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    • 专利摘要:
    Mushroom tyrosinase as an inhibitor of the hepatitis C virus. The present invention refers to the use of white mushroom tyrosinases (Agaricus bisporus), and fragments thereof, which have antiviral activity against the hepatitis C virus (HCV), as a medicine for the treatment of said infection, as well as to pharmaceutical compositions comprising them. These tyrosinases are proteins that can be easily extracted from mushrooms or from commercial mushroom protein extracts and, therefore, make it possible to obtain low-cost drugs for the treatment of hepatitis C. (Machine-translation by Google Translate, not legally binding)
    公开号:ES2843553A1
    申请号:ES202030029
    申请日:2020-01-17
    公开日:2021-07-19
    发明作者:Carmona Jose Miguel Palomo；Tejedor Fco David Lopez；Franco Olga Abian；Campoy Adrian Velazquez
    申请人:Fundacion Instituto De Investig Sanitaria Aragon；INST ARAGONES DE CIENCIAS de la SALUD；Consejo Superior de Investigaciones Cientificas CSIC；Universidad de Zaragoza；Fundacion Agencia Aragonesa para la Investigacion y el Desarrollo ARAID；
    IPC主号:
    专利说明:

    [0002] MUSHROOM TYROSINASE AS AN INHIBITOR OF HEPATITIS C VIRUS
    [0004] The present invention relates to the use of white mushroom tyrosinases, or active fragments thereof, as a medicine, preferably in the treatment of hepatitis C, as well as to pharmaceutical compositions comprising said tyrosinases for the treatment of hepatitis C. Therefore, the invention could be framed in the field of medicine, more specifically in the field of drugs or compositions with antiviral capacity, preferably against the hepatitis C virus.
    [0006] STATE OF THE ART
    [0008] Infection with the hepatitis C virus (HCV) is one of the main health problems in the world. HCV is a small, single-stranded RNA virus that is made up of several proteins involved in its replication cycle, which are processed in the endoplasmic reticulum (ER). Among all these, the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B are of great importance because most of the drugs currently marketed with therapeutic indication, as well as those that are being developed, are based on mechanisms. of inhibition of some type of these proteins.
    [0010] Among them, NS2 is a multifunctional protein essential for assembly and replication due to its function as an autocatalytic cysteine protease. The N-terminal domain of NS3 is the second viral protease that processes the viral polypeptide towards the C-terminus, while the C-terminal domain of NS3 has a helicase function. NS4A is a small hydrophobic protein that serves as a cofactor for the serine NS3 protease. NS4B and NS5A are proteins involved in replication and assembly. NS5B is the viral RNA-dependent RNA polymerase that forms a replication complex together with NS3, NS4A, NS4B and NS5A.
    [0012] Currently, some of the best known systems in the formulation of drugs against the hepatitis C virus consist of a mixture of several molecules. For example, Harvoni®, the last drug registered by Gilead (2014), is a combination of the compounds Sofosbuvir and Ledipasvir, inhibitory compounds against the NS5B and NS5A proteins respectively. Other commercial drugs that consist of a combination of compounds are Epclusa® (Gilead), Vosevi® (Gilead), Zepatier® (MSD) and Maviret® (Abbvie).
    [0014] There are chemical compounds, natural and synthetic, with antiviral capacity against HCV. For example, document CN109574828A describes the use of sesquiterpenoids extracted from the fermentation of the fungus Trichoderma harzianum in the inhibition of HCV. Also, CN109627272A describes a nucleoside phosphate analog for use in the treatment of hepatitis C. Also, CN109503518A describes a pharmaceutical composition comprising a diarylamide compound with anti-HCV activity. JP2018076368A describes a combination of compounds capable of inhibiting the function of the HCV NS5A protein.
    [0016] However, currently available treatments for the hepatitis C virus are too expensive and many patients, especially from less developed countries, do not have access to them. They are treatments that cost around € 60,000, thus, even in developed countries, health budgets are exceeded, forcing governments to establish restrictive criteria for the prescription of these drugs.
    [0018] It is estimated that there are around 150 million people infected with HCV in the world. Therefore, it is necessary to develop effective treatments against the virus that are alternatives to the existing ones that are also low-cost.
