Compositions and methods for treating malaria

Abstract

Compositions and methods for treating malaria are provided, utilizing antimicrobial peptides (AMPs) targeting malaria parasites (particularly Plasmodium falciparum (Pff). The AMPs of the provided compositions and methods penetrate red blood cells infected with the parasites with high selectivity, while sparing non-infected cells, and are characterized by reduced risk of drug resistance. Further provided are novel peptides targeting the malaria parasite Plasmodium falciparum (Pf) which are particularly effective as anti-malaria agents.

Description

[0001] COMPOSITIONS AND METHODS FOR TREATING MALARIA

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to antimicrobial peptides (AMPs) targeting malaria parasites (particularly Plasmodium falciparum (Pf)), which are useful in treating malaria.

[0004] BACKGROUND OF THE INVENTION

[0005] Plasmodium falciparum (Pf) and malaria

[0006] Plasmodium falciparum (Pf), the most virulent causative agent of human malaria, accounts for over 99.7% of all malaria cases in Africa and was responsible for -600,000 deaths in 2022 only. Alarmingly, the rapid development of parasitic resistance to all the commercial antimalarial drugs raises the challenge of controlling malaria worldwide.

[0007] The complex life cycle of Pf alternates between the human host and the female Anopheles mosquito vector. The symptomatic phase of the disease begins when parasites migrate from hepatocytes, the initial replication niche, and invade and replicate within mature red blood cells (RBCs). During the blood stage, the intracellular parasite progresses through ring, trophozoite, and schizont developmental stages in an asexual replication cycle lasting approximately 48 hours (h). At the final asexual stage of the cycle, new daughter cells (merozoites) are formed, egress, and invade into naive host RBCs.

[0008] While numerous antimalarial drugs exist, the ongoing necessity for the development of new therapeutic approaches persists, primarily attributed to the parasite's rapid resistance development to commercial drugs, including frontline treatments and combination therapies. In addition, the use of some antimalarial drugs could lead to severe adverse effects, as in the case of primaquine.

[0009] Antimicrobial peptides (AMPs)

[0010] Antimicrobial peptides (AMPs) play a crucial role in the innate immune system and serve as the initial responders in interactions with various pathogens. While AMPs exhibit diversity in their lengths, sequences, and structures, most of them are short and include a combination of hydrophobic and positively charged residues. Many of these peptides are unstructured in aqueous solutions and adopt amphipathic a-helix or P-sheet structures upon partitioning into membrane environments, which is a prerequisite for their lytic activity. AMPs interact via their hydrophilic residues with the membrane surface of the pathogen. Upon reaching a critical accumulation level of AMPs, the membrane is permeabilized either by transient pore formation or membrane disintegration, leading to cell death. Notably, AMPs' interaction with pathogens is meditated by two common attributes of the pathogens' membrane: i. Negatively charged outer membranal leaflet, compared to the zwitterionic membrane of mammalian cells, and ii. The lack of cholesterol in many pathogen’s membrane, compared to mammalian cells, reduces the lipid bilayers' rigidity and induces membrane perturbation by AMPs. Unlike conventional drugs that target specific molecules, the mode of action (MoA) of AMPs is nonreceptor- mediated (NRM MoA) and relies on electrostatic interactions with the pathogen's membrane.

[0011] Papo et al. (2002) The Journal of Biological Chemistry, 277(37), 33913-33921; Segev-Zarko et al. (2015) Biochem J, 468(2), 259-270; Ben-Hur et al. (2022) J Med Chem, 65(13), 9050-9062; and Saar-Dover et al. (2013) Methods in Molecular Biology, 1033, 173-183 report studies of antimicrobial peptides composed of lysines and leucines.

[0012] Ghosh et al. (1997) Journal of Biological Chemistry, 272(50), 31609-31616; Gelhaus et al. (2008) Antimicrob Agents Chemother, 52(5), 1713-1720; Mor et al. (2009) FEBS Journal, 276(22), 6474-6482; Kaushik et al. (2012) Malar 7, 11, 256; and Vale et al. (2014) Front Pharmacol, 5(DEC): 275) report studies of antimicrobial peptides that exhibit activity on malaria parasites.

