Antifungal oligopeptide compounds and their use

Abstract

The present invention relates to antifungal oligopeptide compounds according to Formula I, Asp–R2–Gly–Leu–R5–R6–R7–Leu–Gly–R10–R11–R12 (Formula I), wherein R2 represents Arg, Lys, Cit, D-Arg or Orn; R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3-phenylpropionate; R6 represents Asp, Pro, Leu, Glu or Lys; R7 represents Phe or Trp; each of R10, R11, and R12 independently represents Lys, Arg or Orn; that are effective in therapeutic and research applications related to human pathogenic fungi, particularly yeasts Candida albicans and Cryptococcus neoformans. Additionally, the invention covers the use of these antifungal oligopeptides in the treatment of fungal infections, particularly those affecting the central nervous system (CNS) – by the administration of a therapeutically effective amount of any of these oligopeptide compounds.

Description

[0001] ANTIFUNGAL OLIGOPEPTIDE COMPOUNDS AND THEIR USE

[0002] The present invention relates to antifungal oligopeptide compounds that are effective in therapeutic and research applications related to human pathogenic fungi, particularly yeasts Candida albicans and Cryptococcus neoformans. Additionally, the invention covers the use of these antifungal oligopeptides in the treatment of fungal infections, particularly those affecting the central nervous system (CNS) - by the administration of a therapeutically effective amount of any of these oligopeptide compounds.

[0003] TECHNICAL BACKGROUND

[0004] Fungal infections pose a significant challenge in clinical settings. These infections can range from superficial skin conditions to life-threatening systemic diseases. The treatment of fungal infections often relies on a limited number of antifungal drugs, many of which have been in use for decades. However, the efficacy of these drugs is increasingly compromised by the rising resistance of fungal pathogens. Additionally, many traditional antifungal agents are associated with high toxicity, thus, their use is limited, especially in vulnerable populations such as immunocompromised patients. Currently used antifungal drugs in clinical practice have limited ability to cross the blood-brain barrier (BBB), making the treatment of such infections challenging and associated with high mortality rates. In light of these considerations, there is an urgent need for novel antifungal compounds that are not only effective and have good BBB penetration ability but also exhibit a lower toxicity profile, thereby ensuring greater safety for clinical use (Galgoczy L., ACS Bio Med Chem Au. 2025, 5, 531-552).

[0005] To enhance public awareness and to address this critical issue, the World Health Organization (WHO) released its inaugural statement on fungal infections in 2022, accompanying the publication of the WHO fungal priority pathogens list (WHO Fungal Priority Pathogens List To Guide Research, Development and Public Health Action; World Health Organization, 2022). This document serves as a strategic framework for guiding research, development, and public health interventions. It lists the fungal pathogens that present the greatest risk to public health and delineates key objectives for scientific inquiry, therapeutic advancements, and policy implementation. The statement underscores the pressing necessity of mitigating fungal infection spread and combating antifungal resistance through the development of innovative treatment strategies, employing fundamentally distinct antifungal agents with novel fungus-specific mechanisms of action. Therefore, the development of new medicinal products is a key priority in this area, especially against those human pathogenic yeasts that are included in the critical group of the WHO fungal priority pathogens list, such as Candida albicans, Candida auris. Cryptococcus neoformans, which are known as causative agents of central nervous system (CNS) infections (Galgoczy L., ACS Bio Med ChemAu. 2025, 5, 531-552).

[0006] In the search for new antifungal therapeutic strategies, peptides have emerged as promising candidates due to their broad-spectrum activity, relatively low toxicity, and good BBB penetration ability. Peptides can be designed to target specific components of fungal cells, thereby reducing the likelihood of resistance development. Despite their potential, the development of peptide-based antifungal agents has been hampered by challenges such as stability, delivery, and production costs. Advances in peptide synthesis and formulation technologies are now making it feasible to explore and develop new peptide-based antifungal agents that could address these challenges and provide more effective treatments for fungal infections.

[0007] Peptides have a wide range of applications. One notable example is their potential as therapeutic agents in drug development. International patent application W02024031022A2 describes oligopeptide compounds with various combinations of natural and non-natural amino acids. It involves peptidomimetics with specific structural formulas. The patent mentions the use of oligopeptides in methods of preventing photoreceptor death and protecting of retinal cells.

[0008] The use of peptides as antifungal agents has been mentioned in a Chinese patent application CN113603749A discloses an antifungal peptide KS-16 effective against plant pathogenic fungi.

[0009] The Japanese patent application publ. no. JPH11106399A describes a novel peptide derivative having potent antifungal properties, particularly useful for treating severe fungal infections such as invasive candidiasis.