    [0020] DESCRIPTION OF THE INVENTION
    [0022] The inventors have shown that tyrosinases present in the white mushroom ( Agaricus bisporus), and fragments thereof, have antiviral activity against the hepatitis C virus (HCV) through an inhibition mechanism based on the selective hydroxylation of a surface tyrosine to L-DOPA and later Dopachrome of the proteins involved in the replication of the virus (NS3, NS4 or NS5). These tyrosinases are proteins that are easily extracted from mushrooms or commercial mushroom protein extracts and, therefore, are proteins that can be easily obtained and are part of of pharmaceutical or therapeutic compositions for the treatment of hepatitis C, thereby obtaining lower-cost drugs for patients diagnosed with said virus.
    [0024] The inventors purified the tyrosinases both from a commercial protein extract of semi-purified Agaricus bisporus from Sigma® and directly from white mushroom. Subsequently, they performed L-DOPA activity assays in vitro, to verify tyrosinase activity, and determined cell toxicity and antiviral activity thereof in cultures of Huh 5-2 liver tumor cells transfected with a plasmid that includes all non-protein proteins. structural elements present in the Hepatitis C virus. Through these tests, the inventors determined that both the native tyrosinase obtained directly from the white mushroom ( A. bisporus), which is a tetramer that comprises two subunits H 1 L 1 H 2 L 2 , where the H subunits weigh 45kDa and the L subunits weigh 12kDa, as the H subunits of said tyrosinase (H and H2 dimer), called in the present invention " Tyr45 or TyrAB tyrosinase", are capable of inhibiting the replication of the virus of the hepatitis C without inducing toxicity in liver cells.
    [0026] Furthermore, the inventors have discovered a post-translational isoform of tyrosinase Tyr45, called "Tyr50" because of its weight of 50kDa, which they have shown to have an even higher antiviral capacity than the other tyrosinases tested (up to 10 times greater than the observed inhibition by Tyr45), and which drugs are known, particularly the commercial drug Ribavirin®.
    [0028] Therefore, a first aspect of the invention is a tyrosinase from Agaricus bisporus for use as a medicament, preferably for the treatment of hepatitis C in a subject, where the subject is preferably a mammal, more preferably a human.
    [0030] The term " Agaricus bisporus tyrosinase" or " A. bisporus tyrosinase" or "mushroom tyrosinase", used interchangeably in the present invention, refers to a copper-containing metalloenzyme that catalyzes the oxidation of a phenol, such as tyrosine, where this enzyme belongs to the species Agaricus bisporus, whose common name is the white mushroom. The enzymatic classification number (EC number) is EC 1.14.18.1.
    [0031] The tyrosinase of A. bisporus comprises a tetrameric structure with a molecular weight of around 120KDa, made up of two dimers, where each dimer comprises a long subunit called "H subunit" with a molecular weight around 45KDa and a small subunit called "L subunit. "With a molecular weight around 12KDa, obtaining the homodimer H 1 L 1 H 2 L 2 .
    [0033] The inventors have shown that A. bisporus tyrosinase, extracted directly from mushrooms, does not show toxicity in liver cells and has antiviral activity against hepatitis C virus (HCV), so that said enzyme can be used for the treatment of infection caused by said virus.
    [0035] The term "hepatitis C treatment" or "hepatitis C virus treatment" or "HCV treatment", used interchangeably in the present invention, refers to combating, inhibiting, arresting, reversing, alleviating and / or stabilizing the consequences, pathological conditions, complications, symptoms and / or effects caused by the hepatitis C virus in a subject, or to slow down the development of the disease.
    [0037] In the context of the present invention, "subject" or "individual" is understood to be any animal, of any race, sex or age. In a preferred embodiment, the subject is a mammal and more preferably a human.
    [0039] The inventors have also shown that the H subunits of tyrosinase, which are presented by forming a H 1 H 2 dimer, also exhibit antiviral activity against HCV. This dimer of the H subunits is called in the present invention Tyr45 or Tyr45 tyrosinase or TyrAB, terms used interchangeably in the present invention. This active tyrosinase dimer comprises the amino acid sequence SEQ ID NO: 1. SEQ ID NO: 1 of the present invention is therefore Tyr45 or TyrAB (H 1 H 2 ).
