USE OF PEPTIDES IN THE TREATMENT OF INFECTIONS CAUSED BY FLAVIVIRUSES AND COMPOSITIONS COMPRISING THEM

Description

/ 32 USE OF PEPTIDES IN THE TREATMENT OF INFECTIONS CAUSED BY FLAVIVIRUSES AND COMPOSITIONS COMPRISING THEM FIELD OF THE INVENTION

[001] The present invention is in the field of virology / immunology. It relates to the use of EL28 and PEP5 peptides and their variations, conjugated or not to the cationic peptide derived from the TAT protein (region 47-57) in the preparation of a pharmaceutical composition / medicine with antiviral activity. The present invention also relates to the pharmaceutical composition comprising said peptides. FUNDAMENTALS OF THE INVENTION

[002] The Zika virus (ZIKV) is an arbovirus of the Flaviviridae family, first isolated in Uganda in 1947 (Dick, G. et al, 1952). The first human isolation of ZIKV was reported in Nigeria in 1953. Since then, ZIKV has expanded its geographic reach to several countries in Africa, Asia, Oceania, and the Americas (Ministry of Health).

[003] Data show that the aforementioned virus is associated with a high rate of primary microcephaly and Guillain-Barré syndrome during ZIKV infection in French Polynesia and Brazil (Schuler-Faccini, L. et al., 2016; Cauchemez, S. et al., 2016; Cao-Lormeau, VM et al., 2016; Ventura, CV et al., 2016).

[004] In addition, ZIKV infection can cause thrombocytopenia, eye and testicular damage, multiple organ failure, and lead to Guillain-Barré Syndrome, representing a serious threat to public health worldwide (Pielnaa et al., 2020).

[005] ZIKV is organized into an icosahedral layer composed of 180 copies of the E protein (Kostyuchenko et al., 2016; Sirohi et al., 2016). The E protein contains three domains: DI, DII, and DIII (Dai et al., 2016). Previously, Ma et al. (2022) demonstrated that DII (Ala268-Leu273) is involved in viral binding. Petition 870250060600, dated 07 / 15 / 2025, page 11 / 50 / 32

[006] The Zika genome (~11 kb) contains two untranslated regions (UTRs) and an open reading frame (ORF) that encodes a polyprotein with more than 3,000 amino acid residues, subsequently cleaved into 10 viral proteins: three structural proteins — capsid (C), precursor membrane (prM), and envelope (E) — and seven non-structural proteins — NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (Shi and Gao, 2017). To date, there are no specific therapies or vaccines approved for the treatment or prevention of ZIKV infection (da Silva et al., 2018; Lin et al., 2018; Woodson and Morobito, 2024).

[007] Despite this, viral proteins have been identified as promising targets for the identification of antiviral molecules (Bollati et al., 2010; Kang et al., 2017; Valente and Moraes, 2019; Feng et al., 2024). During the ZIKV life cycle, viral entry is mediated by the interaction of the E protein with specific host receptors, making this envelope protein a strategic target for the development of anti-ZIKV inhibitors (Wang et al., 2019; Guo et al., 2021; Ma et al., 2022). Furthermore, the essential roles played by NS1, NS2B-NS3, and NS5 proteins in viral replication, assembly, and modulation of the intracellular environment make these non-structural proteins attractive targets for drug discovery (Luo et al., 2015; Kang et al., 2017; Guo et al., 2021).

[008] Flavivirus NS1 is essential for viral genome replication in its dimeric form and plays crucial roles in immune evasion and modulation of the host environment during infection, as secreted hexamers (Gutsche et al., 2011; Mulller et al., 2012; Akey et al., 2014). The NS1 structure is composed of three domains: the N-terminal β-roll domain, the wing domain, and the C-terminal β-ladder domain (Brown et al., 2016). Interestingly, residues Trp28, Trp115, Trp118, Tyr122, Phe123, Val124, and Phe163 form anchoring points for membrane association (Brown et al., 2016; Xu et al., 2016). Petition 870250060600, dated 07 / 15 / 2025, page 12 / 50 / 32

[009] In NS2B-NS3, the NS2B subunit contains transmembrane domains responsible for anchoring the complex to the ER membrane, while the N-terminal of NS3 constitutes a serine protease domain responsible for cleaving the polyprotein, and the C-terminal of NS3 has a helicase domain, being involved in the replication and assembly of viral RNA (Zhang et al., 2016; Li et al., 2017).

