine leukemia viruses, they are processed and activated by protein cleavage in the producer cells.

[0038] Furin plays a particularly important role in Human Immunodeficiency Virus (HIV) infection and pathogenicity.

[0039] Indeed, in the case of HIV-1, the viral envelope protein (Env) precursor gp160 is cleaved into gp120 and membrane-anchored gp41, which remain associated by non-covalent interactions. Proteolytic cleavage of furin occurs in intracellular compartments, prior to virion assembly at the plasma membrane. It is noteworthy that proteolytic processing of Env depends on correct N-linked glycosylation, as aberrant carbohydrate side chains can lead to subcellular processing error or sequestration of Env (Moulard M et al., 2000). Most likely, HIV-1 takes advantage of the redundancy of several proprotein convertases recognizing the polybasic cleavage motif in Env. Furin, PCSK5, PCSK6 and PCSK7 have all been shown to cleave gp160 in cells, although with different efficiencies (Moulard M et al., 2000).

[0040] In the case of influenza A virus hemagglutinins (HA), it is noteworthy that H5 and H7 hemagglutinins of a large number of highly pathogenic avian influenza A viruses (HPAIVs) can be cleaved by furin or PCSK5, which are present in many cell types (Stieneke Gröber A et al., 1992; Horimoto T et al., 1994).

[0041] It is noteworthy that the ability to exploit furin for efficient HA cleavage and the associated increase in pathogenicity are determined by the presence of a consensus furin target site.

[0042] Furin cleaves the glycoproteins (GPs) of Marburg virus (MARV) and all five Ebolavirus species into a large N-terminal subunit (GP1) that mediates receptor binding and a small membrane-anchored C-terminal portion (GP2) that contains the fusion peptide (Volchkov VE et al., 1998; Volchkov VE et al., 2000).

[0043] Thus, the high conservation of the furin cleavage site among different Ebolavirus species is striking and it remains to be determined whether furin-mediated GP processing plays a role in the natural reservoir hosts of these viruses (Wooi-Lewis RJ et al., 1999).

[0044] Notably, the EBOV GP gene harbors an RNA editing site sequence and can express not only full-length GP, but also a soluble form of the glycoprotein (pre-sGP) that lacks the C-terminal transmembrane domain (Volchkov VE et al., 1995).

[0045] Interestingly, pre-sGP harbors another furin recognition site and is cleaved into mature sGP and a so-called short D-peptide. Both are eventually released from infected cells (Volchkova VA et al., 1999). Among other things, sGP is thought to serve as a decoy antigen, to act as a structural substitute for GP1 and to induce apoptosis of uninfected lymphocytes (De la Vega MA et al., 2015).

[0046] In the case of Flaviviruses, the flavivirus RNA is translated into a single large polyprotein that is cleaved by cellular and viral proteases into all the structural and non-structural proteins of the virus. The structural proteins include the envelope proteins prM and E that are incorporated as prM / E heterodimers into budding virions.70 prM acts as a chaperone and facilitates the correct folding of the E glycoprotein (Guirakhoo F et al., 1991).

[0047] Many flaviviruses bud into the lumen of the Endoplasmic Reticulum (ER) and enter the secretory pathway (Pierson TC et al., 2012). In the trans-Golgi network (TGN), a furin cleavage site is exposed and prM can be cleaved into mature pr and M proteins (Yu IM et al., 2008). Thus, furin-mediated cleavage of the viral glycoprotein occurs only after its incorporation into newly formed virions. The pr peptide remains associated with the E protein until the virion is released from the cell, preventing unintentional premature fusion with the membranes of the producer cell (Yu IM et al., 2009). Furin-mediated prM cleavage is essential for the replication of flaviviruses such as tick-borne encephalitis or dengue viruses (Elshuber S et al., 2003; Zybert EA et al., 2008).

[0048] In dengue viruses, large amounts of immature or partially mature viruses are released, most likely due to a conserved acidic residue (defect) in the furin recognition site (Junjhon J et al., 2008). Thus, furin appears to be crucial for the maturity and virulence of dengue viruses. It is also important to note that the prM content in viral particles also affects antibody recognition and, consequently, antibody-dependent stimulation of dengue virus infection (Rodenhuis-Zybert IA et al., 2010).

[0049] Therefore, in dengue viruses in particular, inhibition of furin helps preserve the prM protein complex, which promotes the proper functioning of the host's immune defense response.

[0050] While furin plays a key role in the activation of envelope glycoproteins of a variety of viruses, its activity is also exploited for the cleavage of other viral proteins. The cleavage of the L2 protein of papillomaviruses is an illustration (Richards RM et al., 2006).

[0051] The assembly, consisting of the major capsid protein L1 and this minor capsid protein L2, constructs the viral capsid. The furin cleavage site is located near the N terminus of capsid L2 and is highly conserved among different human papillomavirus (HPV) strains (Day PM et al., 2009). Cleavage is not required for virus assembly or release but is essential for infection of new target cells.

[0052] Papillomavirus L2 is exclusively cleaved on target / host cells (Day PM et al., 2009).

[0053] Thus, it appears that non-enveloped viruses have also developed the ability to exploit furin, particularly in their proliferation strategy on new host cells.

[0054] Another non-viral envelope protein cleaved by furin is the hepatitis B virus (HBV) outer core antigen (HBeAg) (Ito K et al., 2009; Messageot F et al., 2003). Cleaved HBeAg is secreted from infected cells and exerts immunosuppressive effects (Milich DR et al., 1998). It has been suggested to act by preventing the destruction of infected hepatocytes by cytotoxic T lymphocytes (Chen MT et al., 2004). In contrast, uncleaved HBeAg may have the opposite effects when transported to the plasma membrane where it can trigger antiviral immune responses (Schlicht HJ et al., 1989). Thus, furin-mediated cleavage of HBeAg may affect the outcome of infection by promoting the immune response.