    [0041] Therefore, another aspect of the invention relates to a polypeptide ("polypeptide of the invention") comprising the amino acid sequence SEQ ID NO: 1 for use as a medicine. Preferably, said polypeptide comprises SEQ ID NO: 2. SEQ ID NO: 2 of the present invention is the complete sequence of A. bisporus tyrosinase, that is, the tetramer formed by two subunits H 1 L 1 H 2 L 2 .
    [0042] In a more preferred embodiment, said polypeptide of the invention consists of the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.
    [0044] Additionally, the inventors have demonstrated the existence of an isoform of Tyr45 tyrosinase (from SEQ ID NO: 1), called Tyr50, whose difference in weight (50 KDa in Tyr50 versus 45 KDa in Tyr4b) is due to post-translational changes , mainly glycosylation, which is present both in white mushrooms and in commercial semi-purified mushroom protein extracts. This isoform is also capable of inhibiting the replication of the hepatitis C virus, providing an antiviral capacity even higher than the other tyrosinases tested here and than drugs known as Ribavirin, and which is up to 10 times higher than the inhibition observed by Tyr45 , with an EC50 of 0.0012-0.0025 versus 0.006-0.012, respectively.
    [0046] Thus, in another particular embodiment, the polypeptide of the invention consists of a post-translational, glycosylated isoform of the amino acid sequence consisting of SEQ ID NO: 1. This isoform is called "Tyr50" in the present invention.
    [0048] In the present invention the term "post-translational isoform" or "post-translational isoenzyme" refers to a different or equivalent form of Tyr45 tyrosinase of SEQ ID NO: 1, generated from the same gene but subjected to post-translational modifications or differential maturation and / or to alternative splicing. In this way, the Tyr45 tyrosinase and the Tyr50 isoform have the same amino acid sequence (SEQ ID NO: 1) but the second presents post-translational modifications in relation to the first.
    [0050] In the present invention, the term "post-translational modification" refers to the changes, modifications, alterations, variations or transformations that Tyr45 tyrosinase undergoes after its synthesis in ribosomes as one of the final steps of its synthesis and gene expression. Said post-translational modifications comprise the addition or removal of a functional or chemical group or the coupling of two or more proteins. The person skilled in the art knows the types of post-translational modifications that exist. A post-translational modification is selected, for example, from the list consisting, without limitation, of: acylation, phosphorylation, methylation, hydroxylation, glycosylation, glycation, sulfonylation, prenylation, palmitoylation, nitrosylation, nitration, sumoylation, and ubiquitination.
    [0052] In particular, the post-translational modification exhibited by the Tyr50 isoform is glycosylation.
    [0054] Preferably, the glycosylated isoform of the amino acid sequence consisting of SEQ ID NO: 1 to which the present invention refers, that is, the Tyr50 isoform, is obtained by the procedure described in López-Tejedor D. and Palomo José M., 2018, Protein expression and purification, 145: 64-70. Briefly, said isoform is obtained by a process comprising the following steps: commercial lyophilized Agaricus bisporus tyrosinase from Sigma-Aldrich®, more preferably 1 mg of said tyrosinase, is dissolved in distilled water, preferably in 4 ml of distilled water. Subsequently, octadecyl-Sepabeads® (preferably 0.5 g) is added to this solution and, after incubating the mixture for preferably two hours, it is filtered under vacuum, recovering the supernatant to which sodium phosphate buffer is added. , preferably at pH 7, to a final concentration of preferably 100 mM, and Triton ™ X-100 to a final concentration of preferably 0.07% (w / v). Finally, octadecyl-Sepabeads® (preferably 0.5 g) is added to this solution and, after incubating for preferably 3 h, the supernatant is recovered, which contains only the isoform of the pure enzyme.
    [0056] In another more particular embodiment the drug to which the present invention refers is for use in the treatment of hepatitis, Denge or Zika, preferably hepatitis, more preferably hepatitis C, in a subject, where the subject is preferably a mammal, plus preferably a human.