[0013] Kiper et al. (2025) J. Biol. Chem., 301(4): 108298, published after the priority date of the present invention, examined anti-P / activity of a groups of AMPs, synthetic peptides containing lysine (K) and leucine (L) residues in the D and L configurations.

[0014] WO 2004 / 110341 discloses lipophilic conjugates comprising a peptide coupled to a fatty acid, the peptide comprising at least two positively charged amino acid residues, said peptide after conjugation to the fatty acid possessing antibacterial, antifungal, and / or anticancer activity higher than prior to conjugation. The lipophilic conjugates are suitable for treatment of infections caused by pathogenic organisms such as bacteria and fungi. The lipophilic conjugates are also suitable for sanitation, as disinfectants, or for food preservation CN 17599152 discloses the use of the antimicrobial peptides BF15 (VKRFKKFFRKLKKSV) and / or BF15-1 (VKRFKKFFRKFKKFV, C-terminus is amidated) in the preparation of antimalarial drugs. US 7,001,983 discloses non-hemolytic cytolytic agents selected from peptides, complexes of bundled peptides, mixtures of peptides or random peptide copolymers have a selected cytolytic activity manifested in that they have a cytolytic activity on pathogenic cells, being cells which are non-naturally occurring with the body consisting of microbial pathogenic organisms and malignant cells; and are non-hemolytic, having no cytolytic effect on red blood cells. The peptides may be cyclic derivatives of natural peptides such as pardaxin and melittin and fragments thereof in which L-amino acid residues are replaced by corresponding D-amino acid residues, or are diastereomers of linear peptides composed of varying ratios of at least one positively charged amino acid and at least one hydrophobic amino acid, and in which at least one of the amino acid residues is a D- amino acid. Pharmaceutical compositions comprising the non-hemolytic cytolytic agents can be used for the treatment of several diseases caused by pathogens including antibacterial, fungal, viral mycoplasma and protozoan infections and for the treatment of cancer.

[0015] There remains a need for improved anti-malaria drugs, which are effective and also have reduced risk of drug resistance.

[0016] SUMMARY OF THE INVENTION

[0017] The present invention provides compositions and methods for treating malaria, utilizing antimicrobial peptides (AMPs) targeting malaria parasites (particularly Plasmodium falciparum (Pff). The AMPs for use according to the present invention penetrate red blood cells infected with the parasites with high selectivity, and effectively inhibit growth of the parasites. The AMPs for use according to the present invention are further characterized by reduced risk of drug resistance. The present invention therefore provides particularly beneficial and effective anti-malaria compositions and methods.

[0018] AMPs are not a straightforward choice for combating malaria, mainly due to the asexual blood stage, in which the intracellular parasite is enclosed within its host red blood cell and lacks direct contact with the extracellular environment. Hence, there is uncertainty about whether such peptides could penetrate the host cell and access the encapsulated parasite. However, the inventors of the present invention identified AMPs which are surprisingly capable of selectively penetrating red blood cells infected with malaria parasites, while sparing non-infected cells, and effectively inhibiting their growth.

[0019] The present invention further provides novel peptides targeting malaria parasites (particularly Plasmodium falciparum (Pf)) which are particularly effective as anti-malaria agents, characterized by very low IC50 values, while maintaining high selectivity towards infected red blood cells.

[0020] According to one aspect, the present invention provides a peptide of 15-25 amino acids consisting of the amino acid residues L-Lysine (K), D-Lysine (k), L-Leucine (L), and D-Leucine (1), having a net charge of (+7) to (+10) and comprising a sequence selected from kLLL, LLKKL1, 1KL1LK, L1KLL, 1KKLL, K1LLK and kLLA- (wherein K* is a L-Lysine bonded by an isopeptide bond). Each possibility represents a separate embodiment of the present invention.

[0021] In some embodiments, the peptide consists of 15-20 amino acids, of which 3-6 residues are in D-configuration. In additional embodiments, the peptide consists of 15-20 amino acids, of which 3-5 residues are in D-configuration.