[0010] In a recent study, Sebak et al. (ACS Omega 2021, 6, 34470-34484) describes the synthesis of novel cell penetrating peptides (CPPs), i. e. , ERD-A, ERD-B and ERD-C peptides and publish experimental data on the potential of these peptides for delivering small -molecule organic drugs into cells, as well. These lysine-rich peptides are derived from the plant dehydrin ERD14. These peptides were investigated for their potential to facilitate drug delivery, particularly for cancer treatment. Carboxyfluorescein (Cf) labelled peptides, prepared via the conventional synthetic route, were used to monitor their cellular internalization. Furthermore, the impact of (5)-Cf and (6)-Cf conjugation on the structural properties of the peptides was systematically investigated. The results show that the peptides enter cells through energy -dependent pathways with localization in different cellular compartments (e.g., cytosol, lysosomes, and vesicles). Specific amino acids play a critical role in peptide function; notably, aromatic side chains and proline residues have been shown to influence both cellular internalization and efficacy. The plant- derived peptides offer potential advantages over animal -derived counterparts, including reduced allergenicity and cytotoxicity. However, these oligopeptide compounds have not yet been evaluated for antifungal activity.

[0011] Ohkubo T. et al. (Phytochemistry 2020, 173, 112300) describe that highly conserved F- segments of Arabidopsis dehydrins show cryoprotective activities.

[0012] Rosales R. et al. (Phytochemistry 2014, 108, 17-25) highlight the crucial role of O- and K- segments in Vitis vinifera dehydrin DHNla in protecting against various abiotic stresses and their antifungal activity against the fungal pathogen Botrytis cinerea.

[0013] Drida M. et al. (Appl Biochem Biotechnol. 2015, 175, 3310-3321) reveal that wheat dehydrin DHN-5 and its recombinant truncated forms containing two K-segments have antibacterial and antifungal activities. They demonstrated that the inhibition of bacterial and fungal growth is linked to ly sin-rich K-segments.

[0014] However, the antifungal activities of oligopeptides comprising or derived from the F- segment of dehydrins with fundamentally different amino acid sequences have not yet been disclosed.

[0015] Bearing in mind the significant demand for novel antifungal oligopeptide compounds, there is a clear need for agents that can be used safely and effectively in clinical settings due to their fungus-specific mode of action.

[0016] The purpose of the present invention is to provide a new type of antifungal oligopeptides characterized by high efficacy and enhanced safety, suitable for therapeutic application.

[0017] SUMMARY OF THE INVENTION

[0018] Accordingly, the object of the invention is an antifungal oligopeptide compound according to Formula I,

[0019] Asp-R2-Gly-Leu-R5-R6-R7-Leu-Gly-R10-Rl 1-R12 (Formula I) wherein

[0020] R2 represents Arg, Lys, Cit, D-Arg or Orn;

[0021] R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3 -phenylpropionate;

[0022] R6 represents Asp, Pro, Leu, Glu or Lys; R7 represents Phe or Trp; each of RIO, R11, and R12 independently represents Lys, Arg or Orn; for use in therapy.

[0023] Preferably the antifungal oligopeptide compound for use is an oligopeptide compound, wherein

[0024] R2 represents Arg, Cit or D-Arg;

[0025] R5 represents Phe, Arg or Trp;

[0026] R6 represents Asp or Pro;

[0027] R7 represents Phe or Trp; each of RIO, R11, and R12 independently represents Lys or Arg.

[0028] More preferably the oligopeptide compound is one of the following oligopeptides: Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A; SEQ ID NO: 1), an oligopeptide compound which differs in up to 3 amino acid residues from ERD-A, particularly

[0029] Asp-Arg-Gly-Leu-Phe-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A D6P; SEQ ID NO:2), Asp-Arg-Gly-Leu-Trp-Asp-Trp-Leu-Gly-Lys-Lys-Lys (ERD-A F5,7W; SEQ ID NO:3), Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Arg-Arg-Arg (ERD-A K10,l l,12R; SEQ ID NO:4)

[0030] Asp-Cit-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A R2Cit; SEQ ID NO: 5);

[0031] Asp-Arg-Gly-Leu-Arg-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A F5R D6P; SEQ ID

[0032] NO: 6);

[0033] Asp-Arg-Gly-Leu-Trp-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A F5W; SEQ ID NO: 7); or Asp-D-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A R2D-Arg; SEQ ID NO:8).

[0034] In one embodiment, the antifungal oligopeptide compounds are for use in the treatment or prevention of a fungal infection or a disease related to a fungal infection, particularly those affecting the CNS, preferably wherein the fungal infection is caused by a human pathogenic species, particularly an infection of a human pathogenic yeast, more particularly an infection caused by Candida spp., particularly Candida albicans or Candida auris. or an infection of Cryptococcus spp., particularly Cryptococcus neoformans. Moreover, the oligopeptide compounds are intended for use in the treatment of CNS fungal infections, as they exhibit favourable BBB penetration abilities. In a further embodiment, the invention relates a pharmaceutical composition comprising at least one antifungal oligopeptide compound as defined above and a pharmaceutically acceptable carrier, diluent and / or excipient, for use in therapy.