    [0058] Another aspect of the present invention is a pharmaceutical composition ("pharmaceutical composition of the invention") comprising:
    [0059] to. a polypeptide comprising the amino acid sequence SEQ ID NO:
    [0060] 1 or SEQ ID NO: 2, or
    [0061] b. a polypeptide consisting of the amino acid sequence SEQ ID NO:
    [0062] 1 or SEQ ID NO: 2, or
    [0063] c. a polypeptide consisting of a glycosylated post-translational isoform of the amino acid sequence consisting of SEQ ID NO: 1, or d. any combination of (a) to (c),
    [0064] and a pharmaceutically acceptable carrier and / or excipient, for use as a medicament.
    [0066] The term "pharmaceutical composition" or "therapeutic composition", used interchangeably in the present invention, refers to a mixture, solution or solution, natural or artificial, used for the prevention, relief, treatment or cure of diseases, preferably hepatitis C, in a subject, preferably a mammal, more preferably a human. The administration of said composition should be adjusted to a desirable dose of the composition and varies depending on the condition and weight of the subject, the severity of the disease, the form of the drug, the route and the period of administration, and may be chosen by the person skilled in the art.
    [0068] The pharmaceutical composition must comprise a therapeutically effective amount of the polypeptide of the invention, where the term "therapeutically effective amount" refers to that amount of the polypeptide sufficient to cause the prevention and / or treatment of the disease, preferably hepatitis C, in the subject, preferably mammalian and more preferably human. It is routine practice for the person skilled in the art to calculate the therapeutically effective amount based on factors such as the enzymatic stability of the polypeptide, the bioavailability of the same once administered, the age, sex and weight of the subject to be treated, the condition general health of the subject to be treated, the mode and time of administration of the composition, etc.
    [0070] The pharmaceutical composition of the invention can comprise any combination of the polypeptides defined in the present invention.
    [0072] Furthermore, the pharmaceutical composition of the invention comprises one or more pharmaceutically acceptable excipients and / or vehicles.
    [0074] The term "excipient" refers to a substance that helps the absorption of the elements of the composition of the invention, stabilizes said elements, activates or helps the preparation of the composition in the sense of giving it consistency or providing flavors that make it nicer. Thus, excipients could have the function of keeping the ingredients together, as for example is the case of starches, sugars or celluloses, the function of sweetening, the function of coloring, the function of protection of the composition, for example, to isolate it from air and / or humidity, the function of filling a tablet, capsule or any other form of presentation, as for example, is the case of dibasic calcium phosphate, the disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
    [0076] The "vehicle", like the excipient, is a substance that is used in the composition to dilute any of the components included in it to a certain volume or weight. The pharmacologically acceptable vehicle is an inert substance or substance analogous to any of the active principles comprised in the composition. The function of the vehicle is to facilitate the incorporation of other elements, to allow a better dosage and administration or to give consistency and shape to the composition. When the presentation form is liquid, the pharmacologically acceptable carrier is the diluent. The term "vehicle" therefore refers to a diluent, adjuvant or carrier with which the composition of the invention is to be administered; obviously, said vehicle must be compatible with said composition. Such pharmaceutical vehicles can be liquids, such as water, solvents, oils or surfactants, including those of petroleum, animal, vegetable or synthetic origin, such as, for example, and without limitation, peanut oil, soybean oil, mineral oil, oil. of sesame, castor oils, polysorbates, sorbitan esters, ether sulfates, sulfates, betaines, glycosides, maltosides, fatty alcohols, nonoxynols, poloxamers, polyoxyethylenes, polyethylene glycols, digitonin and the like. Other examples of pharmaceutically acceptable carriers include one or more from the list which, without limitation, consists of: water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, lactose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, cellulose, methyl cellulose, sodium carboxymethyl cellulose, hydroxypropylmethyl cellulose, corn starch, wheat starch, rice starch, potato starch, polyvinylpyrrolidone, cross-linked polyvinylpyrrolidone, agar, alginic acid and sodium alginate.
    [0078] The excipient or vehicle also includes any substance that serves to improve the delivery and the efficacy of the active principle within the pharmaceutical composition, in addition they can comprise minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, that improve life usefulness or efficacy of the pharmaceutical composition.