[0022] In some embodiments, the peptide comprises a sequence selected from the group consisting of:

[0023] LKlLKkLlkKLLkLLLkLL (D4) (SEQ ID NO: 52);

[0024] LLKKL1KL1LKL1KK (D2) (SEQ ID NO: 50);

[0025] LLkKLIKLILKLIKK (C4) (SEQ ID NO: 34);

[0026] LLKKL1K11LKL1KK (B2) (SEQ ID NO: 13);

[0027] LLkLLkKllKKLlKLL (Bl) (SEQ ID NO: 12);

[0028] LKILKkLIKKLLkLL (C3) (SEQ ID NO: 33);

[0029] LLkKLIKILLKLIKK (C5) (SEQ ID NO: 35);

[0030] LKlLKkLlkKLLkLLK* (C6) (SEQ ID NO: 36); and

[0031] LLKKL1K11LKL1KK1KK (D3) (SEQ ID NO: 51), wherein K, k, L, 1 and K* are as defined above.

[0032] Each possibility represents a separate embodiment of the present invention.

[0033] In some embodiments, the peptide is selected from the peptides set forth as SEQ ID NO: 52, SEQ ID NO: 50, SEQ ID NO: 34, SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 51. Each possibility represents a separate embodiment of the present invention. In some embodiments, the peptide is characterized by relative hydrophobicity of at least 55% AcN. In some embodiments, such a peptide is selected from the peptides set forth as SEQ ID NO: 52, SEQ ID NO: 50, SEQ ID NO: 34, SEQ ID NO: 13 and SEQ ID NO: 12. Each possibility represents a separate embodiment of the present invention.

[0034] According to a further aspect, the present invention provides a pharmaceutical composition comprising a peptide according to any one of the preceding claims and at least one pharmaceutically acceptable excipient.

[0035] In some embodiments, the pharmaceutical composition is for use in the treatment of malaria.

[0036] In some embodiments, the malaria is caused by a Plasmodium species selected from P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi and P. chabaudi. Each possibility represents a separate embodiment of the present invention.

[0037] In some embodiments, the pharmaceutical composition comprises a plurality of peptides of the present invention.

[0038] In some embodiments, the pharmaceutical composition is for use in the treatment of malaria in combination with at least one anti-malaria drug. In some embodiments, the anti-malaria drug is selected from artemisinin or derivatives thereof, and chloroquine or derivatives thereof. Each possibility represents a separate embodiment of the present invention. In some particular embodiments, the anti-malaria drug is artesunate. In additional particular embodiments, the anti-malaria drug is primaquine.

[0039] According to a further aspect, the present invention provides a conjugate comprising a peptide of the present invention and a heterologous moiety.

[0040] In some embodiments, the heterologous moiety is an anti-malaria drug. In some embodiments, the anti-malaria drug is selected from artemisinin or derivatives thereof, and chloroquine or derivatives thereof. Each possibility represents a separate embodiment of the present invention. In some particular embodiments, the anti-malaria drug is artesunate. In additional particular embodiments, the anti-malaria drug is primaquine.

[0041] According to a further aspect, the present invention provides a pharmaceutical composition for use in the treatment of malaria, comprising a conjugate of the present invention.

[0042] According to yet a further aspect, the present invention provides a pharmaceutical composition for use in the treatment of malaria, comprising at least one peptide of 15-25 amino acids in length comprising one or more positively-charged units and one or more hydrophobic units arranged in the peptide in an alternating order, wherein each positively- charged unit is composed of one or more consecutive positively-charged residues and each hydrophobic unit is composed of one or more consecutive hydrophobic residues, wherein the peptide has a net charge of (+7) to (+10), and wherein the peptide comprises a sequence selected from the group consisting of:

[0043] LKlLKkLlkKLLkLL (AmplD) (SEQ ID NO: 1);

[0044] KKkLLlLllLLLkKK (Seg5D) (SEQ ID NO: 2);

[0045] LLILLkKkkKKLILL (Seg6D) (SEQ ID NO: 3);

[0046] GIGKFLKKAKKFGKAFVKILKK (MSI-78) (SEQ ID NO: 9);

[0047] LLkLLkKllKKLlKLL (Bl) (SEQ ID NO: 12);

[0048] LLKKL1K11LKL1KK (B2) (SEQ ID NO: 13);

[0049] LKILKkLIKKLLkLL (C3) (SEQ ID NO: 33);

[0050] LLkKLIKLILKLIKK (C4) (SEQ ID NO: 34);

[0051] LLkKLIKILLKLIKK (C5) (SEQ ID NO: 35);

[0052] LKlLKkLlkKLLkLL^* (C6) (SEQ ID NO: 36);

[0053] LLKKLIKLILKLIKK (D2) (SEQ ID NO: 50);

[0054] LLKKL1K11LKL1KK1KK (D3) (SEQ ID NO: 51); and LKlLKkLlkKLLkLLLkLL (D4) (SEQ ID NO: 52), wherein K, k, L, 1 and K* are as defined above.