[0010] The NS5 protein contains an N-terminal methyltransferase (MTase) domain for RNA encapsulation and a C-terminal RNA polymerase (RdRp) domain for RNA synthesis (Godoy et al., 2017; Zhao et al., 2017). The RdRp domain is subdivided into three subdomains: fingers, palm, and thumb (Godoy et al., 2017; Zhao et al., 2017). Furthermore, in ZIKV NS5, three channels are found: the central channel and the NTP channel are responsible for RNA template entry, nascent RNA exit, and NTP entry, respectively (Godoy et al., 2017; Zhao et al., 2017). Furthermore, in DENV NS5, a region in the finger subdomain (320-341) is involved in binding to NS3 and plays an important role in viral replication (Tay et al., 2015).

[0011] Intracellular peptides are generated by proteasomes through the breakdown of proteins originating from the nucleus, cytosol, and mitochondria, and can be processed by additional peptidases to create a larger reservoir of peptides within cells. Numerous intracellular peptides have been sequenced in a variety of organisms, including plants, yeast, zebrafish, rodents, and human cells and tissues.

[0012] The relative abundance of these intracellular peptides varies in response to human diseases and cellular stimulation, corroborating their biological roles. However, only a limited number of intracellular peptides have been pharmacologically characterized, and their biological importance and mechanisms of action remain obscure. (de Araujo et al., 2019). Petition 870250060600, dated 07 / 15 / 2025, page 13 / 50 / 32

[0013] In recent years, peptides have attracted attention as promising drug candidates, and a wide variety of pharmacological effects have been demonstrated. Currently, there is great pharmaceutical interest in these compounds, with more than 80 peptides commercially available and about 500 in preclinical studies. Therapeutic peptides currently span diverse medical areas, including pulmonology, urology, metabolism, cardiovascular, and antimicrobials. The COVID-19 pandemic has increased the demand for novel broad-spectrum antivirals (Telenti A., et al 2021 and Da Zhu J., et al 2015). Antiviral peptides have demonstrated antiviral activity by directly inhibiting viruses through different mechanisms of action (Vilas Boas LCP., et al 2020). However, the increase in viral resistance (Musiime V., et al 2013), concomitant infections (Deming P., et al 2011) and outbreaks of diseases such as COVID-19, H1N1, Ebola and Zika (Bontempia E., et al 2020, Marston BJ., et al 2018 and Lowe R., et al 2018) highlight the need for more effective therapeutic alternatives. The application of antiviral peptides is an area of ​​intense research, as demonstrated in recent reviews (Monroe, MK, et al 2022 and Agamennone M., et al 2022).

[0014] In this context, Ayusso et al. (2023) demonstrated that a peptide derived from Bothrops jararacussu venom exhibited antiviral activity against ZIKV, inhibiting different stages of the viral replication cycle. Furthermore, Li et al. (2019) showed that the Ev37 peptide derived from Euscorpiops validus scorpion venom was able to impair ZIKV infection.

[0015] Peptide 5 (pep5; WELVVLGKL and pep5r WELVVL) is a natural intracellular peptide derived from the degradation of Cyclin D2 via the ubiquitin-proteasome system. It was first identified in extracts of HeLa cells, which specifically increased during the S phase of the cell cycle (de Araujo et al., 2014; Chen et al., 2012; Russo et al., 2017). Petition 870250060600, dated 07 / 15 / 2025, page 14 / 50 / 32

[0016] This peptide induced cell death when introduced into MDA-MB-231 breast cancer cells, which expressed low levels of Cyclin D2.6 Pep5 promotes substantial cytoskeletal disruption, likely by binding to Plectin and / or CLIC1, which appears to contribute to cell death pathways (Russo et al., 2017).

[0017] Pep5-cpp also induces cell death in epimastigote, trypomastigote, and amastigote forms of the parasite Trypanosoma cruzi, responsible for Chagas disease. At low doses, Pep5-cpp decreases the percentage of infected cells without any detectable toxic effects on mammalian host cells. The infective form of T. cruzi, i.e., trypomastigotes, pre-treated with Pep5-cpp was unable to infect LLC-MK2 cells (de Araujo, 2019).

[0018] Another important function of the proteasome is the generation of peptides presented as antigens via MHC-I. Under stress or acute immune response, an alternative form of the proteasome can be induced, altering catalytic specificity and improving antigen presentation.

[0019] In HeLa cells treated with interferon-gamma (INF-γ), the peptide el28 was identified, which showed a three-fold increase in the repertoire of intracellular peptides compared to the control. It was found that el28 increases proteasome activities similar to caspases, trypsins, and chymotrypsins, and increases the proliferation of CD8+ T cells (Monte et al., 2017).