[0035] Preferably the said therapy is the treatment or prevention of a fungal infection or a disease related to a fungal infection, particularly those affecting the CNS, more particularly wherein the fungal infection is a human pathogenic infection, more particularly an infection caused by a human pathogenic yeast, more particularly an infection of a Candida spp., particularly Candida albicans or Candida auris. or an infection of Cryptococcus spp., particularly Cryptococcus neoformans.

[0036] In a further embodiment, the object of the present invention is an antifungal oligopeptide compound according to Formula I,

[0037] Asp-R2-Gly-Leu-R5-R6-R7-Leu-Gly-R10-Rl 1-R12 (Formula I) wherein

[0038] R2 represents Arg, Lys, Cit, D-Arg or Om;

[0039] R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine, or 3 -pheny Ipropi onate ;

[0040] R6 represents Asp, Pro, Leu, Glu or Lys;

[0041] R7 represents Phe or Trp; each of RIO, R11, and R12 independently represents Lys, Arg or Orn; provided that the oligopeptide compound is not Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu- Gly-Lys-Lys-Lys (ERD-A).

[0042] In a preferred embodiment, the oligopeptide compound is an oligopeptide compound, wherein

[0043] R2 represents Arg, Cit, or D-Arg;

[0044] R5 represents Phe, Arg or Trp;

[0045] R6 represents Asp or Pro;

[0046] R7 represents Phe or Trp; each of RIO, R11, and R12 independently represents Lys or Arg.

[0047] Preferably the oligopeptide compound is one of the following oligopeptides:

[0048] Asp-Arg-Gly-Leu-Phe-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A D6P; SEQ ID NO:2), Asp-Arg-Gly-Leu-Trp-Asp-Trp-Leu-Gly-Lys-Lys-Lys (ERD-A F5,7W; SEQ ID NO:3), Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Arg-Arg-Arg (ERD-A K10,l l,12R; SEQ ID Asp-Cit-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD- A R2Cit; SEQ ID NO: 5); Asp-Arg-Gly-Leu-Arg-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A F5RD6P; SEQ ID NO: 6);

[0049] Asp-Arg-Gly-Leu-Trp-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A F5W; SEQ ID NO: 7); and

[0050] Asp-D-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A R2D-Arg; SEQ ID NO:8).

[0051] The invention comprises oligopeptide compounds as promising antifungal agents.

[0052] BRIEF DESCRIPTION OF THE FIGURE

[0053] Figure 1. ERD-A binding / uptake of Candida albicans CBS 5982 cells in low cationic medium (LCM). ERD-A was labelled with 5- and 6-carboxyfluorescein, resulting in (5)-Cf- ERD-A and (6)-Cf-ERD-A conjugates. White arrows indicate Candida albicans cells that bind or internalize (5)-Cf-ERD-A or (6)-Cf-ERD-A. Scale bars: 20 pm.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] Abbreviations

[0056] C or Cys Cysteine

[0057] D or Asp Aspartic acid

[0058] E or Glu Glutamic acid

[0059] F or Phe Phenylalanine

[0060] G or Gly Glycine

[0061] H or His Histidine

[0062] K or Lys Lysine

[0063] L or Leu Leucine

[0064] P or Pro Proline

[0065] Q or Gin Glutamine

[0066] R or Arg Arginine

[0067] W or Trp Tryptophan

[0068] Y or Tyr Tyrosine

[0069] Cit Citrullin

[0070] Orn Ornithine Definitions

[0071] Antifungal (effect): the observable outcome of a substance, treatment, or process that inhibits the growth of fungi, damages their structure, or kills them outright. In the context of the experimental systems described herein, a peptide was considered antifungally effective (in in vitro antifungal susceptibility assays) if it completely inhibited fungal growth at a concentration of <100 pg / ml in low cationic medium. In RPMI-1640 medium, antifungal activity was defined as a measurable growth inhibition expressed as a percentage relative to the untreated control (100% growth).

[0072] ERD14: ERD14 is a 185 residue-long, intrinsically disordered plant stress protein from Arabidopsis thaliana that acts as a chaperone, preventing heat-induced aggregation and / or inactivation of various substrates.

[0073] ERD-A: arbitrarily selected Lys-rich region of ERD14 protein with amino acid sequence is: Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (D R G L F D F L G K K K) (SEQ ID NO: 1).

