5-fluorouracil as an antiviral agent against viruses of the family coronaviridae

Abstract

The present invention relates to 5-fluorouracil (5-FU) for use thereof as an antiviral, alone or in combination with peptides that inhibit binding between nsp10 and nsp14 and between nsp10 and nsp16, acting against viruses of the family Coronaviridae, preferably SARS-CoV-2.

Description

[0001] 5-FLUOROURACIL AS AN ANTIVIRAL AGENT AGAINST VIRUSES OF THE CORONAVIRIDAE FAMILY DESCRIPTION FIELD OF THE TECHNIQUE The present invention falls within the field of medicine and virology and focuses on the search for effective antivirals for the treatment of infections caused by viruses of the Coronaviridae family, specifically by SARS-CoV-2. PRIOR STATE OF THE ART Coronaviruses are a family of viruses that cause respiratory diseases that can be mild or fatal. Members of the Coronaviridae family are distributed in several subfamilies within which those traditionally known as coronaviruses, which affect birds and mammals, are grouped within the Orthocoronavirinae subfamily. Most of the known coronaviruses belong to this subfamily, which, in turn, is distributed in four genera: Alphacoronavirus, Betacoronavirus,Gammacoronavirus and Deltacoronavirus. Human coronaviruses that cause common colds (HCoVs) are classified in the genera Alphacoronavirus and Betacoronavirus. Within the Betacoronavirus genus, there are other pandemic zoonotic coronaviruses capable of causing severe disease, such as Severe Acute Respiratory Syndrome (SARS), caused by the viruses SARS-CoV and SARS-CoV-2, and Middle East Respiratory Syndrome (MERS), caused by MERS-CoV. In 2002–2003, SARS affected 8,098 people worldwide and caused 774 deaths, including healthcare workers. Ten years after that, Middle East Respiratory Syndrome coronavirus (MERS or nCoV-2012) emerged in the Middle East and continues to appear with sporadic cases and localized outbreaks, including one in Korea. The outbreak of pneumonia (disease named as COVID-19 by WHO on 11 February 2020) caused by the coronavirus 2019-nCoV or SARS-CoV-2 in Wuhan, Hubei Province, China,at the end of 2019 is a major challenge for public health and clinical treatment. All of these coronaviruses have had significant human morbidity and mortality, especially SARS-CoV-2 with more than 692 million cases worldwide, of which 6.9 million people (1% of those affected) have died. These RNA viruses, named coronaviruses due to the presence of the trimeric Spike protein that protrudes from the envelope giving it a crown shape, are emerging pathogens with outbreak potential due to the inherent propensity of their RNA genomes to rapid mutation and recombination. The RNA synthesis machinery of SARS-CoV, like that of all Betacoronaviruses, is composed of 16 non-structural proteins (nsp1-16) encoded by ORF1a / 1b. The nsp10 subunit is known to interact with both nsp14 and nsp16,stimulating their respective 3'-5' exoribonuclease and 2'-O- methyltransferase activities. Several studies have been carried out to deepen the understanding of nsp10-nsp14 and nsp10-nsp16 interactions, as well as between nsp10-nsp14-nsp16 and their relevance to the viability and viral replication of viruses of the Coronaviridae family, especially SARS-CoV, which point to the interaction surfaces between these proteins as targets for the development of antivirals. Specifically, it has been described how the interruption of the nsp10-nsp14 interaction abrogates the activation of the nsp14 exoribonuclease driven by nsp10 [1]. It has been experimentally proven that the surface of nsp10 that interacts with nsp14 overlaps with the surface involved in nsp10-mediated activation of the 2'-O-methyltransferase activity of nsp16,suggesting that nsp10 is an important regulator of SARS-CoV replication complex function. Reverse genetics experiments supported an essential role for the nsp10 surface interacting with nsp14 in SARS-CoV replication, as several mutations that abolished the interaction in vitro produced a replication-negative viral phenotype. In contrast, mutants in which the nsp10-nsp16 interaction was disrupted were found to be low-fit but viable. This implies that the nsp10 surface interacting with nsp14 and nsp16, and possibly other subunits of the viral replication complex, may be a target for the development of antiviral compounds against pathogenic coronaviruses. Nonstructural protein 16 (nsp16) is a 2'-O-methyltransferase that plays an essential role in immune evasion. Nsp16 has a similar function to its human counterpart, the CMTr1 protein.which methylates mRNA, improving translation efficiency and distinguishing self from non-self. Unlike human CMTr1, nsp16 requires a binding protein, nsp10, to activate its enzymatic activity. Thus, the binding formed by the nsp10-nsp16 complex is key to the activation of nsp16 and therefore to the ability of SARS-CoV-2 to evade the host immune system [2]. The viral ribonuclease nsp14 is one of the major antagonists of SARS-CoV-2 interferon. It is a multifunctional protein with two distinct activities, an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both critical for the life cycle of coronaviruses, suggesting that nsp14 could be a prominent target for the development of antiviral drugs. In coronaviruses,nsp14 ExoN activity is stimulated by interaction with the nsp10 protein. Based on the relevance of nsp10-nsp14 complex interactions and the roles of nsp14 in SARS-CoV-2 activity, the development of peptides that inhibit nsp10-nsp14 binding has been proposed in order to disrupt the assembly of this complex and interfere with coronavirus replication [3]. Along these lines, it has been proposed that interfering with the interaction between nsp10 and nsp16 of SARS-CoV by short peptides could specifically inhibit 2′-O-MTase activity [4]. The N7-MTase domain of the nsp14 protein and the 2′-O-MTase activity of nsp16 protein, respectively, of SARS-CoV, both activated by nsp10, are involved in viral RNA methylation and the formation of the cap-1 structure. The high degree of conservation of the interaction regions between nsp10 and nsp16 in the Coronaviridae family,and especially in viruses of the Orthocoronavirinae subfamily, suggests the use of the