[0020] The present invention discloses the antiviral role of synthetic peptides derived from Cyclin D2 and a 26S protease regulatory subunit 4 (Rpt2) protein. The present invention demonstrates that the peptides inhibit Zika infection and effectively interact with key targets involved in entry, assembly, and replication. Petition 870250060600, dated 07 / 15 / 2025, page 15 / 50 / 32

[0021] The results presented in this invention highlight the potential of intracellular peptides as promising molecules for the development of new molecules against ZIKV infection. SUMMARY OF THE INVENTION

[0022] Considering the absence of specific antiviral therapies for ZIKV infection and the potential for serious outcomes associated with the disease, especially in pregnant women and newborns, the investigation of new therapeutic approaches becomes extremely important. ZIKV infection, in addition to self-limiting symptoms in most cases, can result in neurological complications, such as Guillain-Barré syndrome, and serious congenital anomalies, notably microcephaly and other manifestations of congenital ZIKV syndrome.

[0023] To date, treatment is only symptomatic, based on analgesics and antipyretics, with no specific drugs with proven antiviral action against the virus. Given this scenario, the development of new drugs with antiviral activity targeting ZIKV represents a highly relevant strategy for public health, especially in endemic regions with high social vulnerability.

[0024] The present invention aims to provide peptides for the treatment of viral infections.

[0025] In particular, the use occurs through pharmaceutical compositions comprising the aforementioned peptides.

[0026] In a first embodiment, the present invention provides the use of a peptide in the preparation of a pharmaceutical composition for the treatment of infection by a virus of the Flaviviridae family.

[0027] Specifically, the peptides are those selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0028] Additionally, the virus from the Flaviviridae family is ZIKV. Petition 870250060600, dated 07 / 15 / 2025, page 16 / 50 / 32

[0029] In a second embodiment, the invention provides a composition for use in the treatment of viral infections.

[0030] Specifically, the peptides are those selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

[0031] Additionally, the virus from the Flaviviridae family is ZIKV. BRIEF DESCRIPTION OF THE FIGURES

[0032] Figure 1A: Molecular anchoring against the ZIKV E protein. a. Electrostatic representation of the surface of the ZIKV E protein. The “stem” region and the receptor binding site are highlighted.

[0033] Figure 1B. Illustrative drawing of the ZIKV E protein. Domains I, II, and III are shown in red, blue, and yellow, respectively. Peptides pep5_cpp, pep5r_cpp, and el28_cpp are colored green, orange, and magenta, respectively.

[0034] Figure 1C: Illustrative representation of the protein-peptide linkage of pep5_cpp; hydrophobic, electrostatic, and hydrogen bonding interactions are marked with dashed pink, orange, and green lines. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and orange, respectively. Protein residues are shown as sticks and marked with a 1-letter symbol, followed by the position of the present residue in the primary structure.

[0035] Figure 1D: Illustrative representation of the protein-peptide linkage of el28_cpp, hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and marked with the 1-letter symbol, followed by the position of the present residue in the primary structure. Petition 870250060600, dated 07 / 15 / 2025, page 17 / 50 / 32

[0036] Figure 1E: Illustrative representation of the protein-peptide linkage of pep5r_cpp (e), hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and marked with the 1-letter symbol, followed by the position of the present residue in the primary structure.

[0037] Figure 2A: Molecular anchoring against the ZIKV NS1 dimer. a. Electrostatic representation of the surface of the ZIKV NS1 dimer. The membrane anchoring region is highlighted.

[0038] Figure 2B: Illustrative drawing of the ZIKV NS1 dimer. The β-roll, wing, and β-ladder domains are shown in blue, yellow, and red, respectively. The pep5_cpp, pep5r_cpp, and el28_cpp peptides are colored green, orange, and magenta, respectively.

[0039] Figure 2C: Illustrative representation of the protein-peptide linkage of pep5_cpp. Hydrophobic, electrostatic, and hydrogen bonding interactions are marked with dashed pink, orange, and green lines. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and orange, respectively. Protein residues are shown as sticks and marked with a 1-letter symbol followed by the position of the residue in the primary structure.

[0040] Figure 2D: Illustrative representation of the protein-peptide linkage of el28_cpp. Hydrophobic, electrostatic, and hydrogen bonding interactions are marked with dashed pink, orange, and green lines. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and orange, respectively. Protein residues are shown as sticks and marked with a 1-letter symbol, followed by the position of the present residue in the primary structure. Petition 870250060600, dated 07 / 15 / 2025, page 18 / 50 / 32

[0041] Figure 2E: Illustrative representation of the protein-peptide linkage of pep5r_pp, hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and marked with the 1-letter symbol, followed by the position of the present residue in the primary structure.

[0042] Figure 3A: Molecular fit against the ZIKV NS1 hexamer. Molecular fit against the ZIKV NS1 hexamer. a. Electrostatic representation of the surface of the ZIKV NS1 hexamer. The central channel is highlighted.

[0043] Figure 3B: Illustrative drawing of the ZIKV NS1 hexamer. The β-roll, wing, and β-ladder domains are shown in blue, yellow, and red, respectively. The pep5_cpp, pep5r_cpp, and el28_cpp peptides are colored green, orange, and magenta, respectively.