[0055] In 2013, Aerts and colleagues discovered that protease-activated receptor 1 (PAR1), a G protein-coupled receptor, interferes with furin expression and furin-mediated processing of the human metapneumovirus F protein (Aerts L et al., 2013). Follow-up experiments revealed that PAR1 harbors a motif that mediates interaction with multiple PCSKs (Kim W et al., 2015). Consistent with this, soluble PC5A / PCSK5 and PCSK6 cleave PAR1 and abrogate its ability to induce calcium signaling upon thrombin-mediated cleavage at the plasma membrane. Surprisingly, however, membrane-bound PCSKs such as furin fail to cleave PAR1 at this position. Instead, furin traps PAR1 in the trans-Golgi network and prevents its anterograde transport to the cell surface. At the same time, PAR1 also blocks the proteolytic activity of furin, inhibiting, for example, the maturation of HIV-1 Env.This inhibitory activity is not shared by its paralog PAR2, which is efficiently cleaved by furin (Sachan V et al., 2019). Notably, PAR1 expression is induced in pro-inflammatory environments such as the brain of HIV-1-infected individuals with HIV-associated neurocognitive disorders (HAND) (Kim W et al., 2015).

[0056] Thus, PAR1-mediated furin inhibition may represent an innate immunity mechanism limiting the spread of HIV-1 and potentially other fur-dependent viral pathogens. Similar inhibitory activity has recently been described for two IFNc-inducible GTPases, termed guanylate-binding proteins 2 and 5 (GBP2 and GBP5). Initially, GBP5 was described in a screen for novel HIV restriction factors and shown to interfere with retroviral Env protein maturation. As a result, cleavage of the Env precursor gp160 into mature gp120 and gp41 is reduced, and newly formed virions are only weakly infectious.Since many viral pathogens rely on furin or related PCSKs for the maturation of their own (glyco) proteins, GBP2 and GBP5 exert broad antiviral activity, inhibiting the replication of highly pathogenic avian influenza A, measles, and Zika virus. In contrast, GBP2 and GBP5 do not decrease the infectivity of virions carrying the vesicular stomatitis virus glycoprotein, which does not require a proteolytic activation step. Of note, furin inhibition in infected cells comes at a cost, as furin-mediated processing of matrix metalloproteinases and other cellular substrates is also reduced in the presence of increased levels of GBP2 or GBP5 (Braun E et al., 2019). Of note, increased GBP2 / 5 expression is associated with favorable outcomes in patients with melanoma or breast cancer (Wang Q et al., 2018; Godoy P et al., 2014).

[0057] In summary, among enveloped viruses, a furin inhibitor can block the infection and / or pathogenicity of Herpesviruses, Coronaviruses, Flaviviruses, Togaviruses, Bornaviruses, Bunyaviruses, Filoviruses, Orthomyxoviruses, Paramyxoviruses, Pneumoviruses and Retroviridae, and more particularly in Human Immunodeficiency Virus (HIV), Rous sarcoma virus, murine leukemia viruses, avian influenza A viruses, Marburg virus, all five species of Ebolavirus, tick-borne encephalitis virus and dengue viruses.

[0058] In the event of a viral attack, inhibition of furin can promote the proper functioning of the host's immune defense response, particularly in dengue viruses.

[0059] In non-enveloped viruses, a furin inhibitor can block the infectious spread of papillomaviruses, and promote a good immune response in the case of hepatitis B virus.

[0060] Due to the key role of furin in the pathogenesis of cancer and infectious diseases, its suitability as a therapeutic target has attracted considerable interest for several years. Many laboratories have explored the possibility of limiting tumor growth, viral replication, or bacterial poisoning by reducing the amount or proteolytic activity of furin.

[0061] Initially, most studies focused on peptides or proteins that bind to the furin active site and competitively inhibit substrate binding. For example, a variant of the naturally occurring serine protease inhibitor α-1 antitrypsin was engineered to harbor a consensus furin cleavage site. This variant, termed α-1 antitrypsin Portland (α1-PDX), inhibits furin and PCSK5 and has been shown to prevent processing of HIV-1 Env and measles virus F in vitro (Anderson ED et al., 1993; Watanabe M et al., 1995).

[0062] Hemagglutinin and polyarginines compete with natural furin substrates (Strongin A et al., 2009; Cameron A et al., 2000). Even exogenous addition of the autoinhibitory furin propeptide has been shown to reduce its enzymatic activity, for example limiting MMP9 activation in breast cancer cells (Zhong M et al., 1999; Lapierre M et al., 2007). However, the therapeutic potential of the propeptide has never been evaluated in vivo and the inhibitory effects are most likely limited as it is known to dissociate from furin in the TGN (Golgi compartment). Several approaches, including the incorporation of D- instead of L-amino acids, have been applied to increase the stability and thus the efficacy of furin inhibitors.For example, hexa-D-arginine (D6R), one of the first furin inhibitors, shows good stability and prevents the cytotoxic effects of Pseudomonas exotoxin A in vitro and in vivo (Sarac MS et al., 2002). Similarly, topical application of nona-D-arginine (D9R) reduces corneal damage in mice infected with . Pseudomonas aeruginosa(Karicerla P et al., 2009). D9R has also shown direct bactericidal activity, probably due to its polycationic nature (Karicerla P et al., 2010). Besides D-amino acids, the incorporation of amino acid analogues such as decarboxylated arginine mimetics or 4-amidinobenzylamide (Amba) has been used to increase the stability of peptide-derived furin inhibitors (Becker GL et al., 2010). In addition, the addition of a chloromethyl ketone (CMK) moiety to the C-terminus of a polybasic cleavage motif has been shown to be useful as it results in alkylation of the furin active site which irreversibly blocks its enzymatic activity (Henrich S et al., 2003). However, the cytotoxicity of CMK-based inhibitors and the instability of the CMK moiety may limit their use to topical applications such as the treatment of HPV skin infections (Couture F et al., 2015).