same peptide-based inhibition strategy in other viruses of this family. Based on this high degree of conservation, short peptides have been designed from the mouse hepatitis virus (MHV) to try to inhibit nsp10-nsp16 junctions in SARS-CoV [4]. The peptides obtained were tested in in vitro cultures to verify the inhibition of MHV (in L2 cells) and SARS-CoV (in Vero cells) showing promising results as an inhibitor for SARS-CoV in vitro and for MHV, both in vitro and in vivo. These results show that the nsp10 of coronaviruses are interchangeable in the stimulation of the 2′-O-MTase activity of nsp16 among coronaviruses. It is concluded that the critical residues identified in the interaction interface of nsp10 and nsp16 of SARS-CoV are highly conserved among coronaviruses,Therefore, the nsp10 of one coronavirus is capable of stimulating the nsp16 2′-O-MTase of another virus. Similarly, nsp10 inhibitors that interact with these conserved regions can inhibit the binding of these regions to nsp16 and nsp14 in any virus in the Coronaviridae family. Different strategies have been chosen for the design of peptides that interfere with the nsp10-nsp14 and nsp10-nsp16 interaction, among which the one developed by this group stands out, [5] which comprises the following stages: - select the areas that present greater interaction between proteins from the crystallographic structures of their nsp10-nsp14, nsp10-nsp16 and nsp10-nsp14-nsp16 complexes simultaneously, - define one protein as a ligand and another as a receptor and fragment the ligands in the interaction areas according to the maximum length of the peptides to be designed,- optimize the peptide sequence by mutating all the residues that make up the peptide fragment to alanine and subsequently mutating each residue individually to each of the 20 natural amino acids, - measure the interaction energy of each mutant, - analyze the SARS-CoV-2 quasispecies from clinical samples by sequencing using Illumina and bioinformatic sequence identification using Quasiflow, - select the best peptide sequences according to the most negative interaction energy values, highest number of intra and intermolecular interactions, highest percentage of hydrophobic residues and lowest sequence identity with respect to the original, - characterize the inhibitory capacity of the designed peptides in cells infected by the virus, - sequence by Illumina and analyze with QuasiFlow the quasispecies of the cultures in the presence and absence of peptides,- assess the inhibitory capacity of the peptides against nsp14 and nsp16 based on the mutations that the different quasispecies may present, and - select the peptides with the greatest inhibitory capacity. This procedure can give rise to a very high number of potential inhibitory peptides of the interaction between nsp10-nsp14 and nsp10-nsp16, and nsp10-nsp14-nsp16, so in vitro and in vivo characterization of the peptides obtained is essential to optimize the process. On the other hand, 5-fluorouracil, also known as 5-FU, is a potent antimetabolite used in the treatment of cancer. It belongs to the group of antimetabolites known as fluoropyrimidines. 5-FU intervenes by blocking DNA synthesis and inhibits RNA formation to a lesser extent, due to its analogy with uracil,causing cell apoptosis. It is used for the treatment of cancer due to its effect on the rapid growth of neoplastic cells that preferentially use the uracil molecule for nucleic acid biosynthesis. Its effectiveness lies in the fact that it irreversibly binds to the enzyme thymidylate synthase, essential for the synthesis of thymine nucleotides. The lack of thymine means that DNA cannot replicate, which inhibits cell division. Various studies have assessed the potential capacity of this compound 5-FU as an antiviral for the treatment of COVID-19. A retrospective study [6] states that all attempts made in this regard to date have been unsuccessful, with 5-FU being ineffective in treating any coronavirus infection,probably due to the elimination of this compound due to the exceptional error-correcting capacity of the coronavirus. Other recent studies point to these same conclusions [7] reiterating the ineffectiveness of 5-FU and most of its analogues as potential drugs for the treatment of COVID-19. DESCRIPTION OF THE INVENTION The present invention describes the surprising potential of 5-fluorouracil (5-FU) for use as an antiviral, alone or in combination with peptides that inhibit the binding between nsp10-nsp14 and between nsp10-nsp16, against viruses of the Coronaviridae family, preferably SARS-CoV-2. It has been proven in in vitro cultures that 5-FU has an antiviral effect against SARS-CoV-2. The use of 5-FU together with peptides that inhibit the nsp10-nsp16 and nsp10-nsp14 junctions also presents a synergistic effect that has been confirmed in cell cultures,Therefore, the combined use of these compounds could allow the necessary dose of 5-FU to be reduced, considerably reducing its toxicity, without losing activity against SARS-CoV-2. Treatment with 5-FU together with the aforementioned inhibitory peptides, being a combined therapy, acts against several viral targets simultaneously, so the probability of selecting resistant SARS-CoV-2 variants that overcome the combined treatment would be lower than with other available treatments that act against a single target. Another great advantage of this combined treatment is that it could act against all known coronaviruses. Although the trials have been carried out in cell cultures only with the Omicron variant of SARS-CoV-2, due to the structural homology and high conservation of the sequences of the nsp10, nsp14 and nsp16 proteins of the viruses of the Coronaviridae family, it could be effective against any virus of said family,especially against viruses of the Orthocoronavirinae subfamily. Therefore, a first aspect of the invention relates to 5-fluorouracil for use as an antiviral agent against viruses of the Coronaviridae family, preferably of the Orthocoronavirinae subfamily, more preferably any of the viruses in Table 1, and even more preferably, against SARS-CoV-2. Table 1. List of viruses against which the antiviral treatment of the invention could be effective.