[0044] Figure 3C: Illustrative representation of the protein-peptide linkage of pep5_cpp (c), hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and labeled with the 1-letter symbol, followed by the position of the present residue in the primary structure.

[0045] 3D Figure: Illustrative representation of the protein-peptide linkage of el28_cpp, hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and labeled with the 1-letter symbol, followed by the position of the present residue in the primary structure. Petition 870250060600, dated 07 / 15 / 2025, page 19 / 50 / 32

[0046] Figure 3E: Illustrative representation of the protein-peptide linkage of pep5r_cpp, hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and labeled with the 1-letter symbol, followed by the position of the present residue in the primary structure.

[0047] Figure 4A: Molecular docking against ZIKV NS2B-NS3. a. Electrostatic representation of the surface of ZIKV NS2B-NS3. RNA and NS5 binding sites are highlighted.

[0048] Figure 4B: Illustrative drawing of the ZIKV NS2B-NS3 protein. The NS2B and NS3 domains (protease and helicase) are shown in blue, yellow, and red, respectively. The pep5_cpp, pep5r_cpp, and el28_cpp peptides are colored green, orange, and magenta, respectively.

[0049] Figure 4C: Illustrative representation of the protein-peptide linkage of pep5_cpp, hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and marked with the 1-letter symbol, followed by the position of the present residue in the primary structure.

[0050] Figure 4D: Illustrative representation of the protein-peptide linkage of el28_cpp, hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and marked with the 1-letter symbol, followed by the position of the present residue in the primary structure. Petition 870250060600, dated 07 / 15 / 2025, page 20 / 50 / 32

[0051] Figure 4E: Illustrative representation of the protein-peptide linkage of pep5r_cpp, hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and marked with the 1-letter symbol, followed by the position of the present residue in the primary structure.

[0052] Figure 5A: Molecular docking against ZIKV NS5. a. Electrostatic representation of the ZIKV NS5 surface. The central channel is highlighted.

[0053] Figure 5B: Illustrative drawing of the ZIKV NS5. The methyltransferase (MTase) and RNA polymerase (RdRp) domains — finger, palm, and thumb — are shown in cyan, blue, yellow, and red, respectively. The pep5_cpp, pep5r_cpp, and el28_cpp peptides are colored green, orange, and magenta, respectively.

[0054] Figure 5C: Illustrative representation of the protein-peptide linkage of pep5_cpp; hydrophobic, electrostatic, and hydrogen bonding interactions are marked with dashed pink, orange, and green lines. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and orange, respectively. Protein residues are shown as sticks and marked with a 1-letter symbol followed by the position of the residue in the primary structure.

[0055] Figure 5D: Illustrative representation of the protein-peptide linkage of el28_cpp; hydrophobic, electrostatic, and hydrogen bonding interactions are marked with dashed pink, orange, and green lines. Oxygen, nitrogen, and sulfur atoms are colored red, blue, and orange, respectively. Protein residues are shown as sticks and marked with a 1-letter symbol followed by the position of the residue in the primary structure. Petition 870250060600, dated 07 / 15 / 2025, page 21 / 50 / 32

[0056] Figure 5E: Illustrative representation of the protein-peptide linkage of pep5r_pp, hydrophobic, electrostatic and hydrogen bonding interactions are marked in dashed pink, orange and green lines. Oxygen, nitrogen and sulfur atoms are colored red, blue and orange, respectively. Protein residues are shown as sticks and marked with the 1-letter symbol, followed by the position of the present residue in the primary structure. DETAILED DESCRIPTION OF THE INVENTION

[0057] It should be noted preliminarily that the following description will be based on a preferred embodiment of the invention. As will become evident to anyone skilled in the art, however, the invention is not limited to this specific embodiment.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as understood by a person skilled in the art to which the invention pertains. The terminology used in the description of the invention is intended to describe particular embodiments only and is not intended to limit the scope of the teachings. Unless otherwise indicated, all numbers expressing quantities, percentages, and proportions, and other numerical values ​​used in the descriptive report and claims, should be understood as being modified, in all cases, by the term "about". Thus, unless otherwise indicated, the numerical parameters shown in the descriptive report and claims are approximations that may vary depending on the properties to be obtained.