[0074] MIC (Minimum Inhibitory Concentration): the lowest concentration of a chemical, usually a drug, which prevents the visible in vitro growth of bacteria or fungi. According to this definition, the MIC is defined as the lowest peptide concentration capable of reducing fungal growth to <5% relative to the untreated control, as determined by absorbance measurements. The absorbance of the untreated control is considered 100%, serving as the reference point for assessing the inhibitory effect of the peptide.

[0075] BBB (blood-brain barrier) penetration ability: refers to a capacity of a compound to cross the BBB and reach the brain tissue in therapeutically relevant concentrations. According to this definition, an oligopeptide is considered to exhibit good blood-brain barrier (BBB) penetration ability if its cumulative apparent permeability coefficient and / or translocation percentage exceeds that of the reference peptide, opiorphin.

[0076] Description

[0077] Investigations have been conducted on plant dehydrins or plant dehydrin-derived phytopeptides in accordance with the invention, with the aim of synthesizing and characterizing them as potential drug candidates for drug delivery. The results of these investigations have revealed that these phytopeptides are not only candidates for the previously mentioned objective, but also exhibit antifungal activity, a phenomenon that had not been predicted prior to this discovery. The antifungal efficiency of these oligopeptide compounds exceeds the initial expectations.

[0078] It is important to note, that these oligopeptides are not cytotoxic on human cells, even in high concentrations (up to 200pM) (Sebak F., et al. ACS Omega 2021, 6, 34470-34484). This feature is really unique, because most CPPs are highly cytotoxic in vitro in such concentration (z.e., penetration is cytotoxic in amounts >10pM). Considering the necessity for effective antifungal agents in therapeutic applications, these materials demonstrate considerable potential for development into medicinal products.

[0079] According to the invention, oligopeptide sequences used are derived from plant dehydrin ERD14. ERD14 dehydrin is a 185 -residue-long, disordered plant stress protein, derived from Arabidopsis thaliana and contains several conserved segments. The lysine-rich K-segments are responsible for chaperone activity and cell viability under stress conditions. In the present invention, one of the lysine-rich peptide fragments designated as ERD-A and its variants were prepared and used as antifungal agents.

[0080] Oligopeptides can be synthesized rapidly and in large amounts via established and commonly applied solid-phase peptide synthesis methods.

[0081] As demonstrated in our previous study (Sebak F., et al. ACS Omega 2021, 6, 34470-34484) the internalization of ERD-A oligopeptide is energy-dependent and most likely occurs via active transport. This could be also a receptor-mediated route.

[0082] Therefore, the peptide sequence that binds to the receptor can be modified with amino acids or groups that do not interfere with the binding, and can positively affect the antifungal activity, physicochemical properties and structural stability of ERD-A. This process allows the enhancement of water solubility, stability of the oligopeptide within the bloodstream and improve the BBB penetration property. Modifications to the functional groups can effectively delay its elimination via the kidney, or reduce its accumulation in the kidney. Additionally, oligopeptide compounds, due to their low molecular weight and minimal structural complexity, typically do not elicit an immune response.

[0083] The present invention also provides variants of ERD-A oligopeptide exhibiting antifungal activity.

[0084] ERD-A variants may include amino acid substitutions or mutations that do not significantly alter their biological properties, such as antifungal activity. For instance, conservative amino acid substitutions are well established in the field and unlikely to disrupt functional integrity. However, the disclosed ERD- A variants preferably contain mutations that confer enhanced efficacy and / or improved physicochemical properties compared to ERD-A, making them particularly suitable for application in antifungal therapy, especially in the treatment of central nervous system CNS fungal infections.

[0085] The variants of oligopeptide according of Formula I may contain 1, 2, 3, 4, 5, 6 or 7 amino acid substitutions, preferably not more than 3 substitutions.

[0086] The following conceptions were taken into account when designing antifungal variants of the ERD-A peptide.

[0087] We introduced mutations that affect the variation of the total molecular charge (Lys, Glu, Asp), influence the membrane binding and (BBB) penetrating property, and also mutations that impact the length and polarity of the charged amino acid residue (Leu, Glu). Pro increases conformational heterogeneity and prevents the formation of secondary structural propensities. Substituting amino acids to Lys, Glu, or Asp generally reduces BBB penetration. Substitution with Pro enhances brain retention; however, it does not consistently improve BBB permeability (z.e., higher cumulative apparent permeability coefficients and translocation percentage than that of the parental ERD-A peptide; see Table 3). These amino acid substitutions can increase the antifungal effectiveness of an antifungal peptide and broaden its antifungal spectrum.

[0088] The mutations can fine-tune membrane interaction strength by using various amino acid residues with different aromatic side chains. Arg, Lys, hydrophilic, polar residues also contribute through increased charge, while Trp, Tyr, Phe enhance hydrophobicity; which can improve the antifungal activity and broaden the antifungal spectrum. Substitutions with Arg or Lys hinder passive diffusion across BBB. Unless the peptide is actively transported or chemically modified for targeted delivery, such substitutions typically reduce CNS accessibility. In contrast, incorporation of Trp, Tyr, or Phe may enhance BBB penetration.