    [0079] The mechanism of viral inhibition on HCV described here for the polypeptide of the invention is extrapolated to other hepatitis viruses, Dengue or Zika. Thus, in another particular embodiment, the pharmaceutical composition referred to in the present invention is for use as a medicine in the treatment of hepatitis, Dengue or Zika, preferably hepatitis, more preferably hepatitis C, in a subject, where the subject it is preferably a mammal, more preferably a human.
    [0081] In another preferred embodiment, the pharmaceutical composition of the invention is administered alone or in combination, sequentially or simultaneously, with one or more drugs or active principles used for the treatment of hepatitis C from among those known in the art. Examples of such drugs are, but are not limited to, Harvoni®, Epclusa®, Vosevi®, Zepatier® or Maviret®, or the active substances sofosbuvir, ledipasvir, velpatasvir, voxilaprevir, elbasvir, grazoprevir, glecaprevir, pibrentasvir or rivabirin.
    [0083] Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention.
    [0085] DESCRIPTION OF THE FIGURES
    [0087] FIGURE 1. Shows the inhibition of the Hepatitis C virus (HCV) replicon, evaluation of the potency and cytotoxicity of the protein in cell assays. The replication rate of the HCV replicon is shown with black bars and cell survival with closed squares, both were measured in cell culture by increasing the protein concentration to determine the EC50. The protein is in aqueous solution at pH 5.
    [0089] FIGURE 2. Shows the effect of the presence of Triton X-100 in the inhibition of the Hepatitis C virus (HCV) replicon, evaluation of the potency and cytotoxicity of the protein in cell assays. The replication rate of the HCV replicon is shown with bars black and cell survival with closed squares, both were measured in cell culture increasing the protein concentration to determine the EC50.
    [0091] FIGURE 3. Shows the inhibition of the Hepatitis C virus (HCV) replicon, evaluation of the potency and cytotoxicity of the protein in cell assays. The replication rate of the HCV replicon is shown with black bars and cell survival with closed squares, both were measured in cell culture by increasing the protein concentration to determine the EC50. The purified protein sample (0.25 mg / ml) contained <0.05% Triton X-100.
    [0093] FIGURE 4. Shows the inhibition of the Hepatitis C virus (HCV) replicon, evaluation of the potency and cytotoxicity of the protein in cell assays. The replication rate of the HCV replicon is shown with black bars and cell survival with closed squares, both were measured in cell culture by increasing the protein concentration to determine the EC50. Purified TyrAB sample (0.25 mg / ml) (A) and Tyr50 isoform sample (0.5 mg / ml) in 10 mM sodium phosphate pH 7 (B).
    [0095] FIGURE 5. Shows Hepatitis C virus (HCV) replicon inhibition, potency evaluation, and cytotoxicity of the temperature-inactivated Tyr50 isoform in cell assays. A) Inactivated at 37 ° C (partially active). B) Inactivated at 80oC (inactive). The replication rate of the HCV replicon is shown with black bars and cell survival with closed squares, both were measured in cell culture by increasing the protein concentration to determine the EC50. Proteins at 0.5 mg / ml in 10 mM sodium phosphate pH 7.
    [0097] EXAMPLES
    [0099] The invention will now be illustrated by tests carried out by the inventors that show the antiviral activity of mushroom tyrosinase against the hepatitis C virus.
    [0100] MATERIALS AND METHODS
    [0102] Purification of inases from a semi-purified commercial extract of Agaricus bisporus from Sigma®.
    [0104] Through this protocol it was possible to purify two different isoforms of active tyrosinase in dimeric form (H subunits) (Tyr45 and Tyr50) from the commercial raw Sigma-Aldrich® powder.
    [0106] 1 mg of commercial lyophilized tyrosinase from Sigma-Aldrich® was dissolved in 4 ml of distilled water. 0.5 g of octadecyl-Sepabeads® (C18-1) was added to this solution and the mixture was incubated for 2 h. The mixture was then vacuum filtered, the supernatant was recovered and sodium phosphate buffer was added at pH 7 to a final concentration of 100 mM. Subsequently, Triton ™ X-100 was added to a final concentration of 0.07% (w / v). 0.5 g of fresh octadecyl-Sepabeads® (C18-2) was added to this supernatant and the mixture was incubated for 3 h.