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y 3 N 6L N A second aspect of the invention relates to a composition, hereinafter "composition of the invention", comprising 5-fluorouracil for use as an antiviral agent against viruses of the Coronaviridae family, preferably of the Orthocoronavirinae subfamily, more preferably any of the viruses in Table 1, and even more preferably against SARS-CoV-2. A third aspect of the invention relates to the composition of the invention, which also comprises peptides that inhibit nsp10-nsp16 and / or nsp10-nsp14 binding. Preferably, these inhibitors are selected from: SEQ. ID. NO: 1 with amino acid sequence ELKIRMRWK, SEQ. ID. NO: 6 with amino acid sequence QLQIYLMRK, and SEQ. ID. NO: 7 with amino acid sequence KIEKTFEYYMVR individually or in any of their combinations. Preferably, the composition of the invention thus defined is a pharmaceutically acceptable composition.As used herein, the term "pharmaceutical composition" refers to any substance used for the diagnosis, prevention, alleviation, treatment, or cure of a disease in humans or animals. The pharmaceutical composition of the invention may be used alone or in combination with other pharmaceutical compositions. In a particular embodiment, the pharmaceutical composition of the invention further comprises a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable excipient" refers to a substance that aids in the absorption of the pharmaceutical composition comprising the composition of the invention, stabilizes said pharmaceutical composition, or aids in its manufacture by giving it consistency, shape, flavor, or any other specific functional characteristic.Thus, excipients could have the function of keeping the ingredients together, such as starches, sugars or celluloses, a sweetening function, a coloring function, a protective function, such as isolating it from air and / or moisture, a filling function for a tablet, capsule or any other form of formulation, such as dibasic calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption, without excluding other types of excipients not mentioned in this paragraph. A "pharmaceutically acceptable carrier" (or "pharmacologically acceptable") refers to any substance, or combination of substances, known in the pharmaceutical sector, used in the manufacture of pharmaceutical administration forms and includes, among others, solids, liquids, solvents or surfactants.The carrier may be an inert substance or have an action similar to any of the compounds of the present invention, having the function of facilitating the incorporation of the drug as well as other compounds, allowing for better dosage and administration, or providing consistency and shape to the pharmaceutical composition. When the pharmaceutical form is liquid, the carrier is the diluent. The term "pharmacologically acceptable" refers to the fact that the compound it refers to is permitted and evaluated so as not to cause harm to the organisms to which it is administered. The pharmaceutical composition of the invention can be administered through any route of administration, and as such, said composition will be formulated in the pharmaceutical form appropriate to the chosen route of administration. Thus, the pharmaceutical composition of the invention can be administered orally, nasally, ocularly, topically, intradermally, intracranially, intravenously, or intraperitoneally.The pharmaceutical composition may further comprise another compound useful in the treatment of infections caused by viruses of the Coronaviridae family, preferably of the Orthocoronavirinae subfamily, more preferably any of the viruses in Table 1, and even more preferably against SARS-CoV-2. The pharmaceutical composition may include a single composition or separate compositions. The pharmaceutical composition may include an effective amount of the 5-fluorouracil inhibitor in combination with nsp10-nsp16 and / or nsp10-nsp14 binding inhibitor peptides.The term "effective amount" as used herein refers to an amount sufficient to inhibit replication of viruses of the Coronaviridae family, preferably of the Orthocoronavirinae subfamily, more preferably any of the viruses listed in Table 1, and even more preferably SARS-CoV-2, and to prevent or treat infections caused by said viruses in an individual in need of such prevention or treatment. The effective amount can be suitably selected based on a cell or individual selected by a person skilled in the art.For example, the effective amount may be determined based on the severity of the disease, the patient's age, body weight, health conditions, sex, patient's sensitivity to the drug, duration of administration, route of administration, excretion rate, duration of treatment, and other factors, including the use of a drug in combination with or at the same time as the pharmaceutical composition, and other factors known in the field of medicine. The term "prevention", as used herein, refers to the ability of the pharmaceutical composition of the invention to avoid, minimize, or hinder the progression of infections caused by viruses of the Coronaviridae family, preferably of the Orthocoronavirinae subfamily, more preferably any of the viruses in Table 1, and even more preferably by SARS-CoV-2.The term "treatment", as used herein, refers to the ability of the pharmaceutical composition of the invention to reduce, reverse, minimize or render asymptomatic infections caused by viruses of the Coronaviridae family, preferably of the Orthocoronavirinae subfamily, more preferably any of the viruses in Table 1 and even more preferably by SARS-CoV-2. A fourth aspect of the invention relates to the composition of the invention which also comprises peptides that inhibit nsp10-nsp16 and / or nsp10-nsp14 binding, for use as an antiviral agent. Preferably, the inhibitors of nsp10-nsp16 and / or nsp10-nsp14 binding are selected from SEQ ID NO: 1, SEQ ID NO: 6 and SEQ ID NO: 7, individually or in any of their combinations.A fifth aspect of the invention relates to the composition of the invention which also comprises peptides that inhibit nsp10-nsp16 and / or nsp10-nsp14 binding, for use as a medicament, preferably for the prevention and treatment of infections caused by viruses of the Coronaviridae family, preferably of the Orthocoronavirinae subfamily, more preferably any of the viruses in Table 1 and even more preferably, caused by the SARS-CoV-2 virus. Preferably, the inhibitors of nsp10-nsp16 and / or nsp10-nsp14 binding are selected from SEQ ID NO:1, SEQ ID NO: 7 and SEQ ID NO: 6, individually or in any of their combinations.The term "patient" or "subject" as used herein refers to any animal, preferably a mammal, and includes, but is not limited to, domestic and