[0063] The elucidation of the crystal structure of furin has enabled the targeted modeling of non-peptide inhibitors such as streptamine-based compounds. Upon addition of guanidine residues, streptamine derivatives mimic the cleavage site of cationic furin and inhibit its enzymatic activity in the nanomolar range in vitro (Jiao GS et al., 2006). Dahms and colleagues describe an interesting example of a 2,5-dideoxystreptamine-derived inhibitor, where two molecules of the inhibitor form a complex with furin (Dahms SO et al., 2017). While the first inhibitor molecule directly interferes with the conformation of the catalytic triad, the second molecule binds to an adjacent planar peptide stretch. To inhibit anthrax toxin activation, the inhibitor does not need to enter cells, because furin cleaves the toxin precursor on the cell surface.In contrast, inhibitor penetration into the cell is essential to prevent maturation of HIV-1 Env and other viral glycoproteins that are cleaved intracellularly. While some inhibitors (e.g., HA-derived peptides) efficiently penetrate cells, others have been modified to increase their intracellular availability. For example, the addition of a decanoyl moiety to CMK inhibitors increases their ability to penetrate cells (Garten W et al., 1989).

[0064] While many of the inhibitors described above potently reduce furin activity in vitro And in vivo,Most of them also inhibit other proprotein convertases recognizing the same or similar polybasic cleavage sites. This limitation is inherent in competitive inhibitors that aim to mimic the furin target sequence and can be overcome by allosteric inhibitors that bind to specific furin motifs outside the active site. One example is the nanbody Nb14, which binds to the C-terminal domain of furin, thereby blocking access of larger substrates to the active site. Notably, Nb14 binds specifically to the P domain of furin and does not recognize other PCSKs (Zhu J et al., 2012).

[0065] The inventor has demonstrated that a family of compounds, pentacyclic triterpenes, can be used as active ingredients inhibiting PCSK (proprotein convertase subtilisin / kexin type), and more particularly PCSK3 also called furin.

[0066] Unexpectedly and in a novel manner, the inventor has shown that pentacyclic triterpenes and more particularly mixtures of α and β amyrins, whose chemical structure is very robust and devoid of nitrogen, and therefore of a non-protein nature can be used as PCSK inhibitors, and more particularly PCSK3.

[0067] Thus, a first object of the invention relates to a pentacyclic triterpene chosen from α and β amyrins, for its use as an active ingredient inhibiting PCSK3 also called furin for the treatment of infectious diseases in mammals to block microbiological toxins of type AB and keep them passive and without virulence, to stop anthrax, as an anti-viral in enveloped viruses, blocking the infection and pathogenicity of Coronaviruses, Flaviviruses, Togaviruses, Bornaviruses, Bunyaviruses, Filoviruses, Paramyxoviruses, Pneumoviruses and Retroviridae, and more particularly of the Human Immunodeficiency Virus (HIV), the Rous sarcoma virus, murine leukemia viruses, the Marburg virus, the five species of Ebolavirus, the tick-borne encephalitis virus and the dengue viruses, to promote the proper functioning of the host immune defense response in dengue viruses,as an antiviral in non-enveloped viruses by blocking the infectious spread of papillomaviruses.

[0068] In recent years, several peptide and non-peptide inhibitors have been developed to block the activation of bacterial toxins, prevent the maturation of viral proteins, and suppress tumor growth.

[0069] In particular, the article “Analysis of antioxidant and antiviral biomarkers β-amyrin, β-sitosterol, lupeol, ursolic acid in Guiera senegalensis leaves extract by validated HPTLC methods” by Parvez et al. Saudi Pharmaceutical Journal vol.26 n°5 pages 685-693 discloses on page 692, left column, first paragraph that β-amyrin is known for its antiviral properties against influenza A virus and the article “Triterpenoid Saponin Biosynthetic Pathway Profiling and Candidate Gene Mining of the Ilex asprella Root Using RNA-Seq” by Xiasheng Zheng et al. International Journal of molecular Sciences vol.15 n°4 pages 5970-5987 discloses on page that the Ilex asprellawhich contains a large amount of α-amyrin is a medicinal plant known for its antiviral properties against influenza A virus. These articles do not mention α and β amyrins, for their use as an active ingredient inhibiting furin for the treatment of infectious diseases according to the present invention.

[0070] The active ingredient used according to the invention is a pentacyclic triterpene, chosen from purified amyrins and natural extracts containing amyrins and amyrin derivatives, in particular extracts of Burseraceae, and more particularly extracts of Protium heptaphyllum.

[0071] Advantageously, said pentacyclic triterpene consists of α amyrin (100% or viminalol), or β amyrin (100%), or a mixture of α and β amyrins containing from 20% to 80% of α amyrin and from 20% to 80% of β amyrin, preferably a mixture of α and β amyrins containing from 20% to 80% of α amyrin and from 20% to 80% of β amyrin and preferably a mixture comprising 60% of α amyrin and 40% of β amyrin.

[0072] Preferably, the active ingredient comprises α and β amyrins and more particularly purified amyrins from Protium heptaphyllum" which is a complex composed of 60% α amyrin and 40% β amyrin.

[0073] According to a first implementation of the invention, the pentacyclic triterpene is used according to the invention to block microbiological toxins of type AB and keep them passive and without virulence.

[0074] According to a second implementation of the invention, the pentacyclic triterpene is used according to the invention to stop anthrax.

[0075] According to a third implementation of the invention, the pentacyclic triterpene is used according to the invention as an antiviral in enveloped viruses, blocking the infection and pathogenicity of Coronaviruses, Flaviviruses, Togaviruses, Bornaviruses, Bunyaviruses, Filoviruses, Paramyxoviruses, Pneumoviruses and Retroviridae, and more particularly of the Human Immunodeficiency Virus (HIV), the Rous sarcoma virus, the murine leukemia viruses, the Marburg virus, the five species of Ebolavirus, the tick-borne encephalitis virus and the dengue viruses.

[0076] According to a fourth implementation of the invention, the pentacyclic triterpene is used according to the invention to promote the proper functioning of the host's immune defense response, in particular in dengue viruses. According to a fifth implementation of the invention, the pentacyclic triterpene is used according to the invention as an antiviral in non-enveloped viruses by blocking the infectious propagation of papillomaviruses.

[0077] The furin activity inhibitor according to the invention therefore makes it possible to avoid the activation of numerous bacterial toxins (themselves, possibly of viral origin), in particular the group of AB toxins with an intracellular inhibitor which keeps the toxins passive and without virulence. The furin activity inhibitor according to the invention also makes it possible to stop and avoid toxins such as anthrax in the extracellular environment.