[0059] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, recombinant DNA techniques and immunology, within the knowledge of the art. Such techniques are fully explained in the literature. See, for example, Fundamental Virology, 2nd Edition, vols. I & II (BN Fields and DM Petition 870250060600, dated 07 / 15 / 2025, p. 22 / 50 / 32 Knipe, eds.); Handbook of Experimental Immunology, Vols. I-IV (DM Weir and CC Blackwell eds., Blackwell Scientific Publications); TE Creighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); AL Lehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

[0060] As used throughout this application, the term “amino acid” denotes the α-amino acid group that directly or in the form of a precursor can be encoded by a nucleic acid. Individual amino acids are encoded by nucleic acids consisting of three nucleotides, known as codons or base triplets. Each amino acid is encoded by at least one codon. The fact that the same amino acid is coded by different codons is known as "degeneracy of the genetic code".The term “amino acid”, as used in this application, denotes the naturally occurring α-amino acids, comprising alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

[0061] The terms “peptide,” “polypeptide,” or “protein” may be used interchangeably, and refer to a polymer of amino acids connected by peptide bonds, regardless of the number of amino acid residues that constitute this chain. Polypeptides, as used herein, include “variants” or “derivatives” thereof, which refer to a polypeptide that includes variations or modifications, for example, substitution, deletion, addition, or chemical modifications in its amino acid sequence relative to the reference polypeptide. Examples of chemical modifications are glycosylation, G-alkylation, PEG-alkylation, phosphorylation, acetylation, amidation, etc. Polypeptides can be artificially produced from nucleotide sequences cloned using the technique Petition 870250060600, dated 07 / 15 / 2025, page 23 / 50 / 32 of recombinant DNA or it can be prepared through a known chemical synthesis reaction.

[0062] The term “cell-penetrating peptide” or “CPP” refers to short amino acid sequences that facilitate the entry of the parent peptide into the intracellular environment and thus act as a mode of peptide transport into the intracellular environment. The preferred CPP in the present application is defined by the sequence YGRKKRRQRRR. Also included in the context of the present patent application are known hydrophilic tails from the prior art, such as the TAT tail, SynB1, SynB3, PTD-4, PTD-5, DTat, R9-Tat, among others. Amphiphilic tails such as the MAP, SBP, FBP tails, among others, are also included.

[0063] In a first aspect, the present invention provides el28 and pep5 peptides and their variations, conjugated or not to the cationic peptide derived from the TAT protein (region 47-57) for use in the preparation of a pharmaceutical composition for the treatment of viral infections.

[0064] The terms “pep5-cpp” and “el28-cpp” refer to the synthesized pep5 and el28 peptides coupled to the cell penetrating peptide (cpp - cell penetrating peptide - sequence YGRKKRRQRRR, TAT 47-57), covalently linked to the C-terminal of the aforementioned peptide.

[0065] Pep5r represents the portion of minimal pharmacological activity of this peptide, composed of six amino acids (WELVVL).

[0066] In this sense, the peptides of the present invention comprise the following sequences: (i) SEQ ID NO: 1 pep5r-cpp (WELVVL-YGRKKRRQRRR); (ii) SEQ ID NO: 2 el28-cpp (VGSELIQKYYGRKKRRQRRR); (iii) SEQ ID NO: 3 cpp-el28 (YGRKKRRQRRRVGSELIQKY); (iv) SEQ ID NO: 4 el28 alone (VGSELIQKY); Petition 870250060600, dated 07 / 15 / 2025, p. 24 / 50 / 32 (v) SEQ ID NO: 5 pep5-cpp (WELVVLGKLYGRKKRRQRR); and (vi) SEQ ID NO: 6 pep5 alone (WELVVLGKL).

[0067] The term “treatment” refers to a method for relieving or eliminating a disease and / or its accompanying symptoms.

[0068] “Viral infections” are pathological disorders caused by viruses, microscopic infectious agents that invade host cells to replicate, and can affect different organisms and tissues. Broadly speaking, this term encompasses infections caused by various viral families, such as Flaviviridae (e.g., dengue virus, yellow fever virus, Zika virus), Orthomyxoviridae (such as influenza viruses), Retroviridae (including the human immunodeficiency virus - HIV), Herpesviridae (such as herpes simplex virus and Epstein-Barr virus), Adenoviridae, Coronaviridae (such as SARS-CoV-2), among others. Each family comprises viruses with distinct morphological, genomic characteristics and infection mechanisms, and can cause anything from acute and self-limiting infections to chronic and debilitating conditions.In the context of this patent application, although the term "viral infections" is used in its broad sense, the invention described herein is specifically directed to infections caused by ZIKV, an arbovirus of the Flaviviridae family, associated with clinical manifestations such as fever, rash, conjunctivitis and, in more severe cases, neurological complications and congenital anomalies.

[0069] In a first embodiment, the present invention discloses the use of one or more peptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, in the preparation of a pharmaceutical composition for the treatment of infection by a virus of the Flaviviridae family.