[0089] The antifungal oligopeptides need to maintain a constant positive charge, to maintain or increase the antifungal effectiveness. Arg retains its zwitterion structure over a wider pH range than Lys. Thus, an Arg^Lys substitution results in a stronger antifungal effect, but generally reduces BBB penetration. However, this engineered mutation increases the peptide uptake by other cells, selectivity and solubility. Also facilitates the peptide pore forming ability on fungal membranes, and therefore broaden the antifungal spectrum.

[0090] Since Trp among the amino acids with aromatic side chains tends to interact more strongly with the membrane, it can be preferable to substitute phenylalanine for tryptophan (Phe^Trp). This is indirectly demonstrated by the stronger antifungal activity of the Trp variants (z.e., lower MIC; see Table 1). Substituting an amino acid to Trp can enhance blood-BBB penetration (z.e., higher cumulative apparent permeability coefficients and translocation percentage than that of the parental ERD-A peptide; see Table 3).

[0091] Pro provides greater structural heterogeneity due to the cis / trans isomerism. A stronger antifungal effect was achieved by the D^P substitution. The presence of the amphypatic Pro instead of D (Asp) increases cellular uptake and fungal selectivity and the antifungal effectiveness; however, it does not consistently improve BBB permeability (z.e., higher cumulative apparent permeability coefficients and translocation percentage than that of the parental ERD-A peptide; see Table 3).

[0092] Furthermore, D-amino acids have stabilizing effect and, in the three-dimensional structure, the charge is relocated, allowing for the collection of additional structural information during a research and therapeutic application. Substituting amino acids to D-amino acids can enhance BBB penetration in some cases.

[0093] Cit is used to replace arginine to study the effect of changes in the charge of the amino acid sequence. Cit also acts as a more efficient precursor to arginine, as contrary to arginine does not show pre-systemic degradation in the gut and liver. Citrulline substitution can reduce antifungal potency by weakening membrane interactions, but it may also enhance stability and selectivity, especially in serum-rich environments. Substituting an amino acid to citrulline may have a neutral or slightly favorable effect on BBB penetration.

[0094] Orn can be considered an indirect way to increase arginine levels. This is due to the fact, that L-omithine can be metabolized to L-arginine in the body. Orn substitution can preserve or even enhance antifungal activity, while potentially improving selectivity, stability and protease resistance. However, substituting an amino acid to ornithine generally reduces BBB penetration.

[0095] His substitution can provide targeted delivery, low toxicity, and stability under acidic conditions. Substituting an amino acid to His has a moderate and context-dependent effect on BBB penetration.

[0096] 4-aminophenylalanine can boost stability and hydrophobicity. Substituting an amino acid to 4-aminophenylalanine may reduce BBB penetration compared to its parent residue, Phe.

[0097] 3 -phenylpropionate can boost membrane activity and stability, and compromise selectivity and charge balance. Substituting an amino acid to 3 -phenylpropionate may enhance BBB penetration, depending on the overall peptide structure. EXAMPLES

[0098] Example 1: Peptide synthesis

[0099] The designed oligopeptides were synthesized using solid-phase peptide synthesis, applying the Fmoc / tBu strategy using a CEM Liberty Blue microwave-assisted fully automated peptide synthesizer. The crude products were purified using a RP-18 HPLC on a PerfectSil 100 ODS-3 5 pm (250 x 10 mm) column.

[0100] The homogeneity and purity were verified by HPLC. MS and NMR measurements identified and clarified the solution structure of the compounds. In all cases, the measured masses coincided with the calculated value and a purity of at least 97% was detected.

[0101] The following oligopeptides were synthesized:

[0102] ERD-A, ERD-A D6P, ERD-A F5,7W, ERD-A K10,l 1,12R, ERD-A R2Cit, ERD-A F5R

[0103] D6P, ERD-A F5W and ERD-A R2D-Arg.

[0104] Example 2: Biophysical properties, structural characterization, and cellular uptake of the synthesized compounds - Cellular internalization, cell viability, intracellular localization

[0105] In the context of our previously conducted research, the cell penetration abilities of the ERD oligopeptides on A431 skin squamous cell carcinoma cell lines were already characterized (Sebak F., et al. ACS Omega 2021, 6, 34470-34484). The calculated LogP values, obtained using the Chemicalize online platform, indicate that ERD-A fragment is hydrophilic, and is predominantly localized within the cytoplasm.