[0078] In the fight against bacterial toxins and for their neutralization, the furin activity inhibitors according to the invention are substances to be integrated into the prevention and treatment of infectious diseases involving these toxins.

[0079] According to the invention, the use of α and β amyrins as inhibitors of furin activity in enveloped viruses blocks or participates in blocking the infection and pathogenicity of Coronaviruses, Flaviviruses, Togaviruses, Bornaviruses, Bunyaviruses, Filoviruses, Paramyxoviruses, Pneumoviruses and Retroviridae, and more particularly of the Human Immunodeficiency Virus (HIV), the Rous sarcoma virus, the murine leukemia viruses, the Marburg virus, the five species of Ebolavirus, tick-borne encephalitis and dengue viruses.

[0080] In case of viral attack, the use of α and β amyrins as inhibitors of furin activity promotes the proper functioning of the host's immune defense response, particularly with dengue viruses.

[0081] In non-enveloped viruses, the use of α and β amyrins according to the invention as an inhibitor of furin activity blocks and promotes the cessation of the infectious spread of papillomaviruses. The use of α and β amyrins according to the invention is particularly implemented according to local therapeutic applications, in mammals and more particularly in humans, such as the topical treatment of bacterial and viral infections. PCSK inhibitors, and more particularly PCSK3, are still rare and at the heart of cutting-edge therapeutic strategies in the fields mentioned above. One of the main drawbacks of the inhibitors conventionally used is their fragility and ability to degrade rapidly in biological environments due to their peptide nature. Indeed, conventional inhibitors are mainly of a peptide nature even in the case of the allosteric inhibition strategy. Non-peptide inhibitors are a priori a little more robust but remain structured with an amine or amide function (4-amidinobenzylamide, streptamine, 2,5 dideoxystreptamine) which makes them mimetic to the amino bases of peptides.

[0082] α and β Amyrin are the components whose furin activity modulation activity is more particularly at the heart of the invention.

[0083] According to Vasquez (Vasquez LH et al., 2012), pentacyclic triterpenes are ubiquitous in the plant kingdom, in their free aglycone forms or in their combined forms. α and β amyrins and their analogues are very common in the Burseraceae family and more particularly in the genus Protium. The chemical structure of α amyrin is C 30 H 50 O, its melting point is between 184°C and 186°C (Sirat, et al., 2010); the chemical structure of β amyrin is C 30 H 50 O, its melting point is between 189°C and 191°C (Lin et al., 2011).

[0084] α amyrin is a naturally occurring triterpene isolated from various sources, including plant resins. The highest amounts of this triterpene are found in resins of the Burseraceae and especially in the genre Protium. Other known sources of α amyrin include Mexican copal (5 g / kg) Bursera glabrifolia (Burseraceae) (Hernández-Vázquez, et al., 2010), Cassia obtusifolia (Caesalpinaceae) (140 mg / kg) (Sob et al., 2010) and the resin of Commiphora holtziana (syn . Commiphora erythraea ) (Burseraceae) (200 mg / kg) (Manguro et al., 2009).

[0085] The most important sources of β amyrin include lotus bee pollen Nelumbo nucifera (Nelumbonaceae) (3 g / kg) (Xu. Et al., 2011), the bark of “cuachalalate” Amphipterygium adstringens (Anacardiaceae) (2.4 g / kg) (Rosas-Acevedo et al., 2011), isolation of resin from various Protium sp. (Burseraceae) (Alpha-amyrin 1g / kg and Beta-amyrin 1.7 g / Kg) (Dias et al., 2011), biomass residues of Eucalyptus globulus (Myrtaceae)from the pulp industry (326 mg / kg) (Domingues et al., 2010), latex Ficus carica (Moraceae) (1.2 g / kg) (Oliveira et al., 2010), root bark of Ficus cordata (Moraceae) (20 mg / kg), vapor bark Ficus cordata (200 mg / kg) (Kuete et al., 2008) and the leaves and bark of Byrsonima crassifolia Or crassus (Malpighiaceae) (1.3 g / kg) (Higuchi et al., 2008). Mixtures of α and β amyrin were obtained from steam bark residues of Byrsonima crassifolia (Malpighiaceae) (9 g / kg) (Hernández-Vázquez et al., 2010), leaves of Byrsonima fagifolia (Malpighiaceae) (2,3 g / kg) (Higuchi et al., 2008) and leaves of Pouteria sp. (Sapotaceae) yielded α, β amyrin and other triterpenes (Silva et al., 2009). Below is a non-exhaustive list of plants known to possess α amyrin, β amyrin and a mixture of α, β amyrin in small amounts (detected and isolated):

[0086] α Amyrin was isolated from the resin of Boswellia carterii (Burseraceae)(Wang et al., 2011), detected in stem wood and bark of Populus sp. (Salicaceae) (Xu et al., 2010), isolated (65 mg / kg) from n-hexane extracted from leaves of Melastoma malabathricum (Melastomataceae) (Sirat et al., 2010), identified in the methanolic extract of the stem bark of Poncirus trifoliate (Rutaceae ) (Feng et al., 2010), isolated (1 mg / kg) from methanol extracted from stem bark of the African tree Antiaris sp. (Moraceae) (Vouffo et al., 2010), detected in Saskatoon berry seed oil Amelanchier alnifolia (Rosaceae) (Bakowska-Barczak et al., 2009), isolated (23 mg / kg) from methanol extract of stem bark and leaves of Ficus pandurata (Moraceae) (Ramadan et al., 2009), dried rhizomes of Nelumbo nucifera (Nelumbonaceae ) (Chaudhuri et al., 2009), and detected in bread wheat Triticum aestivum (Poaceae) (Nurmi et al., 2008).