[0070] In a second embodiment, the present invention further reveals that the virus of the Flaviviridae family is the Zika virus. Petition 870250060600, dated 07 / 15 / 2025, p. 25 / 50 / 32

[0071] The present invention relates to a composition incorporating synthetic peptides with specific pharmacological properties, aimed at therapeutic applications. These peptides are obtained by chemical synthesis and may be modified at their N- and / or C-terminal ends to improve their stability, bioavailability, or affinity for biological targets. The concentration of peptides in the formulation may vary according to the intended application, being adjusted to optimize efficacy and minimize adverse effects (1uM to 150uM). The composition may include suitable pharmaceutical excipients, such as liposomes, biodegradable polymers, or controlled-release systems, to facilitate administration and sustained release of the peptide, as well as pharmaceutically acceptable vehicles.

[0072] The aforementioned compositions may also include buffers, such as neutral buffered saline solution, phosphate-buffered saline solution and the like; carbohydrates, such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0073] The compositions of the present invention are formulated for enteral and parenteral administration, such as oral, sublingual and rectal routes or intravenous, intramuscular, subcutaneous routes, among others.

[0074] The term “therapeutically effective amount” refers to the amount of the compound to be administered that is sufficient to prevent the development of, or alleviate to some extent, one or more of the symptoms of the condition or disorder being treated.

[0075] The term “pharmaceutically acceptable vehicle” refers to a non-toxic, inert, semi-solid liquid excipient, diluent, auxiliary formulation of any kind, such as saline solution, and water. Some examples of materials that can serve as pharmaceutically acceptable vehicles are sugars, such as lactose, glucose, and sucrose, and starches, such as starch of Petition 870250060600, dated 07 / 15 / 2025, page 26 / 50 / 32 corn and potato starch, cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate, cyclodextrin; oils, such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol, polyols, such as glyceryl glycol, sorbitol, mannitol and polyethylene; esters, such as ethyl laurate, ethyl oleate, agar; buffering agents, such as aluminum hydroxide and magnesium hydroxide; alginic acid; isotonic saline, Ringer's solution; Ethyl alcohol and phosphate buffer solutions, oily emulsion in water containing heat-killed mycobacteria or components of their cell wall (Freund's complete adjuvant), as well as other compatible non-toxic substances used in pharmaceutical formulations.

[0076] In a third embodiment, the present invention discloses a composition comprising a therapeutically effective amount of one or more peptides selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 and a pharmaceutically acceptable carrier.

[0077] In a fourth embodiment, the present invention further discloses that the composition can be used in the treatment of infection by a virus of the Flaviviridae family.

[0078] In a fifth embodiment, the present invention also reveals that the virus of the Flaviviridae family is the Zika virus. METHODOLOGY

[0079] Peptide synthesis: Peptides were synthesized (Shigeri Y, et al 2001 and Borgia Já., et al. 2000) coupled or not to a cell-penetrating peptide (cpp: YGRKKRRQRRR; TAT47-57) at its C or N terminus (de Araujo, 2014; Monte et al., 2017). All peptides showed purity >95%, confirmed by mass spectrometry and liquid chromatography. Petition 870250060600, dated 07 / 15 / 2025, page 27 / 50 / 32, refers to high-efficiency electrolytic capacitors (HPLC), and were acquired from Proteimax Biotecnologia LTDA, São Paulo, Brazil.

[0080] The peptides were diluted in Milli-Q water and stored at -80 °C. Tests were performed in fetal bovine serum (FBS)-free medium at concentrations ranging from 1 to 150 μM.

[0081] Antiviral potential: VERO cells (CCL-81, ATCC) cultured in VP medium (Thermo Scientific) were inoculated at 200 μL / well at a concentration of 5.0E+04 cells / mL in 96-well plates and incubated at 37°C with a 5% CO2 atmosphere. After 48 hours of growth, the wells reached 80% confluence and the test was performed. Initially, the culture medium was replaced with 150 μL of VP in columns 2 to 10.

[0082] The antivirals were diluted to 100 μM in VP medium and placed in column 1 (300 μL). Serial dilutions from 1 to 10 were performed by passing 150 μL of medium (1:2). The plates were incubated at 37°C for 2 hours. The Zika virus seed was previously described (Oliveira et al., 2018).

[0083] It was established as a working virus seed (WVS) and for each test was diluted in VP medium to the desired final infection rate of MOI 0.1 in 100 μL of VP and added to the wells. Plates were incubated for 96 hours and checked every 24 hours for cytotoxicity or cytopathic effect. Plates were washed (PBS pH 7.2) and stained with Naphthol Blue-Black solution (0.1% Naphthol Blue Black, 1.6% sodium acetate, 6% acetic acid) for 30 minutes.