[0055] Each possibility represents a separate embodiment of the present invention.

[0056] In some embodiments, the peptide is 15-20 amino acid in length.

[0057] In some embodiments, the peptide is selected from the peptides set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52. Each possibility represents a separate embodiment of the present invention.

[0058] In some embodiments, the peptide is selected from the peptides set forth as: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 9. Each possibility represents a separate embodiment of the present invention.

[0059] In some embodiments, the pharmaceutical composition comprises a plurality of peptides. In some embodiments, the malaria is caused by a Plasmodium species selected from P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi and P. chabaudi. Each possibility represents a separate embodiment of the present invention.

[0060] In some embodiments, the pharmaceutical composition is for use in combination with at least one anti-malaria drug. In some embodiments, the anti-malaria drug is selected from artemisinin or derivatives thereof, and chloroquine or derivatives thereof. Each possibility represents a separate embodiment of the present invention. In some particular embodiments, the anti-malaria drug is artesunate. In additional particular embodiments, the anti-malaria drug is primaquine.

[0061] According to a further aspect, the present invention provides a conjugate comprising a peptide and an anti-malaria drug, wherein the peptide is 15-25 amino acids in length comprising one or more positively-charged units and one or more hydrophobic units arranged in the peptide in an alternating order, wherein each positively-charged unit is composed of one or more consecutive positively-charged residues and each hydrophobic unit is composed of one or more consecutive hydrophobic residues, wherein the peptide has a net charge of (+7) to (+10), and wherein the peptide comprises a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52. Each possibility represents a separate embodiment of the present invention.

[0062] In some embodiments, the anti-malaria drug is selected from artemisinin or derivatives thereof, and chloroquine or derivatives thereof. Each possibility represents a separate embodiment of the present invention. In some particular embodiments, the antimalaria drug is artesunate. In additional particular embodiments, the anti-malaria drug is primaquine.

[0063] According to a further aspect, the present invention provides a pharmaceutical composition comprising the conjugate, for use in the treatment of malaria.

[0064] According to another aspect, the present invention provides a method for treating malaria in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a peptide as defined above.

[0065] According to a further aspect, the present invention provides a method for treating malaria in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising a conjugate as defined above.

[0066] A "subject" according to the present invention is a mammal, particularly human.

[0067] Other objects, features and advantages of the present invention will become clear from the following description, examples and drawings.

[0068] BRIEF DESCRIPTION OF THE FIGURES

[0069] Figure 1A-1F. AMPs demonstrate potent antimalarial activity and low cytotoxicity. (1A) Helical wheel projections of AmplD, LL-37, MSI-78 and Seg5D. The asterisk (*) is an indication of the D-enantiomers. The helical wheel projections were generated in HeliQuest. (IB) Trophozoite- stage parasites were incubated with 12.5 pM of a selected panel of AMPs (Table 1), and the relative growth of the parasites was measured 24 hours post treatment (hpt) using FACS (light gray columns). In addition, naive RBCs were incubated with 12.5 pM of the same AMPs panel, and the relative hemolysis level was measured 24 hpt (black columns). Statistical analysis: Two-way ANOVA, with batch effect followed by a Dunnett’s test p<0.05 *, p<0.01 **, p< 0.001 ***. (1C) Relative hemolysis assay with a serial dilution of potent anti-P / AMPs at 1 hpt 24 hpt both N=3. (ID) Dose-response assay of anti-P / AMPs (AmplD, MSI-78, LL-37, Seg5D) and Melittin as a control. Trophozoite-stage parasites were incubated with different AMPs at decreasing concentration levels, and the parasites' relative growth was measured 24 hpt. Statistical analysis of three biological replicates: IC50 values were obtained using the package "dr4pl" for "R". (IE) Ring and trophozoite stage parasites were exposed to a short (3h) pulse of 5 pM AmplD. The parasitemia was measured by flow cytometry after the next P / invasion cycle. Growth (%) was measured after invasion of the treated Ring and Trophozoite- stage parasites and was compared to untreated Ring and Trophozoite- stage parasites respectively. Statistical analysis: t-test, p<0.05 *, p<0.01 ** N=3. (IF) Dose-response assay of anti-P / AMPs Amp4D and AmplD+LN. Trophozoite- stage parasites were incubated with different AMPs at decreasing concentration levels, and the parasites' relative growth was measured 24 hpt. Statistical analysis of three biological replicates: IC50 values were obtained using the package "dr4pl" for "R".