[0087] β amyrin has been isolated and detected in various plants: an ethanolic fraction of oleogum resin from Ferula gummosa (Apiaceae) (Jalali et al., 2011), the plant Carpobrotus edulis (Aizoaceae), (Martins et al., 2011), the leaves of Clerodendrum inerme (Verbenaceae) (22.5 mg / kg) (Parveen et al., 2010), chloroform extract of the aerial parts of the plant or callus of Euphorbia tirucalli (Euphorbiaceae) (Uchida et al., 2010), seed oil of Capparis spinosa (Capparaceae) (Tlili et al., 2011), chloroform extract of the leaves of Ficus benjamina (var. camosa) (Moraceae) (Simo et al., 2009), leaves (2 mg / kg) of Pyrenacantha staudii (Icacinaceae), (Falodun et al., 2009), ethanol extract of leaves of Olea europaea (Oleaceae) (Wang et al., 2009), ethyl acetate extract of apple peels of the Red Delicious variety Malus domestica (Rosaceae) (He et al., 2008), air-dried leaves of Tectona philippinensis (Lamiaceae), an endemic Philippine medicinal plant (Ragasa et al., 2008), a mixed extract of benzene and chloroform from the leaves of Rhus alata (Anacardiaceae) (Parveena et al., 2008), and a stem bark extract of African Piptadenia Or Piptadeniastrum africanum (Mimosaceae) from Cameroon (Mbouangouere et al., 2008).

[0088] A mixture of α and β amyrin was detected in the following plants: n-hexane and chloroform extracts of the epicuticular wax layer of Mandevilla guanabarica And Mandevilla moricandiana (Apocynaceae) (Cordeiro et al., 2011), an ethanolic extract of roots of Salacia amplifolia (Celastraceae) (Wang et al., 2011) and chloroform extracts of blue honeysuckle Lonicera caerulea (Caprifoliaceae) (Palikova et al., 2008). By genetic and molecular engineering, the expression of an oxidosqualene cyclase (OSC) and β-amyrin synthase, which are involved in the cyclization of 2,3-oxidosqualene to produce β-amyrin, has been successfully given in Saccharomyces cerevisiae β-amyrin (Sun R, 2017).

[0089] Still through genetic and molecular engineering, the production of α amyrin is now possible in Saccharomyces cerevisiae. Indeed, 2 Alpha amyrin synthases α-AS, EjAS and MdOSC1 of Japanese Eriobotrya And Malus × domestic were expressed in yeast S . cerevisiae.Yeasts with EjAS produced α-amyrin and β-amyrin in a ratio of 17:3. Yeasts with MdOSC1 produced α-amyrin, β-amyrin, and lupeol in a ratio of 86:13:1 (YU Y, 2018).

[0090] Unexpectedly and in a novel manner, the inventor discovered that pentacyclic triterpenes selected from α and β amyrin can be used as active ingredients for the inhibition of PCSK3.

[0091] In this text, the “pentacyclic triterpene selected from α and β amyrin” used according to the invention is also called “the PCSK3 inhibitor” and “the active ingredient”.

[0092] PCSK inhibition, particularly furin, can halt many infectious phenomena by inhibiting protein cleavage, which activates the toxic protein or the pathogenic microorganism that carries it. The anti-infectious activity of PCSK inhibitors, particularly furin, is therefore not biocidal in nature.

[0093] The furin inhibitor according to the invention is an active ingredient advantageously chosen from purified amyrins, but also from natural extracts containing amyrins and amyrin derivatives and in particular extracts of Burseraceae, and more particularly extracts of Protium heptaphyllum. But more generally, in the context of this invention, the amyrins inhibiting furin activity which constitute the new active ingredient according to the invention, can be derived from the plant kingdom, from plants capable of producing terpenoids of the pentacyclic triterpene type and more particularly α and β amyrins and their derivatives, from families as diverse as Aizoaceae, Anacardiaceae, Apiaceae, Apocynaceae, Asclepiadaceae, Bombacaceae, Burseraceae, Caesalpinaceae, Capparaceae, Caprifoliaceae, Celastraceae, Clusiaceae, Cucurbitaceae, Euphorbiaceae, Fabaceae, lcacinaceae, Irvingiaceae, Lamiaceae, Malpighiaceae, Malvaceae, Melastomataceae, Mimosaceae, Moraceae, Myrsinaceae, Myrtaceae, Nelumbonaceae, Oleaceae, Poaceae, Rosaceae, Rutaceae, Salicaceae, Sapotaceae, Theaceae, Verbenaceae, Vitaceae; More particularly in the context of this invention, the activity-inhibiting amyrins may be derived from the plant kingdom, from plants capable of producing terpenoids of the pentacyclic triterpene type and more particularly α and β amyrins and their derivatives, from species as diverse as Acacia sp or Senegalia visco (Fabaceae), Amelanchier alnifolia (Rosaceae), Amphipterygium adstringens (Anacardiaceae), Antiaris sp (Moraceae), Ardisia elliptica (Myrsinaceae), Bombax ceiba (Bombacaceae), Bombax malabaricum (Bombacaceae), Boswellia carterii (Burseraceae), Bursera glabrifolia (Burseraceae), Bursera simaruba (Burseraceae), Byrsonima crassifolia Or crassa (Malpighiaceae), Byrsonima fagifolia (Malpighiaceae), Calotropis gigantea (Asclepiadaceae), Camellia japonica (Theaceae), Capparis spinosa (Capparaceae), Carpobrotus edulis (Aizoaceae), Cassia obtusifolia (Caesalpinaceae), Clerodendrum inerme (Verbenaceae), Commiphora holtziana (syn. Commiphora erythraea) (Burseraceae), Duranta repens (Verbanaceae), Eucalyptus globulus (Myrtaceae), Euphorbia tirucalli (Euphorbiaceae), Ferula gummosa (Apiaceae), Ficus benjamina (Moraceae), Ficus carica (Moraceae), Ficus cordata (Moraceae), Ficus pandurata (Moraceae), Garcinia subelliptica (Clusiaceae), Klainedoxa gabonensis (Irvingiaceae), Lonicera caerulea (Caprifoliaceae), Ligustrum sp (Oleaceae), Malus domestica (Rosaceae), Melia azedarach (Meliaceae), Mandevilla guanabarica et Mandevilla moricandiana (Apocynaceae), Melabath malabath (Melastomataceae), Nelumbo nucifera (Nelumbonaceae), Olea europaea (Oleaceae), Piptadeniastrum africanum (Mimosaceae), Poncirus trifoliate (Rutaceae), Populus sp (Salicaceae), Pouteria sp (Sapotaceae), Protium sp. (Icacinaceae), Rhus alata (Anacardiaceae), Salacia amplifolia (Celastraceae), Siraitia grosvenorii (Cucurbitaceae), Tectona philippinensis (Lamiaceae), Triticum aestivum (Poaceae), and Vitis vinifera (Vitaceae).