farm animals, primates, and humans, e.g., humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents such as rats and mice. In a preferred embodiment, the subject is a human of any age or ethnicity. In a particular embodiment, the subject is suffering from an infection caused by viruses of the Coronaviridae family, preferably of the Orthocoronavirinae subfamily, more preferably any of the viruses in Table 1, and even more preferably by SARS-CoV-2.A sixth aspect of the invention relates to the composition of the invention which also comprises peptides that inhibit the nsp10-nsp16 and / or nsp10-nsp14 binding, characterized in that the inhibitory peptides are obtainable by the method that comprises: a) structurally analyzing the areas of interaction between both proteins, b) locating the binding zones with greater specificity and affinity to design peptides derived from nsp10 using computational algorithms for protein design.c) test the 20 natural amino acids at each position individually, using Alanine as a reference, d) sequence clinical samples with Illumina and analyze the quasispecies with Quasiflow to identify SARS-CoV2 mutations in the peptide interaction zones, e) preselect the best candidates according to the interaction energy, molecular interactions, the content of apolar amino acids and the similarity with the virus sequence, f) characterize the inhibitory capacity of the designed peptides in cells infected by the virus, g) Sequence by Illumina and analyze the quasispecies of the cultures with QuasiFlow in the presence and absence of peptides, h) assess the inhibitory capacity of nsp14 and nsp16 of the peptides based on the mutations that the different quasispecies may present, and i) select the peptides that present the greatest inhibitory capacity.Unless otherwise defined, all technical and scientific terms used herein have the same meaning commonly understood by one skilled in the art to which this invention pertains. Methods and materials similar or equivalent to those described herein can be utilized in the practice of the present invention. Throughout the description and claims, the word "comprises" and variations thereof are not intended to exclude other technical features, additives, components, or steps. Other objects, advantages, and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples, drawings, and sequence listing are provided by way of illustration and are not intended to be limiting of the present invention.BRIEF DESCRIPTION OF THE FIGURES Figure 1: Logarithmic scale bar graph of the virus titer (plaque forming units or pfu / ml) under 5-FU treatment. Three replicates were used per experiment. The mean ± standard deviation of each experiment is represented. Figure 2: Curve graph representing the normalized value (0-1) of the inhibitory effect (Fa) of 5-FU and the IC50 dose of 5-FU in assays against SARS-CoV-2. Figure 3: Curve graph representing the normalized value (0-1) of the inhibitory effect (Fa) of 5-FU on viruses from passages 3 [FU (3)] and 4 [FU (4)], and the IC50 doses of each assay against SARS-CoV-2. Figure 4: Logarithmic bar graphs of virus titer from passages 3 (A) and 4 (B) under 5-FU treatment. Three replicates were used per experiment. The mean ± standard deviation of each experiment is represented. Figure 5: Example of the results of the analyses using the FoldX program (https: / / foldxsuite.crg.eu / ).Heat maps were obtained from the 3D structures (center). Energy values ​​were normalized from 0 (best interactions, in blue) to 2 (worst interactions, in red). Below are the logos with the letters, whose sizes indicate the degree of difference in interaction of each amino acid in the peptide at a specific position of the target protein. Figure 6: Logarithmic scale bar graph of the virus titer under treatment with peptides #1 to #8 at different concentrations and control without peptide. Three replicates were used per experiment. The mean ± standard deviation of each experiment is represented. Figure 7: Curve graph representing the normalized value (0-1) of the inhibitory effect (Fa) of peptides #1 to #8 and the IC50 doses of each assay against SARS-CoV-2.Figure 8 Logarithmic bar graph of the virus titer (plaque-forming units or pfu / ml) under treatment with peptide #7 at different concentrations, in untreated cultures (control) and treated with the VFA peptide. Three replicates were used per experiment. The mean ± standard deviation of each experiment is represented. Figure 9: Logarithmic bar graph of the virus titer (plaque-forming units or pfu / ml) under treatment with peptide #6 at different concentrations and in untreated cultures (control). Three replicates were used per experiment. The mean ± standard deviation of each experiment is represented. Figure 10: Logarithmic bar graph of the virus titer under treatment with peptide #7 at concentrations of 0, 6, 12 and 24 µM and 5-FU at 61, 122 and 244 µM. Three replicates were used per experiment. The mean ± standard deviation of each experiment is represented.Figure 11: Logarithmic bar graph of the virus titer (plaque-forming units or pfu / ml) under treatment with peptides #1 and #6 in combination with 5-FU at different concentrations and control without peptides. Three replicates were used per experiment. The mean ± standard deviation of each experiment is represented. Figure 12. (A) Curve graph depicting the normalized value (0-1) of the inhibitory effect (Fa) of 5-FU and peptide #7 on viruses from passage 3. The green symbols represent the combinatorial effect of both antivirals. (B): Dot plot depicting the combined effect of peptide #7 and 5-FU on virus replication. Each dot represents a treatment with the two compounds at different concentrations. If the dot is below 1, below the line that intersects the graph, the effect of the combination was synergistic. If it is above the line, the effect was antagonistic.If the point falls on the line, the effect was additive. Figure 13: (A) Curve plot representing the normalized (0-1) inhibitory effect (Fa) of 5-FU and peptide #1 on viruses from passage 3. The green symbols represent the combinatorial effect of both antivirals. (B) Dot plot representing the combined effect of the peptide and 5-FU on virus replication. Each point represents a treatment with the two compounds at different concentrations. If the point falls below 1, below the line that intersects the graph, the effect of the combination was synergistic. If it falls above the line, the effect was antagonistic. If the point falls on the line, the effect was additive. Figure 14: (A) Curve graph representing the normalized value (0-1) of the inhibitory effect (Fa) of 5-FU and peptide #6 on viruses from passage 3. The green signs represent the combinatorial effect of both antivirals.