    [0108] By SDS-PAGE, the inventors confirmed that the supernatant only contained the Tyr50 isoform, while the solid contained Tyr45 and a slight amount of Tyr50. A previous semi-purification was carried out by desorption of the C18-2 derivative using different concentrations of Triton X-100. Finally, soluble protein samples were purified to reduce the amount of detergent in the sample by ultrafiltration with Amicon Ultra 10kDa (<0.05%).
    [0110] Procedure for extraction of inase shots directly from the pinion shark.
    [0112] The inventors cut into small pieces 25 g of white mushrooms ( A. bisporus) (from Eroski supermarkets) and added to 50 ml of cold acetone. They kept the mixture under paddle agitation for 30 minutes in an ice bath and then centrifuged it at 7000 rpm for 20 minutes and recovered the sediment, discarding the solution. They suspended this solid in 25 ml of distilled water and incubated the mixture for 1 hour, then centrifuged it at 8000 rpm for 40 min. After filtration, they carried out the saline precipitation process adding 60% (w / v) of ammonium sulfate, adding it little by little, followed by 1 h incubation. Afterwards, they centrifuged the mixture at 8000 rpm for 40 minutes, recovered the solid and stored it at -20oC.
    [0114] In vitro L-Dopa activity assay.
    [0116] The inventors checked the tyrosinase activity in the presence of 2 ml of 1 mM L-DOPA in 0.1 M phosphate buffer at pH 7 and room temperature, using a V-730 spectrophotometer (Jasco), measuring the increase in the absorbance of aminochromes. at 475 nm caused by 40 µl of enzymatic solution and taking the initial speed between 10 and 70 seconds of the reaction. One unit of enzyme activity (U) was defined as the amount of enzyme that causes an increase in absorbance by 0.001 / min at 25 ° C.
    [0118] Determination of cell toxicity and antiviral activity.
    [0120] In these tests, the inventors used a Huh 5-2 cell type, liver tumor cells (Lunet) that were transfected with a plasmid with a replicon system that includes all the non-structural proteins present in the Hepatitis C virus ( NS2, NS3, NS4, NS5A and NS5B). As it does not contain structural proteins, it is not infective, but it allows quantifying by introducing a reporter sequence (which codes for the luciferase enzyme), the replication rate of the system within cells. In the event of inhibition of replication, it will manifest itself in the reduction of the luminescence signal that is obtained.
    [0122] The inventors performed the cell culture of Huh 5-2 using DMEM with Phenol Red (RF) as the culture medium, supplemented with Fetal Bovine Serum (SFB), Penicillin / Glutamine / Streptavidin, non-essential amino acids (NEAA) and 500 pg / ml Geneticin (G418) from Invitrogen (selection of cells with a replicon system inside them). The culture conditions were: temperature 37 ° C, 5% CO 2 , 95% air and 1 atm of pressure.
    [0124] To determine both the antiviral activity of tyrosinases and their cellular toxicity, they used 96-well plates seeded with 7000 cells per well (using DMEM without RF) in which they tested serial increasing concentrations of tyrosinases (from 0 to 0.2 mg / ml) in triplicate for each of the concentrations (100 pl per well). They incubated the cells for 72 hours with tyrosinases, after which they evaluated both their cytotoxicity and their antiviral activity (in duplicate plates).
    [0125] For cytotoxicity, in one of the plates they determined the cellular metabolism of each of the wells using the Cell Titer 96®AQueous reagent from Promega. They removed the supernatant from the wells and added 20 µl of Cell Titer 96®AQueous diluted 1: 4 in the same DMEM culture medium but without RF, and incubated it for 3h at 37 ° C, 5% CO 2 and 1 atm of pressure. . After this time, they measured the absorbance at 490nm and 800nm. The difference in these absorbance values at these wavelengths corresponds to the substrate that is metabolized by viable cells in culture. They determined each tyrosinase concentration in triplicate, and averaged the values obtained. They represented in a graph the relationship between the concentration of tyrosinase and the percentage of absorbance (taking as a value 100%, the values in the blank wells without treatment with tyrosinase). They calculated the LC50 as the concentration of tyrosinase at which cell viability is reduced by 50% (compared to that obtained in the absence of treatment).