[0094] In the context of this invention, the amyrins inhibiting the activity of furin which constitute the new active principle according to the invention, may also be derived from biotechnology, that is to say produced via bacteria or yeasts, or even by enzymes.

[0095] Still within the framework of this invention, the amyrins inhibiting the activity of furin which constitute the new active principle according to the invention may be derived from any synthetic organic chemistry process.

[0096] Finally, within the framework of this invention, the amyrins inhibiting the activity of furin which constitute the new active principle according to the invention may be derived from natural extracts from the animal kingdom, preferably extracted from arthropods, in particular from insects of the genus Platyphora from leaf beetle species.

[0097] For the implementation of therapeutic products within the framework of the invention, the active principle according to the inventor can be previously solubilized or vectorized so as to be able to use it and dose it in various preparations.

[0098] In the context of the present invention, the fatty substances which solubilize or vectorize the active principle may comprise hydrocarbon oils, butters and waxes of animal or vegetable or mineral origin, silicone oils, or their mixture, but also lipophilic additives. As hydrocarbon oils or butters, we can notably cite: vegetable or animal oils or butters, and more particularly triglycerides; synthetic ethers; linear or branched hydrocarbons, of mineral or synthetic origin such as petroleum jelly; synthetic esters such as isopropyl myristate; fatty alcohol benzoates; heptanoates, octanoates, decanoates or ricinoleates of alcohols or polyalcohols; hydroxylated esters such as isostearyl lactate, polyol esters; fatty alcohols such as octyldodecanol; higher fatty acids such as linolenic acid; polymethylsiloxane silicone oils and their mixtures.Examples of vegetable wax include carnauba wax, candelilla wax, jojoba wax, or any other plant compound consisting of an ethylene glycol ester and two fatty acids or a monoester of fatty acid and long-chain alcohol; examples of animal wax include beeswax.

[0099] Other fatty substances include: essential oils; natural and synthetic lipophilic aromatic compounds; natural or synthetic fat-soluble vitamins such as tocopherol or alpha-tocopheryl acetate.

[0100] The active ingredient according to the invention is characterized by its amyrin content. Amyrins are either 100% α amyrin, also called viminalol, or 100% β amyrin, but preferably a mixture of α and β amyrin. The preferred mixture of amyrins will be between 20%-80% α amyrin and 20%-80% β amyrin.

[0101] The amyrins as active ingredient according to the invention may be biotechnological, plant or animal extracts titrated in α and / or β amyrins.

[0102] Amyrins as active ingredient according to the invention can also be the result of an organic chemical synthesis titrated in α and / or β amyrins.

[0103] According to the invention, amyrins are significantly active from a dosage of 25 ppm, they are very active at 250 ppm and reach their maximum activity plateau around 1000 ppm. Nevertheless, the toxicity of amyrins being very low according to current knowledge, it is possible to use amyrins in therapeutic products at high concentration. Thus, therapeutic products can contain as active ingredient from 10 ppm (0.001%) to 100% amyrin.

[0104] The present invention also relates to a therapeutic product comprising a pentacyclic triterpene, intended for mammals, comprising amyrin or derivatives in a content of between 0.001% and 100% by weight of amyrin or derivatives relative to the total weight of the therapeutic product.

[0105] The active ingredient according to the invention is prepared and used by those skilled in the art in therapeutic preparation, pure or after pre-solubilization in one of the vector or solvent compounds described above.

[0106] For example, it is possible to solubilize amyrins in 96° ethanol at approximately 55% in order to obtain a homogeneous liquid active solution that is easy to measure and dilute.

[0107] Another example is to solubilize amyrins in an ester of the rapeseed methyl ester type (methyl canolate) up to 45% so as to obtain a homogeneous liquid active solution that is easy to dose and dilute.

[0108] Another example is to solubilize amyrins in sunflower oil up to 30% in order to obtain a homogeneous liquid active solution that is easy to measure and dilute.

[0109] According to another patent of the inventor FR2977169B1, the capacity of the active ingredient, as the majority compound of plant resin, to create an interface in an aqueous or polar medium between the polar mobile phase and the dispersed phase, namely its vector or its rather apolar solvent, so as to create a pseudo emulsion where the active ingredient is preferentially positioned at the interface of the dispersed and dispersing phase.

[0110] The active ingredient according to the invention can be used by those skilled in the art, in a therapeutic composition of any polarity in the form of a solid product of the lozenge, suppository or topical balm or wax type.

[0111] The active ingredient according to the invention can be used in a therapeutic composition of any polarity in the form of a liquid product of the subcutaneous injectable type, intravenous injectable type, eye drops, spray, serum or oral syrup.

[0112] The active ingredient according to the invention can be used by a person skilled in the art, in a therapeutic composition of any polarity in the form of a topical application product in the form of lotion, gel, serum, emulsion, patch, oil, washing or disinfectant solution.

[0113] This text also describes that the active ingredient according to the invention, as an inhibitor of furin activity, can be used in biological culture media, as an additive or reference or control agent for a test or trial; as a technical or target additive in a medical diagnostic system, but also in a blood or environmental screening system;

[0114] This text also describes that, in addition to the therapeutic activity, the active ingredient according to the invention, as an inhibitor of furin activity, can be used in an environmental sanitizing spray or aerosol type product or for any phyto- and / or zoo-technical product in any form whatsoever; by spraying in a liquid form to film on surfaces so as to generate a bioactive barrier of the anti-viral type. As a non-exhaustive example, we can consider spraying an alcoholic solution of amyrins or resinous extract rich in amyrins and derivatives on face masks so as to increase the viral barrier effect of these devices, because in fact the alcoholic solvent dries quickly, the amyrins are quickly transferred and deposited, they then film the impregnated surfaces.