(B) Dot plot depicting the combined effect of the peptide and 5-FU on viral replication. Each dot represents a treatment with the two compounds at different concentrations. If the dot is below 1, below the line that intersects the graph, the effect of the combination was synergistic. If it is above the line, the effect was antagonistic. If the dot is on the line, the effect was additive. DETAILED DESCRIPTION OF THE INVENTION Materials To study the antiviral effect, in vitro assays were carried out in cultures of the Vero E6 cell line. The assays were carried out in the P3 biosafety level laboratory at the facilities of the Severo Ochoa Molecular Biology Center (CMBSO) in Madrid. Viral samples were selected that presented several mutations that interfered with peptide-receptor binding, and some samples that did not have mutations in those regions.Additionally, new samples were collected from patients infected with the Delta and Omicron variants of SARS-CoV-2. To prepare viral stocks, Vero E6 cultures were infected with the following samples: Sample S26_M_77_M_AH With resistance mutations Sample S7_M_40_M_AH Without resistance mutations Sample S45_M_X_S Omicron Ct = 18 Sample S46_M_X_S Delta Ct = 27 Cytopathic effect was observed in all cultures, except in cultures with the Delta variant (due to contamination). Lysis plaques were only observed in sample S45_M_X_S (Omicron variant), with a very low titer (200 pfu / ml). To increase the virus titer, a second passage was carried out and cytopathic effect was only observed in the sample with the omicron variant. Therefore, sample S45_M_X_S was used for the culture assays. Dimethyl sulfoxide (DMSO) was used to dissolve the cultures and reagents.5-FU Assays Cell cultures were pre-treated for 16 h with increasing concentrations of 5-FU, infected with SARS-CoV-2 and incubated in the presence of these concentrations of 5-FU for 48 h. According to the results of this third passage reflected in Figure 1, there was a very significant decrease in the titer when treating the cultures with doses of 400 µM. At 800 µM, where the presence of the virus disappears in all cultures. The IC50 dose of 5-FU in these assays against SARS-CoV-2 is estimated at 148.12 µM (Figure 2). Differences in the results of 5-FU assays were observed with virus passage 4, whose stock titer was much higher than that of passage 3. These differences were attributed to the adaptation of the inoculated virus from a patient to Vero E6 cell cultures, which could have altered the inhibitory effect of the 5-FU analogue. As can be seen in Figure 3, the decrease in viral load due to 5-FU treatment decreased.To test whether this was indeed due to increased virus fitness, experiments were performed with passage 3 and 4 viruses treated with 5-FU at different concentrations (300, 400, and 500 µM). It was observed that 5-FU, at the lowest concentration (300 µM), had a slightly lower effect on the passage 4 virus than on passage 3 (Figure 4). Thus, the effect of 5-FU against passage 4 viruses required a higher dose than on passage 3 viruses, but was achieved at high concentrations (400 and 500 µM). The 5-FU dose for the IC50 in the passage 4 virus was 231.982 µM, higher than in the passage 3 virus (182.426 µM) (Figure 3). However, at higher concentrations (500 µM), virus inhibition at passage 4 was greater, as can be seen in Figure 4. Therefore, the results suggest that the increase in virus fitness after four passages in VeroE6 cell cultures does not diminish the inhibitory effect of the 5-FU analogue.Design of inhibitory peptides The design of inhibitory peptides for the binding of nsp10 to nsp14 and nsp16 was based on the crystallographic structure of SARS-CoV-2 and SARS-CoV-1, and on the SARS-CoV-2 genomic sequence from the spectra of mutants analyzed from patients. Peptides of between 6 and 15 amino acids were developed. First, the crystallographic structures of the nsp10-nsp14 and nsp10-nsp16 protein complexes of SARS-CoV-2 and two other coronaviruses (SARS-CoV and MERS-CoV) were analyzed to elucidate the residues of each protein that constitute the interaction zones that form the protein complexes. Once the interaction zones were detected, the most suitable ones were selected to design the peptides.The selection of peptides was based on the following factors: (1) peptide length, the longer the length, the greater the interaction, but the higher the manufacturing cost; (2) secondary structure of the peptide and (3) interaction energy (the lower the interaction energy, the greater the interaction strength). As shown in Table 2, two nsp10 peptide sequences were selected. The first is a nsp10 peptide sequence that interacts with nsp14, and the second with nsp16. Table 2: Positions of nsp10 that interact with nsp14 and nsp16. Peptide sequence that interacts with Peptide sequence that interacts with nsp14 – SEQ ID NO: 9 nsp16- SEQ ID NO: 10 Position Amino acid Position Amino acid 11 S 39 T 12 T 40 N 13 V 41 C 14 L 42 V 15 S 43 K The first peptide that interacts with the nsp14 protein presents a strong interaction (interaction energy = -15.45 kcal / mol).The second peptide can interfere with the nsp14 and nsp16 proteins, inactivating them by preventing the formation of protein complexes. The interaction of the peptide with nsp14 is weaker than that of the first peptide (interaction energy = -9.63 kcal / mol). The interaction with nsp16 is stronger (interaction energy = -12.71 kcal / mol). The peptides were optimized using the FoldX program (https: / / foldxsuite.crg.eu / ), which is based on computational protein design algorithms, in which the peptide is mutated to a poly-alanine chain as a reference. The program modifies each individual position to the remaining 19 natural amino acids and repairs the environment to avoid collisions, in order to find the most suitable interactions between the peptide (based on nsp10) and the receptor (the nsp14 and nsp16 proteins).The program calculates the interaction energies between peptides (ligands) and the protein, generating a two-dimensional matrix containing all the calculated energies for the 20 natural amino acids at all ligand positions. The matrices are corrected by adding internal Van der Waals clashes to the interaction surface and normalized with respect to the smallest value (which is forced to be zero). The lower the energy value, the better the interaction between the ligand and the protein. For a given matrix, the binding energy of a peptide can be predicted as the sum of the interaction energies at each of the positions, which are assumed to be independent. The results of the analyses with the FoldX program for the design of inhibitory peptides can be seen in Figure 5. The result was a list of peptides