[0084] After that, the staining solution was removed and the wells were rinsed with water. Absorbance was measured at 450 nm in a microplate reader (Multiskan EX 355 Thermo Scientific, MA, USA). The average absorbance (abs) values ​​of each peptide dilution and analytical triplicates of infected and uninfected controls were used to calculate the infection inhibition rate for each experiment, defined by Equation (1): % / níb íção = Average dilution of peptide absorption — Average absorption control of infected cells — Average absorption control of uninfected cells — Average absorption control of infected cells Petition 870250060600, dated 07 / 15 / 2025, page 28 / 50 / 32

[0085] Inhibition rates were plotted against the respective peptide concentration, and the resulting curve was used to calculate EC50 (effective concentration that inhibits 50% of the infection). The three EC50 values ​​obtained were expressed as mean ± standard deviation. Each round of antiviral analysis accounted for 36 uninfected wells (simulation), which were used to establish the mean absorbance of 100% viable cells, and 42 infected wells (positive control), which were used to establish the mean absorbance of 100% infection. The infection kinetics were previously carefully studied to establish MOI, TOI, and experimental duration, resulting in a readable signal for the 100% infection control, since ZIKV has lytic behavior and tends to generate loose adherent cells at late infection times.

[0086] Cytotoxicity analysis: Cytotoxicity was assessed by incubating serial dilutions of the peptides with Vero cells under the same conditions used in the antiviral experiments. After 96 h, the number of cells was indirectly assessed by reading the absorbance at 450 nm after performing the staining protocol.

[0087] Cytotoxicity was defined as the ratio between the mean absorbance of wells treated with the peptide dilution and the mean absorbance of untreated wells (100% viable cells).

[0088] The CC50 (cytotoxic concentration for 50% of cells) was defined as the lowest cytotoxic concentration, i.e., the lowest dose that caused a decrease in cell viability, represented by an absorbance lower than the expected value considering the standard deviation of the absorbance of the untreated wells.

[0089] Molecular docking: To gain insights into anti-ZIKV activity, amino acid sequences of the structural and non-structural proteins E, NS1, NS2B-NS3, and NS5 of ZIKV were first collected from UniProt (Q32ZE1) (UniProt Consortium, 2025). In Petition 870250060600, dated 07 / 15 / 2025, page 29 / 50 / 32. Subsequently, the three-dimensional structures of the ZIKV targets were predicted using the AlphaFold3 software (Abramson et al., 2024). Then, molecular docking simulations were performed to evaluate the binding affinity of the peptides against the ZIKV target proteins using HEPDOCK v. 2.0 (Zhou et al., 2018). The interactions were visualized in Discovery Studio Visualizer v. 24.1.0.23298 (BIOVIA, 2024) and PyMOL v. 2.5.8 (Schrodinger, Inc., NY, USA), and the electrostatic surface was determined using the APBS Electrostatics plugin.

[0090] Prediction of antiviral activity and mechanism of action: An Artificial Intelligence approach was used in conjunction with the identification and classification of antiviral peptides (AVPs). The methodology integrates a multi-layered bidirectional Long Short-Term Memory (LSTM) neural network and a Random Forest (RF) classifier, combining their predictions to increase accuracy (Abukawa, FM et al., 2025, submitted to Bioinformatics).

[0091] The LSTM model processes peptide sequences in multiple layers and neurons in a bidirectional manner, while the RF model classifies peptides based on physicochemical properties. Both models were trained with a negative and positive set of 1,695 experimentally validated peptides and independently optimized, with final predictions determined by a weighted combination of their results.

[0092] The predicted AVPs were classified into three categories for mechanism of action — membrane-acting agents, replication inhibitors, and viral assembly destabilizers — using a machine learning method, Support Vector Machine (SVM) with a One-vs-All classification scheme. A curated dataset containing 1,399 AVPs was used for training, with class imbalance addressed using the SMOTE (Synthetic Minority Oversampling) technique. Petition 870250060600, dated 07 / 15 / 2025, page 30 / 50 21 / 32 (Technique). Feature extraction included physicochemical and compositional descriptors, and model optimization was performed by cross-validation.

[0093] This methodology was applied to predict new AVPs and classify their mechanisms of action, which were synthesized and validated through experimental trials. The integration of computational and experimental approaches demonstrated the model's effectiveness in identifying promising AVP candidates with potential therapeutic applications. EXAMPLES Example 1

[0094] Evaluation of cell viability and anti-ZIKV activity: It was observed that the peptides inhibited ZIKV multiplication at different concentrations and to different extents (Table 1). Table 1. Antiviral activity of peptides against ZKV. Antiviral and cytotoxicity assays were performed using peptide dilutions to treat Vero cells 2 h before ZKV infection. The effective dose (EC50) and cytotoxic dose (CC50) for 50% of the cell culture were calculated. The selectivity index (SI) represents the ratio between CC50 / EC50, describing the window between cytotoxicity and antiviral activity. The higher the SI, the safer the compound. Sample Maximum Infection Inhibition (%) ECso (μM) CCso (μM) SI iPP 82.4 3.88 50 12.89 cpp_el28 65.69 23.42 100 4.27 el28_cpp 75.62 24.25 100 4.12 el28 53.24 49.18 100 2.03 pep5r cpp 83.23 3.26 25 7.68