[0115] The following examples are intended to illustrate the invention without limiting its scope. EXAMPLES Example 1 Inhibition of Furin Enzyme Activity

[0116] The experiment is performed using recombinant human furin, which catalyzes the cleavage of a specific fluorescent substrate. Reference Product

[0117] Decanoyl-Arg-Val-Lys-Arg-CMK at 100 nm was used as a reference inhibitor of furin activity. Incubation protocol

[0118] Furin is pre-incubated for 10 minutes at room temperature in the absence (control) or presence of the reference product or increasing concentrations of the test compound: “Purified Amyrins from Protium heptaphyllum”; 25 ppm; 250 ppm and 1000 ppm (v / v). The product “Purified Amyrins from Protium heptaphyllum”, AMY at 1000 PPM corresponds to a 0.5% volume solution of the HTRE product marketed by the company Ephyla.

[0119] At the end of the pre-incubation step, furin substrate was added and the experimental conditions were incubated again at room temperature protected from light for 5 minutes. All experiments were performed in triplicates. Preparation of compound :

[0120] The test compound "Purified Amyrins from Protium heptaphyllum" was pre-solubilized at 10,000 ppm in DMSO. The solution was then diluted in assay buffer to achieve the concentrations described above. Assessment protocol

[0121] Cleavage of the fluorescent furin substrate was monitored for 5 min after substrate addition by reading fluorescence at 485 nm / 535 nm. Statistics

[0122] Results are expressed in RFU (relative fluorescent units) + / - SD (Standard deviation).

[0123] The statistical significance of the difference observed between the “Control” and “Reference Product” groups was assessed by a Student t-test (p <0.001).

[0124] The statistical significance of the observed difference between the “Control” and “Test Compound” groups was assessed by one-way ANOVA analysis, followed by a HolmSidak test ( p < 0.05). Results

[0125] The compound "Purified Amyrins from Protium heptaphyllum", designated as "AMY" (below) under the chosen experimental conditions significantly inhibits furin activity: Furin activity (in % of control) "AMY" at 25 ppm (v / v) 84,6% (p <0,001) "AMY" at 250 ppm (v / v) 12,0% (p <0,001) "AMY" at 1000 ppm (v / v) 0,7% (p <0,001) Reference product and vehicle

[0126] The reference furin inhibitor named Decanoyl-Arg-Val-Lys-Arg-CMK, tested at 100 nm, significantly inhibited furin activity by 97.9% (p < 0.001).

[0127] This result was expected and validates the study. Conclusion

[0128] At 250 ppm, “Purified Amyrins from Protium heptaphyllum” inhibits 88% of the activity of the enzyme Furin

[0129] At 1000 ppm, “Purified Amyrins from Protium heptaphyllum” inhibits 99.3% of the activity of the Furin enzyme. Example 2 : Anti-infectious effect against a SARS-CoV-2 crown virus

[0130] The experiment was performed using a 0.5% HTRE solution which corresponds to the 1000 PPM AMY complex described in Example 1 above.

[0131] The test uses 2 main components.

[0132] On the one hand, a recombinant human HEK293 cell strain expressing full-length human ACE2 (Genbank # NM_021804.3), with surface expression of ACE2 confirmed by flow cytometry.

[0133] On the other hand, SARS-CoV-2 Spike pseudotyped lentiviruses produced with SARS-CoV-2 Spike (Genbank accession number QHD43416.1) as envelope glycoproteins instead of the commonly used VSV-G. These pseudovirions also contain the firefly luciferase gene driven by a CMV promoter, therefore, spike-mediated cell entry can be conveniently measured via luciferase reporter activity. Protocol:

[0134] Step 1: ACE2-HEK cells are placed in culture wells. For this purpose, ACE2-HEK cells are thawed in thawing medium 1, expanded in 1N growth medium, then harvested and cultured in white and transparent flat-bottomed 96-well culture plates at 10,000 cells / well in 50 µl of thawing medium 1. The cells are incubated overnight at 37°C. Step 2: Pseudotyped lentiviral infection test

[0135] The next day, visual control of homogeneity and integrity of the cell layer was validated using an inverted microscope and the following test ingredients were prepared: Three HTRE solutions were prepared C1 (0.5%), C2 (0.25%) and C3 (0.1%); then these solutions were mixed with DMSO according to the ratio of 75% HTRE solution and 25% DMSO.

[0136] Upon addition of DMSO, HTRE forms two phases of similar volume called “up” and “down”. The “down” phase displays a marked yellow color. HTRE “up” is tested at the three concentrations 0.5 (C1), 0.25 (C2) and 0.1% (C3), while HTRE “down” is tested at two concentrations, C1 and C2. The final concentration of DMSO in the corresponding analysis wells is 0.5%.

[0137] Extracts are diluted to an intermediate concentration 11x in Thawing Medium 1, for subsequent transfer of 5 µl to the assay plate. After a 30 min incubation period at 37 °C, 5 µl of undiluted pseudotyped lentivirus (Bald or S1-Spike) is added to the corresponding assay wells. As additional negative controls, 5 µl of Thawing Medium 1 containing either 5.5% DMSO or PBS is used. As a positive control, the ACE2 blocking mAb is used at a final concentration of 0.5 µM in the assay wells.

[0138] The bald pseudovirion is an additional witness (virus without crown "Spike proteins") which shows that without Spike proteins the Bald virus does not have the capacity to infect cells.

[0139] The results obtained are presented in the Figure 1 attached. Conclusion

[0140] Bald vs Spike: Greater than 57-fold increase in RLU signal upon infection with S1 Spike pseudotyped lentivirus, validating the experiment. Spike vs anti-ACE2 Spike: 41.91% drop in RLU signal induced by the blocking antibody used at concentrations close to the IC50 values ​​measured by the kit supplier, validating the experiment. HTRE “down”: 96.93% drop in RLU signal induced by the blocking antibody used at 0.5% concentration; 98% drop in RLU signal induced by the blocking antibody used at 0.25% concentration; 96.62% drop in RLU signal induced by the blocking antibody used at a concentration of 0.1% The anti-infectious effect against a SARS COV2 crown virus is thus demonstrated and conclusive from the dose of 0.1% of HTRE (i.e. 200 PPM AMY equivalent) with a 96.62% reduction in viral infection.HTRE “up”: 58.66% drop in RLU signal induced by the blocking antibody used at 0.5% concentration; 54.58% drop in RLU signal induced by the blocking antibody used at 0.25% concentration. The HTRE “up” fraction is simply HTRE less well solubilized in the medium because it is very little bound to DMSO; this poorer solubilization results, as expected, in a significant but less conclusive score. BIBLIOGRAPHY

[0141] Braun E et al., 2019: “Furin-mediated protein processing in infectious diseases and cancer” Elisabeth Braun & Daniel Sauter, Clinical & Translational Immunology 2019; 8: e1073. Unless otherwise stated, the references of all articles cited in this text are presented at the end of the article Braun E et al., 2019 cited above.