[0106] The uptake mechanism of the studied peptides primarily involves energy-dependent pathways, which can be chemically inhibited. At low peptide concentration, endocytosis is the predominant route of internalization; however, when Cf-peptides are applied at higher concentrations, they are capable of directly translocating across the plasma membrane. The intracellular localization of the Cf-peptides also indicates that, following internalization, they predominantly traffic through endocytic pathways toward late endosomes and lysosomes, where they may undergo degradation within a lysosomal compartment.

[0107] The intracellular fate of plant-derived cationic peptides is highly sensitive to changes in their amino acid sequence. These peptides exhibit favourable safety profiles, as they are non- cytotoxic and do not exert cytostatic effects.

[0108] Example 3: Antifungal susceptibility test - Antifungal properties An in vitro microdilution susceptibility test was applied to determine the antifungal activity of ERD-A, and its amino acid substituted variants (ERD-A D6P, ERD- A F5,7W, ERD-A K10,l l,12R, ERD-A R2Cit, ERD-A F5R D6P, ERD-A F5W and ERD-A R2D-Arg) against human pathogenic yeasts (Candida albicans WO1, Candida albicans SC5314, Candida auris 0381, and Cryptococcus neoformans IFM 5844) and a The susceptibility tests were conducted in a low cationic medium [(LCM) 5 g / 1 D-glucose, 0.25 g / 1 yeast extract, 0,125 g / 1 peptone] for indicating the potential antifungal effect of peptides (Varadi G., et al. ACS Omega, 2024, 9, 7206-7214). Briefly, 100 pl peptide (0.39-400 pg / ml in twofold serial dilutions in LCM) was mixed with 100 pl of 2 * 105 / ml mid-log phase yeast cells in a flat-bottom 96-well microtiter plate (TC Plate 96 Well, Suspension, F; Sarstedt, Numbrecht, Germany) in LCM. A mixture of 100 pl LCM without peptide and 100 pl of cell suspension served as the untreated growth control, whereas 200 pl LCM was used for background calibration. The microtiter plates were incubated statically for 48 h at 30°C. Following a 5-second shaking period, the absorbance (OD620) of each well was determined using a microtiter plate reader (SPECTROstar Nano; BMG Labtech, Ortenberg, Germany). The absorbance of the untreated control was taken as 100% growth for the minimum inhibitory concentration (MIC) calculation. MIC was defined as the lowest peptide concentration that reduced fungal growth <5% in comparison to the growth observed in the untreated control. Antifungal susceptibility test was repeated with ERD- A in RPML1640 medium (Sigma-Aldrich, St Louis, MO, USA) against Candida albicans CBS 5982, Cryptococcus neoformans IFM 5844, and Cryptococcus neoformans IFO 410. RPML 1640 is a standard medium recommended for clinical susceptibility tests, which simulates the composition of the human extracellular environment. In this test, the maximum concentration of ERD-A was 200 pg / ml. The susceptibility tests were performed in two technical replicates and repeated at least twice.

[0109] Microdilution susceptibility tests indicated the potent in vitro antifungal activity of ERD-A in LCM against human pathogenic yeasts, such as Candida albicans^ and Cryptococcus neoformans with MICs of 25 pg / ml (18 pM) and 50 pg / ml (35 pM), respectively (Table 1). It was ineffective against Candida auris (Table 1). Amino acid substations could keep or enhance the antifungal efficacy (Table 1). Among the peptide variants designed, ERD-AD6P, ERD-A F5,7W, and ERD-A K10,l 1,12R demonstrated the highest efficacy. Peptides were classified as ineffective if their MIC exceeded 100 pg / ml in LCM in the applied experimental setup. ERD- A D6P variant kept the antifungal efficacy against Candida albicans (MIC: 25 pg / ml, 18 pM), made ERD-A effective against Candida auris (MIC: 100 pg / ml, 71 pM), and increased the efficacy on Cryptococcus neoformans (MIC: 12.5 pg / ml, 9 pM). ERD-A F5,7W variant proved to be effective against Candida albicans (MIC: 25 pg / ml, 17 pM) and showed increased efficacy against Cryptococcus neoformans (MIC: 12.5 pg / ml, 8 pM). ERD-A K10,l l,12R variant showed increased activity against Candida albicans (MIC: 12.5 pg / ml, 8 pM), Cryptococcus neoformans (MIC: 12.5 pg / ml, 8 pM), and was effective against Candida auris (MIC: 100 pg / ml, 66 pM).

[0110] Table 1: Minimum inhibitory concentrations (MICs) of ERD-A and its amino acid substituted variants against human pathogenic yeasts in LCM.