that was classified into three groups.1: the group derived from the 11-22 peptide sequence of nsp10 that is involved in the formation of the nsp10-nsp14 complex, and consisting of 12-amino acid peptides; 2 and 3: the groups of 9-amino acid peptides derived from the 39-47 sequence of nsp10. For their design, the 3D structure of the nsp10-nsp14 complexes stored in the databases was used as a template (group 2) and the 3D templates of the nsp10-nsp16 complexes were used in the other (group 3). The most suitable peptides were selected following the following recommendations: - select peptides with efficient binding to the target protein (lower DG_mut), - avoid peptides that are too hydrophobic, since they can cause solubility problems, which would hinder experimental tests.Select peptides with a net positive charge, - avoid the amino acids aspartic acid (Asp) and cysteine ​​(Cys), since these amino acids can cause potential problems during peptide synthesis or generate redox behavior, and - select those peptides least similar to the virus sequence, taking into account mutations that had appeared in SARS-CoV-2 quasispecies from clinical samples. As a result, a total of 8 peptides (Table 3) of size 9 or 12 amino acids were selected. During their synthesis, modifications were introduced at the N-terminal (acetylation) and C-terminal (amidation) ends, which increase their half-life within cells. Table 3: Inhibitory peptides. Peptide Inhibits binding Weight Sequence SEQ ID NO nps10 with molecular #1 ELKIRMRWK nsp16 1300.63 #2 ELKILMMWK nsp16 1232.61 #3 QLQILMMKK nsp14 1173.54 #4 ELKILLMKK nsp14 1156.53 #5 QLQIIMMRK nsp14 1201.56. #6 QLQIYLMRKnsp14 1233.53#7 KIEKTFEYYMVR nsp14 1647.94 #8 KIERTFQYYMVR nsp14 1674.97 Selection of inhibitory peptides The synthesized peptides were lyophilized (preservation by dehydration), so that they were dissolved until a 5 mg / ml solution was obtained for each peptide. The net dry weight of each peptide was 9 mg, therefore the solutions were diluted in 1.8 ml of DMSO. As shown in Table 4, a 1 ml stock solution of each peptide was prepared at a concentration of 3 millimolar (mM). These stocks were prepared from the 5 mg / ml solutions until a concentration of 3 mM was reached, adjusting the volume with phosphate-buffered saline (PBS). Table 4: Adjustment of peptide concentrations to obtain stock solutions.Concentration # Stock Concentration Peptide# in mM (5 mg / ml) Vol # (µl) 5 mg / ml PBS Vol (µl) # in mg / ml (3 mM) #1 3,844 780 220 3,902 #2 4,056 740 260 3,698 #3 4,261 704 296 3,521 #4 4,323 694 306 3,470 #5 4,161 721 279 3,605 #6 4,053 740 260 3,701 #7 3,034 989 11 4,944 #8 3,000 1000 0 5,025 From the stock solutions, solutions at different concentrations were prepared to carry out the works: 0.7, 7 and 70 µM. For some peptides, in specific assays, higher concentrations were used: 6, 12, 24, 39, and 54 µM. IC50 inhibitory effect of the peptides In the culture assays, the inhibitory effect of the peptides was estimated by determining the viral titer and the IC50 index. All assays were performed in triplicate for each experiment. The first experiments were performed with virus passage 2, from sample S45_M_X_S.An experimental control (culture with virus untreated with any antiviral) and a plating control (to check if the titration was correct) were used, and the stock virus (original Wuhan variant) was used as a positive control. After a two-hour incubation with the peptide, the cell cultures were infected with the viral stock. After one hour of adsorption, each peptide was added at the concentration determined in its corresponding culture. The supernatants were collected after 48 hours and titrated in confluent monolayers of Vero E6 cells. The inhibitory effect of peptides #1 to #8 was estimated. As shown in Figure 6, in the initial experiments, all peptides showed a significant inhibitory effect at 70 µM, where a significant decrease in the SARS-CoV-2 viral titer was detected. Treatment with peptide #2 was the least effective in inhibiting viral replication and therefore decreasing its viral load.Peptides designed to bind to nsp14 were most effective in inhibiting SARS-CoV-2 replication at 70 µM, especially peptides #7 and #8. The Compusyn program was used to estimate the IC50 value. The IC50 values ​​of the peptides varied greatly (4.5–110 µM) (Figure 7). The most extreme values ​​were observed for peptide #1, where the lowest IC50 value was estimated (4.5 µM), and peptide #2, which required a very high concentration to decrease the viral titer to 50% (110 µM). For peptides 3–8, the IC50 values ​​ranged from 15.81 to 65.19 µM. These results show that each peptide exerts a specific effect on nsp16 and nsp14 of the virus, depending on the unique characteristics of each peptide according to its amino acid sequence, as well as the composition of mutations in each viral quasispecies, which can alter the peptide-protein interaction.New experiments were carried out with peptides #7 and #6, which gave good results in viral titer and IC50 index. This second experiment, carried out with peptide #7, in addition to using the untreated virus as a control, included a new control, the virus treated with a specific antiviral peptide against the foot-and-mouth disease virus (FMDV), a peptide of a similar size that was used at a concentration of 70 µM. With this control, we can verify whether the action against SARS-CoV-2 of the designed peptides is due to a specific activity against the virus, and not a chemotoxic effect due to a high concentration of peptide. The results (Figure 8) show that the cultures treated with the FMDV peptide did not undergo changes in viral titer with respect to the untreated viruses (control).The viral titer values ​​obtained in this experiment were considerably higher than in previous assays, but this was because the experiments were performed with the virus obtained in passage 3, starting from a viral stock concentration much higher than that of passage 2. In another assay, the efficacy of peptide #6 in inhibiting viral infectivity was tested (Figure 9). Cytotoxic effect of 5-FU at different concentrations combined with inhibitory peptides. The cytotoxic effect on Vero E6 cells of each of the 8 peptides in Table 3 was analyzed at two concentrations (70 µM and 7 µM), of 5-FU alone, and in combination with one of the designed peptides, specifically peptide #7, using CC50 assays. Different concentrations of 5-FU were prepared: 50, 61, 100, 122, 200, 244, 300, 400, 500 and 800 µM, and peptide #7 at 0.7, 7 and 70 µM. Table 5: Cytotoxicity analysis in infected Vero E6 cultures treated with the antivirals at different concentrations.Células / ml Viabilidad celular (%) Mortalidad celular (%) Control W / 0 1.135 100.0 0.0 # 1 (70 