    [0127] For antiviral activity, in another of the plates the inventors determined the luminescence of each of the wells using the Bright-Glo ™ Luciferase Assay System reagent from Promega. Without removing the supernatant, they added 30 µl of reagent per well and determined the luminescence signal on a Biotek Synergy HT Multi-Modal Microplate Reader (Gen5 ™ Data Analysis software). The luminescence obtained is proportional to the amount of RNA expressed by the reporter gene. They determined the concentration of tyrosinase in triplicate, and made the average of the values obtained. They represented in a graph the relationship between the concentration of tyrosinase and the percentage of luminescence (taking as a value 100%, the values in the blank wells without treatment with tyrosinase). They calculated the EC50 as the tyrosinase concentration at which viral replication is reduced by 50% (compared to that obtained in the absence of treatment).
    [0129] RESULTS
    [0131] First, both the toxicity and antiviral activity against liver tumor cells (Huh 5-2) of the commercial Sigma extract of Agaricus bisporus tyrosinase were evaluated. Tyrosinase is a tetramer (homodimer) made up of two H 1 L 1 H 2 L 2 subunits, where the H subunits weigh 45kDa and the L subunits 12kDa. This extract is semi-purified and presents only the H (active) subunits of 45kDa (H 1 H 2 dimer) ( TyrAB). Furthermore, this semi-purified preparation contains, in addition to TyrAB, another protein with tyrosinase activity of 50 kDa ( Tyr50 ) (tyrosinase activity determined by activity assay in native electrophoresis).
    [0133] The semi-purified Sigma extract was titrated at pH 5 and 7, obtaining in both cases similar results, the extract did not show toxicity towards liver cells giving a surprising value of inhibition of virus replication (EC50 approximately 0.02 mg / ml) (Figure 1).
    [0135] Once this was observed, we proceeded to evaluate whether said result corresponded to tyrosinase, and for this purpose, it was purified. First, they proceeded to a semi-purification to separate the tyrosinases from the rest of the proteins of the commercial extract and evaluate their antiviral activity.
    [0137] In this first preliminary process, whose purification required the use of a detergent (Triton X-100), it was observed that at concentrations> 0.25% (v / v) of Triton X-100 in the eastern medium it was toxic to the cell (Figure 2). Purified samples containing <0.05% detergent, where only tyrosinases are present, resulted in an even better result than the first time, no cell toxicity, and an antiviral inhibition EC of 0.001 mg / ml (Figure 3). This demonstrated that the active tyrosinases are responsible for the antiviral activity of the protein sample.
    [0139] In this way, to be completely sure that the results were due to the enzymes and to find out which of them was responsible for the activity, both enzymes {Tyr45 and Tyr50) present in the commercial extract were purified, obtained separately and their effectiveness evaluated. antiviral ability. In both cases, the inventors did not observe cellular toxicity and found that both enzymes exhibited antiviral activity, although the Tyr50 isoform was responsible for most of the activity, with an inhibition capacity up to 10 times higher than that observed by Tyr45, obtaining an EC50 0.0012-0.0025 versus 0.006-0.012, respectively. (Figure 4, Table 1).
    [0140] With this result, the inventors demonstrate that not only the H 1 H 2 homodimer ( Tyr45) of mushroom tyrosinase is an excellent inhibitor of the hepatitis C virus, but that the Tyr50 isoform is responsible for most of this antiviral activity. . By analysis and sequencing of the electrophoresis band corresponding to the pure protein Tyr50, the inventors demonstrated that it corresponds to an isoform of Tyr45 tyrosinase with some post-translational change, particularly glycosylation, since its sequence exactly corresponded to that of the Tyr45 tyrosinase of A. bisporus (SEQ ID NO: 1).
    [0142] In this way, these results seem to demonstrate a virus replication inhibition mechanism based on enzymatic activity, in this case on the selective oxidation of tyrosines to L-DOPA and later dopachrome.