[0111] The antifungal activity of ERD-A against human pathogenic yeasts decreased in RPMI- 1640 medium, however, not full but significant slight (Candida albicans and Cryptococcus neoformans IFO 410) or remarkable (Cryptococcus neoformans IFM 5844) growth inhibition was detected at 200 pg / ml (140 pM) or 400 pg / ml (280 pM) (Table 2). It has to be noted that the composition of the test medium can influence the activity of an antifungal peptide, and higher MICs are usually detected in RPMI-1640 than in LCM. Therefore, peptide was considered antifungally active if a measurable growth inhibition expressed as a percentage relative to the untreated control (100% growth). Table 2: Minimum inhibitory concentrations (MICs) of ERD-A against human pathogenic yeasts in RPMI-1640.

[0112] Growth percentages (mean ± SD) are indicated in brackets in comparison with the untreated control, which was considered as 100%.

[0113] *: Significant growth reduction (p < 0.05) according to paired t-test.

[0114] Example 4: Fungal uptake investigation

[0115] The uptake of green fluorescent, carboxyfluorescein-labeled ERD-A [(6)-Cf-ERD-A and (5)-Cf-ERD-A] by Candida albicans was analysed by fluorescence-activated cell sorting (FACS) analyses. Candida albicans CBS 5982 cells (2 x 107) were treated with 50 pg / ml peptide in LCM (30°C, 16 h, 160 rpm, then washed two-times in phosphate buffered saline and suspended in it). Cf-ERD-A positive cells were detected by a flow cytometer equipped with lasers at 405 (violet), 488 (blue), and 642 nm (red) (Amins, Merck Millipore, Billerica, MA, USA). Calibration controls were used to avoid overexposure of the positive events in specific fluorescent channels, which cause false-positive staining signals in other fluorescent channels. A calibration control was prepared by treating cells with 2 pg / ml fluorescein diacetate (Thermo Fischer Scientific, Waltham, MA, USA) for 20 minutes at room temperature at 16 rpm. A total of 5000 cells per run were detected. Cf-ERD-A was detected at 488 nm, with excitation lasers and emission in channel 2 window. The gating was adjusted to reach at least 96% of the untreated cells and debris was excluded during data acquisition. Data analysis was performed with Image Data Exploration and Analysis software (IDEAS; Amins, Millipore, Billerica, MA, USA). The FACS experiment was repeated two times.

[0116] FACS analysis quantified the ERD-A binding / uptake ability of Candida albicans cells in LCM which can be connected with the antifungal effect. According the results 98±1% and 99±1% of the treated cells bounded / took up (6)-Cf-ERD-A and (5)-Cf-ERD-A, respectively.

[0117] Example 5: Microscopic observations

[0118] Candida albicans CBS 5982 cells (2 x 107) were treated with 12.5 pg / ml (6)-Cf-ERD-A and (5)-CfERD-A in LCM (30°C, 4 and 16 h, 160 rpm, then washed two-times in phosphate- buffered saline and suspended in it), then they were visualized by light and fluorescence microscopy (Carl Zeiss Axiolab LR 66238C; Zeiss, Oberkochen, Germany) and photographed with a microscope camera ERc 5s; Zeiss, Oberkochen, Germany (Zeiss AxioCam), Fluorescence microscopy investigation confirmed the ERD-A binding / uptake ability of Candida albicans as cells showed intensive green fluorescence under microscope (Figure 1).

[0119] Example 6: Blood-brain barrier penetration

[0120] For the BBB permeability assessment, a contact co-culture model of the BBB was employed, wherein brain endothelial cells and brain pericytes were co-cultured in a Transwell system, as previously described (Meszaros et al., Cells, 2023, 12, 503). The two cell types were maintained in co-culture for six days prior to initiating permeability measurements in 24-well culture plates. Cumulative apparent permeability coefficients (Papp) were determined according to established protocols (Bocsik et al., J. Pharm. Sci., 2016, 105, 754-765), providing a quantitative measure of compound translocation across the BBB. The extent of translocation reflects the percentage of the initial peptide amount that successfully traversed the barrier.

[0121] Table 3: Calculated cumulative apparent permeability coefficients (Papp) of peptides.

[0122] The parental oligopeptide ERD-A was assessed for its capacity to cross BBB. The native sequence demonstrated superior penetration and translocation characteristics compared to the reference compound, opiorphin, as reported in the literature. This was substantiated by the elevated Pappand increased translocation percentage, as shown in Table 3. Moreover, amino acid-substituted variants of ERD-A (specifically, ERD-A D6P and ERD-A F5-7W) designed to modulate hydrophobicity and membrane interaction potential, also exhibited enhanced performance relative to opiorphin, and showed elevated Papprelative to the parental oligopeptide, ERD-A (Table 3).

Claims

WHAT IS CLAIMED IS:

1. An antifungal oligopeptide compound according to Formula I,Asp-R2-Gly-Leu-R5-R6-R7-Leu-Gly-R10-Rl 1-R12 (Formula I) whereinR2 represents Arg, Lys, Cit, D-Arg or Orn;R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3 -phenylpropionate;R6 represents Asp, Pro, Leu, Glu or Lys;R7 represents Phe or Trp; each of RIO, R11, and R12 independently represents Lys, Arg or Orn; for use in therapy.