µM) 1.377 121.3 -21.3 # 2 (70 µM) 1.497 131.9 -31.9 # 3 (70 µM) 1.440 126.8 -26.8 # 4 (70 µM) 1.932 170.2 -70.2 # 5 (70 µM) 1.663 146.5 -46.5 # 6 (70 µM) 1.250 110.1 -10.1 # 7 (70 µM) 1.409 124.1 -24.1 # 8 (70 µM) 1.242 109.4 -9.4 # 1 (7 µM) 0.945 83.3 16.7 # 2 (7 µM) 1.271 111.9 -11.9 # 3 (7 µM) 1.132 99.7 0.3 # 4 (7 µM) 1.075 94.7 5.3 # 5 (7 µM) 1.022 90.0 10.0 # 6 (7 µM) 0.862 76.0 24.0 # 7 (7 µM) 0.764 67.3 32.7 # 8 (7 µM) 0.919 80.9 19.1 5-FU 800 µM 1.935 170.5 -70.5 5-FU 800 µM + # 7 (70 µM) 1.405 123.8 -23.8 5-FU 800 µM + # 7 (7 µM) 2.139 188.4 -88.4 5-FU 400 µM + # 7 (0.7 µM) 2.099 184.9 -84.9 5-FU 400 µM 1.791 157.8 -57.8 5-FU 400 µM + # 7 (70 µM) 0.500 44.1 55.9 5-FU 400 µM + # 7 (7 µM) 1.790 157.7 -57.7 5-FU 800 µM + # 7 (0.7 µM) 2.084 183.6 -83.6 5-FU 200 µM 1.294 114.0 -14.0 5-FU 200 µM + # 7 (70 µM) 1.554 136.8 -36.8 5-FU 200 µM + # 7 (7 µM) 1.484 130.7 -30.7 5-FU 200 µM + # 7 (0.7 µM) 1.447 127.4 -27.4 # 7 (0.7 µM) 0.800 70.5 29.5 5-FU 100 µM 1.000 88.1 11.9 5-FU 50 µM 0.890 78.4 21.6 According to the results compiled in Table 5, in general, no significant cytotoxic effect was observed in cells treated with the antivirals. In most treated cultures the number of cells increased with respect to the control. Surprisingly, the highest cell death rates were observed in samples with the lowest concentration of peptide (0.7 and 7 µM), and 5-FU (50 µM). At higher concentrations, cell viability is higher than in the control. Antiviral effect of 5-FU at different concentrations combined with inhibitory peptides The combined antiviral action of 5-FU with certain peptides was analyzed at different concentrations. Three peptides were selected: #1 (9aa, nsp16), #6 (9aa, nsp14) and #7 (12aa, nsp14) according to previous results, the size of each peptide and the target protein.To measure the inhibitory capacity IC50, different concentrations of 5-FU were prepared: 61, 122 and 244 and of peptides #1, #6 and #7 at 0, 6, 12 and 24 µM. The assays were performed with the virus 35163916_AG (S7_M_40_M_AH). First, assays were performed with peptide #7. Peptide #7 was treated separately and in combination at 0, 6, 12 and 24 µM and 5-FU at 61, 122 and 244 µM. The cultures were infected with the virus from passage 3. According to the results shown in Figure 10, the virus titer decreased as the dose of 5-FU and peptide #7 was increased. Generally, the use of peptide #7 at increasing concentrations across each 5-FU concentration range resulted in a slight decrease in titer, except at 122 µM 5-FU, where there appeared to be no synergistic effect between the peptide and the analogue. The same assays were subsequently performed with peptides #1 and #6. Viruses from passage 3 were used.In this case, only the peptides were tested at 70 µM. The 5-FU concentrations were 300, 400, and 500 µM. At first glance, the combined use of each peptide at 70 µM with 5-FU at different concentrations produced a significant decrease in the virus titer, especially with peptide #1 (Figure 11). To estimate the existence of a possible synergistic effect between 5-FU and the peptides, that is, that the combination of both causes a stronger effect on virus replication, the Compusyn program was used. According to the results obtained using the software, if the combination index (CI) value is less than 1, it means that the combination of the peptide and 5-FU was synergistic, that is, the inhibition of the virus was greater than with the separate treatments.If the CI value is greater than 1, the combination produced an antagonistic effect, suggesting that the combined action of the antivirals negatively affects the virus, decreasing its inhibition. If the value is 1, there is no difference between adding the peptide and 5-FU separately or in combination, which is known as an additive effect. When studying the combinatorial effect of each of the peptides with 5-FU, a considerable synergistic effect was confirmed. According to the results in Figure 12B, there was a trend towards antagonism in the combination of peptide #7 and 5-FU at the highest concentrations, with a synergistic effect observed at the lowest concentrations of 5-FU (61 µM) and #7 (6 µM) (CI = 0.831). At the other concentrations, the combination index ranged from 1.013 to 1.591 (Figure 12A).The combination of peptide #1 and 5-FU generated a synergistic effect at all tested concentrations (CI = 0.471-0.932) (Figure 13). Between 5-FU and peptide #6 there was an antagonistic effect at the lowest concentration (CI = 1.451) (Figure 14). However, at higher concentrations, a trend towards synergism between both compounds was detected (CI = 0.511-0.977). References: 1. Bouvet M, Lugari A, Posthuma CC, Zevenhoven JC, Bernard S, Betzi S, Imbert I, Canard B, Guillemot JC, Lécine P, Pfefferle S, Drosten C, Snijder EJ, Decroly E, Morelli X. “Coronavirus Nsp10, a critical co-factor for activation of multiple replicative enzymes”. J Biol Chem.2014 Sep 12;289(37):25783-96. doi: 10.1074 / jbc.M114.577353. 2. Vithani N, Ward MD, Zimmerman MI, Novak B, Borowsky JH, Singh S, Bowman GR. “SARS-CoV-2 Nsp16 activation mechanism and a cryptic pocket with pan-coronavirus antiviral potential.” bioRxiv [Preprint]. 2020 Dec 10:2020.12.10.420109. doi: 10.1101 / 2020.12.10.420109. 3.Saramago M, Barria C, Costa VG, Souza CS, Viegas SC, Sundays S, Lousa D, Soares CM, Arraiano CM, Matos RG. “New targets for drug design: importance of nsp14 / nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2.” FEBS J.2021 Sep;288(17):5130-5147. doi: 10.1111 / feb.15815. [ PMC free article ] [ PubMed ] [ Cross Ref ] 4. Wang Y, Sun Y, Wu A, Xu S, Pan R, Zeng C, Jin X, Ge X, Shi Z, Ahola T, Chen Y, Guo D. J Virol. 2015 Aug;89(16):8416-27. doi: 10.1128 / JVI.00948-15. Epub 2015 Jun 3. PMID: 26041293; PMCID: PMC4524257. 5. Sergio Ortega-Del Campo, Ana Maria Fernandez-Escamilla, Gregorio Joaquin Fernandez-Ballester, Maria Clara Blanes-Mira, Pedro Seoane-Zonjic, Josefa Gomez- Maldonado, Francisco Jose Villena-Gonzalez, Maria Isabel Viciana-Ramos, Encarnación Clavijo-Frutos, Jesus L.Santos-González, Enrique Viguera-Mínguez and Ana Grande- Pérez, “Design of non-immunogenic peptides that prevent the activation of SARS-CoV-2 nsp14 and nsp16 proteins” 6 de septiembre de 2022 6. Fatemeh Arabi-Jeshvaghani, Fatemeh Javadi-Zarnaghi and Mohamad Reza Ganjalikhany. “Analysis of critical protein–protein interactions of SARS-CoV-2 capping and proofreading molecular machineries towards designing dual target inhibitory peptides”. Sci Rep.2023. Vol.13. DOI: 10.1038 / s41598-022-26778-8 7. Ahmad SI. “5-Fluorouracil in combination with deoxyribonucleosides and deoxyribose as possible therapeutic options for the Coronavirus, COVID-19 infection”. Med Hypotheses.2020 Sep; 142:109754. doi: 10.1016 / j.mehy.2020.109754.