[0095] The peptides showed low overall toxicity to cells, exhibiting cytopathic effects in short periods after treatment and infection. This behavior is frequently characteristic of Petition 870250060600, dated 07 / 15 / 2025, p. 31 / 50 / 32 cytotoxicity attributed to the vehicle. Higher concentrations of the starting material will be needed to better assess the actual toxicity of the compounds. However, antiviral activity was evident for the five peptides tested, as they successfully inhibited the cytopathic effects of ZKV, especially cpp. Example 2

[0096] Molecular docking simulations: The present invention demonstrated that the pep5r-cpp peptide bound to the E protein near the host receptor binding site (Fig. 1). In parallel, it was observed that the pep5-cpp and el28-cpp peptides bound to the “stem” region of the E protein (Fig. 1), which is relevant for viral assembly and membrane fusion in flaviviruses (Allison et al., 1999; Zhang et al., 2003; Mukhopadhyay et al., 2005; Klein et al., 2013, Jablunovsky A et al., 2024).

[0097] The present invention demonstrated that peptides bind to the “wing” domains and interact with residues near the membrane anchoring region of the NS1 dimer (Fig. 2). Furthermore, it was observed that binding to the central channel of the NS1 hexamer (Fig. 3), involved in structural stability via interaction with lipids (Gutsche et al., 2011; Shu et al., 2022), occurred.

[0098] The present invention demonstrated that the peptides pep5r-cpp and el28-cpp interact with the helicase domain at the RNA binding site (Fig. 4) (Jain et al., 2016; Tian et al., 2016), while pep5-cpp binds to the helicase domain at the NS3-NS5 interface (Fig. 4), a region functionally involved in viral replication (Tay et al., 2015; Xu et al., 2019).

[0099] The present invention has demonstrated that pep5_cpp binds to the central channel near the active site of NS5, pep5r_cpp binds to the template channel, and el28_cpp interacts with residues of the NS3-NS5 interface (Fig. 5). Example 3

[00100] Prediction of antiviral peptides and classification of the mechanism of action: Table 2 presents the predicted probability scores for peptides to be classified as antiviral peptides (AVPs), together Petition 870250060600, dated 07 / 15 / 2025, page 32 / 50 / 32 with its respective classification into one of three mechanisms of action: membrane targeting, replication inhibition, or assembly disruption. The probability scores for each mechanism of action indicate the likelihood of the peptide functioning through that specific antiviral mechanism. Table 2. Results and probabilities obtained from the EnAVPClass prediction for the peptide to be an AVP based on the highest score (%) and the probability split across the three mechanisms of action. ID SCORE CLASS Membrane Replication Assembly Sequence el28-cpp 0.97559 Membrane 0.756 0.241 0.003 VGSELIQKYYG RKKRRQRRR el28 0.80046 Membrane 0.785 0.215 0 VGSELIQKY cpp-el28 0.97561 Membrane 0.737 0.26 0.003 YGRKKRRQRR RVGSELIQKY pep5r-cpp 0.97692 Replication 0.431 0.565 0.004 WELVVLYGRK KRRQRRR pep5-cpp 0.97831 Replication 0.438 0.556 0.006 WELVVLGKLY GRKKRRQRRR

[00101] The high AVP prediction scores (above 80%) indicate strong confidence in the antiviral activity of these peptides. The classification results suggest that el28-cpp, el28, and cpp-el28 function primarily by interacting with viral membranes, while pep5r-cpp acts as a replication inhibitor, corroborated by molecular docking simulations against specific proteins. The minimum probability for the Assembly category reinforces that it is unlikely that these peptides act through this mechanism. Petition 870250060600, dated 07 / 15 / 2025, page 33 / 50 / 32 References

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Claims

CLAIMS 1. Use of one or more peptides selected from the group consisting of SEQ ID NO: 1 to 6, characterized by being in the preparation of a pharmaceutical composition for the treatment of infection by a virus.

2. Use in accordance with claim 1, characterized in that the virus is from the Flaviviridae family.

3. Use according to claim 1 or 2, characterized in that the virus of the Flaviviridae family is the Zika virus.

4. Composition, characterized in that it comprises a therapeutically effective amount of one or more peptides selected from the group consisting of SEQ ID NO: 1 to 6 and a pharmaceutically acceptable vehicle.

5. Composition according to claim 4, characterized by being for use in the treatment of infection by a virus of the Flaviviridae family.

6. Composition according to claim 5, characterized in that the virus of the Flaviviridae family is the Zika virus. Petition 870250060600, dated 07 / 15 / 2025, pp. 43 / 50

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