2. The antifungal oligopeptide compound for use according to Claim 1, whereinR2 represents Arg, Cit or D-Arg;R5 represents Phe, Arg or Trp;R6 represents Asp or Pro;R7 represents Phe or Trp; each of RIO, R11, and R12 independently represents Lys or Arg.

3. The antifungal oligopeptide compound for use according to Claim 1 or 2, wherein the oligopeptide compound is one of the following oligopeptides:Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A; SEQ ID NO: 1), an oligopeptide compound which differs in up to 3 amino acid residues from ERD-A, particularlyAsp-Arg-Gly-Leu-Phe-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A D6P; SEQ ID NO:2), Asp-Arg-Gly-Leu-Trp-Asp-Trp-Leu-Gly-Lys-Lys-Lys (ERD-A F5,7W; SEQ ID NO:3), Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Arg-Arg-Arg (ERD-A K10,l l,12R; SEQ ID NO:4)Asp-Cit-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A R2Cit; SEQ ID NO: 5);Asp-Arg-Gly-Leu-Arg-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A F5RD6P; SEQ ID NO: 6); Asp-Arg-Gly-Leu-Trp-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A F5W; SEQ ID NO: 7); or Asp-D-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A R2D-Arg; SEQ ID NO:8).

4. The antifungal oligopeptide compound according to any one of the claims 1 -3 for use in the treatment or prevention of a fungal infection or a disease related to a fungal infection, particularly those affecting the CNS.

5. The antifungal oligopeptide compound for use according to claim 4, wherein the fungal infection is a human pathogenic fungal infection, particularly an infection of a human pathogenic yeast, more particularly an infection caused by Candida spp., particularly Candida albicans or Candida auris. or an infection of Cryptococcus spp., particularly Cryptococcus neoformans, particularly those affecting the CNS.

6. Pharmaceutical composition comprising at least one antifungal oligopeptide compound defined in any one of claims 1-3 and a pharmaceutically acceptable carrier, diluent and / or excipient, for use in therapy.

7. Pharmaceutical composition for use according to claim 6 in the treatment or prevention of a fungal infection or a disease related to a fungal infection, particularly those affecting the CNS.

8. Pharmaceutical composition for use according to claim 7, wherein the fungal infection is a human pathogenic infection, particularly an infection caused by a human pathogenic yeast, more particularly an infection of a Candida spp., particularly Candida albicans or Candida auris, or an infection of Cryptococcus spp., particularly Cryptococcus neoformans, particularly those affecting the CNS.

9. An antifungal oligopeptide compound according to Formula I,Asp-R2-Gly-Leu-R5-R6-R7-Leu-Gly-R10-Rl 1-R12 (Formula I) whereinR2 represents Arg, Lys, Cit, D-Arg or Orn;R5 represents Phe, Arg, Lys, His, Trp, Tyr, D-Tyr, D-Phe, Orn, 4-aminophenylalanine or 3 -phenylpropionate;R6 represents Asp, Pro, Leu, Glu or Lys;R7 represents Phe or Trp; each of RIO, R11, and R12 independently represents Lys, Arg or Orn; provided that the oligopeptide compound is not Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly- Lys-Lys-Lys (ERD-A).

10. The oligopeptide compound according to Claim 9, whereinR2 represents Arg, Cit or D-Arg;R5 represents Phe, Arg or Trp;R6 represents Asp or Pro;R7 represents Phe or Trp; each of RIO, R11, and R12 independently represents Lys or Arg.

11. The oligopeptide compound according to Claim 10, wherein the oligopeptide compound is one of the following oligopeptides: Asp-Arg-Gly-Leu-Phe-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A D6P; SEQ ID NO:2), Asp-Arg-Gly-Leu-Trp-Asp-Trp-Leu-Gly-Lys-Lys-Lys (ERD-A F5,7W; SEQ ID NO:3), Asp-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Arg-Arg-Arg (ERD-A K10,l l,12R; SEQ ID NO:4)Asp-Cit-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A R2Cit; SEQ ID NO: 5); Asp-Arg-Gly-Leu-Arg-Pro-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A F5RD6P; SEQ ID NO: 6); Asp-Arg-Gly-Leu-Trp-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A F5W; SEQ ID NO: 7); andAsp-D-Arg-Gly-Leu-Phe-Asp-Phe-Leu-Gly-Lys-Lys-Lys (ERD-A R2D-Arg; SEQ ID NO:8).

12. Pharmaceutical composition comprising at least one oligopeptide compound according to any one of claims 9-11 and a pharmaceutically acceptable carrier, diluent and / or excipient.

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