Claims

CLAIMS 1. 5-fluorouracil for use as an antiviral agent against viruses of the Coronaviridae family.

2. 5-fluorouracil for use as an antiviral agent against SARS-CoV-2.

3. Composition comprising 5-fluorouracil for use as an antiviral agent against viruses of the Coronaviridae family.

4. The composition according to the preceding claim for use as an antiviral agent against SARS-CoV-2.

5. Composition comprising 5-fluorouracil which further comprises peptides that inhibit nsp10-nsp16 and / or nsp10-nsp14 binding.

6. Composition comprising 5-fluorouracil further comprising peptides that inhibit nsp10-nsp16 and / or nsp10-nsp14 binding selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, individually or in any combination thereof.Composition according to the preceding claim, characterized in that the nsp10-nsp16 and / or nsp10-nsp14 binding inhibitor peptides are selected from: SEQ ID NO: 1 ELKIRMRWK, SEQ ID NO: 7 KIEKTFEYYMVR and SEQ ID NO: 6 QLQIYLMRK, individually or in any of their combinations.

8. Composition according to any of claims 5 to 7 which is pharmaceutically acceptable.

9. Composition according to any of claims 5 to 8 for use as an antiviral agent against viruses of the Coronaviridae family.

10. Composition according to the preceding claim for use as an antiviral agent against SARS-CoV-2.

11. Composition according to any of claims 5 to 8 for use as a medicament.

12. Composition according to the preceding claim for use as a medicament for the prevention and / or treatment of infections caused by a virus of the Coronaviridae family.

13. Composition according to the preceding claim for use as a medicament for the prevention and / or treatment of infections caused by the SARS-CoV-2 virus.

14. Composition according to claim 5, characterized in that the nsp10-nsp16 and / or nsp10-nsp14 binding inhibitor peptides are obtainable by the method comprising: a) structurally analyzing the areas of interaction between both proteins, b) locating the binding zones with greater specificity and affinity to design nsp10-derived peptides using computational protein design algorithms, c) testing the 20 natural amino acids at each position individually, using Alanine as a reference,d) sequence clinical samples with Illumina and analyze the quasispecies with Quasiflow to identify SARS-CoV2 mutations in the peptide interaction zones, e) preselect the best candidates according to the interaction energy, molecular interactions, the content of apolar amino acids and the similarity with the virus sequence, f) characterize the inhibitory capacity of the designed peptides in cells infected by the virus, g) Sequence by Illumina and analyze the quasispecies of the cultures with QuasiFlow in the presence and absence of peptides h) assess the inhibitory capacity of nsp14 and nsp16 of the peptides based on the mutations that the different quasispecies may present, and i) select the peptides that present the greatest inhibitory capacity.,

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