    [0144] To verify this new mechanism of inhibition of the hepatitis C virus, the inventors evaluated the inhibitory effect of partially and totally inactivated tyrosinase. For this, the purified Tyr50 tyrosinase was incubated at 37 ° C for 24 h, or at 80 ° C for the same time. Tyrosinase incubated at 37 ° C gave an EC50 value of 0.01 (ten times higher) while that inactivated at 80 ° C did not inhibit virus replication at all (Table 1, Figure 5). This result shows that the mechanism of action of tyrosinase passes through its enzymatic activity.
    [0146] An important result is that, under the same conditions, ribavirin (a drug used in addition to other drugs such as Harvoni® in the treatment of patients with cirrhosis) presented an EC50 of 0.01 mg / ml. This means that Tyr50 was almost 10 times more potent than ribavirin.
    [0150] Table 1. EC50 and LC50 of the different isoforms of tyrosinase purified from the extract of A. bisporus from Sigma-Aldrich®. ND: not determined at the concentrations tested.
    [0152] Considering these results, the inventors evaluated the antiviral activity of a protein extract directly produced from the mushroom ( A. bisporus). In this case, the extraction and semi-purification method that the inventors used allowed to obtain tyrosinase in its tetrameric form with all its subunits, in this way the possible effect of the presence or absence of the L subunits of the homodimer could be assessed. This semi-purified extract did not show cellular toxicity, giving a similar value in inhibition of virus replication to that obtained previously using the Sigma enzyme, again corroborating the greater inhibitory capacity of the virus compared to ribavirin (Table 2).
    [0157] Table 2. EC50 and LC50 of the extract purified directly from mushrooms ( Tyr extract). ND: not determined at the concentrations tested.
    [0159] The inventors have thus shown, in view of all these results, that the tyrosinases present in mushrooms ( A. bisporus) show antiviral activity against the hepatitis C virus through a catalytic mechanism of selective hydroxylation of tyrosine, where one of they, Tyr50, is 10 times more active than the drug Rivabirin.
    [0161] In addition, they have shown that the antiviral activity of these proteins is found in a similar way both in commercial purified extracts and in an extract directly obtained from mushrooms.
    [0162] Furthermore, this new mechanism of viral inhibition action discovered by tyrosinase could also be effective in other hepatitis viruses, Denge or Zika, thus being a possible broad spectrum pharmacological agent.
    权利要求:
    Claims (7)
    [1]
    1. A polypeptide comprising the amino acid sequence SEQ ID NO: 1 for use as a medicine.
    [2]
    2. The polypeptide for use according to claim 1, comprising SEQ ID NO: 2.
    [3]
    3. The polypeptide for use according to any of claims 1 or 2, consisting of the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 2.
    [4]
    The polypeptide for use according to any of claims 1 or 3, consisting of a glycosylated isoform of the amino acid sequence consisting of SEQ ID NO: 1.
    [5]
    The polypeptide according to any one of claims 1 to 4, for use in treating hepatitis C in a subject, wherein the subject is preferably a mammal, more preferably a human.
    [6]
    6. A pharmaceutical composition comprising:
    to. a polypeptide comprising the amino acid sequence SEQ ID NO:
    1 or SEQ ID NO: 2, or
    b. a polypeptide consisting of the amino acid sequence SEQ ID NO:
    1 or SEQ ID NO: 2, or
    c. a polypeptide consisting of a glycosylated isoform of the amino acid sequence consisting of SEQ ID NO: 1, or
    d. any combination of (a) to (c),
    and a pharmaceutically acceptable carrier and / or excipient, for use as a medicament.
    [7]
    7. The pharmaceutical composition according to claim 6, for use in treating hepatitis C in a subject, wherein the subject is preferably a mammal, more preferably a human.
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    同族专利:
    公开号 | 公开日
    ES2843553B2|2022-01-17|
    WO2021144494A1|2021-07-22|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    US20120082719A1|2010-10-05|2012-04-05|Sam Poon Ang|Compositions For Treating Chronic Viral Infections|
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    PCT/ES2021/070023| WO2021144494A1|2020-01-17|2021-01-18|Mushroom tyrosinase as a hepatitis c virus inhibitor|
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