Claims

1. A pentacyclic triterpene selected among α and β amyrins for use as a PCSK3-inhibiting active substance, also called furin, for the therapeutical treatment of infectious diseases among mammals, to inhibit AB-type microbiological toxins and keep them passive and without virulence, to stop anthrax, as an antiviral drug against enveloped viruses, hence inhibiting the infection and pathogenicity of Coronaviruses, Flaviviruses, Togaviridae, Bornaviridae, Bunyaviruses, Filoviridae, Paramyxoviridae, Pneumoviruses and Retroviridae, in particular of the Human Immunodeficiency Virus (HIV), of the Rous sarcoma virus, of the virus murine leukemia viruses, of the Marburg virus, of the five species of ebolavirus, of the tick-borne encephalitis virus and of the dengue viruses, to improve the function of the defensive immune response of the host for the dengue viruses, as an antiviral drug against naked viruses by inhibiting the infectious spread of the papillomaviruses.

2. A pentacyclic triterpene for use as an active substance according to claim 1, characterized in that said pentacyclic triterpene is selected among purified amyrins and natural extracts containing amyrins, in particular extracts of Burseraceae, more specifically extracts of Protium heptaphyllum.

3. A pentacyclic triterpene for use as an active substance according to claim 2, characterized in that the natural extracts are plant extracts selected among Acacia sp or Senegalia visco (Fabaceae), Amelanchier alnifolia (Rosaceae), Amphipterygium adstringens (Anacardiaceae), Antiaris sp (Moraceae), Ardisia elliptica (Myrsinaceae), Bombax ceiba (Bombacaceae), Bombax malabaricum (Bombacaceae), Boswellia carterii (Burseraceae), Bursera glabrifolia (Burseraceae), Bursera simaruba (Burseraceae), Byrsonima crassifolia or crassa (Malpighiaceae), Byrsonima fagifolia (Malpighiaceae), Calotropis gigantea (Asclepiadaceae), Camellia japonica (Theaceae), Capparis spinosa (Capparaceae), Carpobrotus edulis (Aizoaceae), Cassia obtusifolia (Caesalpinaceae), Clerodendrum inerme (Verbenaceae), Commiphora holtziana (syn. Commiphora erythraea) (Burseraceae), Duranta repens (Verbanaceae), Eucalyptus globulus (Myrtaceae), Euphorbia tirucalli (Euphorbiaceae), Ferula gummosa (Apiaceae), Ficus benjamina (Moraceae), Ficus carica (Moraceae), Ficus cordata (Moraceae), Ficus pandurata (Moraceae), Garcinia subelliptica (Clusiaceae), Klainedoxa gabonensis (Irvingiaceae), Lonicera caerulea (Caprifoliaceae), Ligustrum sp (Oleaceae), Malus domestica (Rosaceae), Melia azedarach (Meliaceae), Mandevilla guanabarica and Mandevilla moricandiana (Apocynaceae), Melastoma malabathricum (Melastomataceae), Nelumbo nucifera (Nelumbonaceae), Olea europaea (Oleaceae), Piptadeniastrum africanum (Mimosaceae), Poncirus trifoliate (Rutaceae), Populus sp (Salicaceae), Pouteria sp (Sapotaceae), Protium sp (Burseraceae), Pyrenacantha staudii (Icacinaceae), Rhus alata (Anacardiaceae), Salacia amplifolia (Celastraceae), Siraitia grosvenorii (Cucurbitaceae), Tectona philippinensis (Lamiaceae), Triticum aestivum (Poaceae), and Vitis vinifera (Vitaceae).

4. A pentacyclic triterpene for use as an active substance according to claim 2, characterized in that the natural extracts are animal extracts, preferably from arthropods, in particular from insects of the genus Platyphora of the chrysomelidae species.

5. A pentacyclic triterpene for use as an active substance according to claim 1, characterized in that said pentacyclic triterpene is selected among biotechnological amyrins, which means produced from bacteria or yeasts, or from enzymes.

6. A pentacyclic triterpene for use as an active substance according to claim 1, characterized in that said pentacyclic triterpene is selected among amyrins from organic synthesis processes.

7. A pentacyclic triterpene for use as an active substance according to any one of previous claims, characterized in that said pentacyclic triterpene is made of α amyrin (100 % or viminalol), or of β amyrin (100 %), or of a mix of α and β amyrins comprising 20 % to 80 % of α amyrin and 20 % to 80 % of β amyrin, preferably of a mix of α and β amyrins comprising 20 % to 80 % of α amyrin and 20 % to 80 % of β amyrin.

8. A therapeutical product comprising at least one pentacyclic triterpene for use according to any one of claims 1 to 7 on mammals, characterized in that it comprises between 0.001 % and 100 % amyrin in weight of amyrin relatively to the total weight of the therapeutical product.

9. A therapeutical product comprising at least one pentacyclic triterpene for use according to any one of claims 1 to 8 on mammals, characterized in that it is in form of a liquid product of any polarity, such as subcutaneously injectable, intravenously injectable, eye drops, spray, serum or drinking syrup.

10. A therapeutical product comprising at least one pentacyclic triterpene for use according to any one of claims 1 to 8 on mammals, characterized in that it is in form of a solid product such as a lozenge, a suppository, a balm or a topic wax.

11. A therapeutical product comprising at least one pentacyclic triterpene for use according to any one of claims 1 to 8 on mammals, characterized in that it is in form of a topic application product, such as a lotion, a gel, a serum, an emulsion, a patch, an oil, a cleaning or disinfectant solution.