[0070] Figure 2A-2F. Anti-P / AMPs bind selectively to P / -iRBC. (2A) Illustration of the experimental setup. NBD-labelled peptides were incubated with a synchronized culture of 2% trophozoite- stage parasites. Using flow cytometry, the binding of the peptide was tracked and the percentage of NBD+naive RBCs and P / -iRBCs was quantified. (2B) Binding selectivity analysis of AmplD, LL-37, MSI-78 and Seg5D (tagged with NBD fluorophore) to P / -iRBCs (light gray) or naive RBCs analysis (dark gray). Trophozoitestage parasites were incubated with tagged AmplD or LL-37 at IC50 concentrations (top panel) or IC25 concentrations (bottom panel), or with tagged MSI-78 or Seg5D at IC25 concentrations for 24h, and the binding selectivity was measured at 5 time points. (2C) Binding load analysis of tagged AmplD and LL-37. Trophozoite- stage parasites were incubated with NBD tagged AmplD or LL-37 at IC50 concentrations. (2D) NBD- AMPs interaction with P / -iRBCs and naive RBCs. Trophozoite stage parasites were incubated with NBD-AMPs (IC50 - top panel; IC25 - bottom panel) for 8h, and images were obtained using imaging flow cytometry. Bright field (BF), NBD-AMP signal, DNA (Hoechst), and merged channels are shown. (2E) Localization analysis of Rho-AmplD within Pf parasites using imaging flow cytometry. Pf parasites were synchronized using magnetic column separation at the trophozoite stage. Parasites were incubated with Rho- AmplD for 30 min. BF, Pf Plasma Membrane, [Rhodaminel23 (R123) 1 pM, blue], Rho-AmplD signal, DNA (Hoechst staining), and merged channels are shown. Arrows are examples of the accumulation of Rho-AmplD on the membrane of P / -iRBCs. (2F) Localization analysis of Rho-AmplD. The ratio between the signal of Rho-AmplD cosignals with Hoechst stained-DNA signal (RhoInside nucleus) and Rho-AmplD not- overlapping with Hoechst stained-DNA signal (RhoOutelde nucleus).

[0071] Figure 3A-3D. AmplD selectively induces structural and mechanical changes in P / -iRBCs. (3A) A representative example of AFM maps of naive and P / -iRBCs cells marked with asterisks. RBCs treated with (+AmplD) or without (NT) treatment. Bright field (BF), AFM 3D topography map with BF overlay, and the Young’s modulus map of the cells are presented. (3B) Comparison of the mean (vertical lines) height of total pixels from naive RBCs and P / -iRBCs with treatment (+AmplD, for 2h) or without (NT). (3C) Representative 3D AFM-topography images of P / -iRBCs and Naive RBCs with and without treatment of AmplD (2 h), and a profile curve of the cells’ height (from the images on the left). Roman numerals correlate the image of each cell and its height profile. (3D) Comparison of the mean (vertical lines) Young's modulus of total pixels from naive RBCs and P / -iRBCs with (+AmplD treatment for 2 h) or without (NT). Data represent three biological repeats. Statistical analysis was done using the packages "ImerTest" and "emmeans" for "R". The means were compared by Two-way ANOVA with random batch effect analysis. p< 0.001 ***.

[0072] Figure 4A-4D. Amp ID disrupts the structure in the cytoskeleton of P / -iRBCs. (4A) Left: Representative images, in increasing magnification, of the cytoskeleton of P / -iRBCs with (AmplD) or without (NT), exposure to AmplD for 30 min (5 pM) after isolation via a magnetic column. Boxes represent an example of flattening of the filaments after treatment. Right: Giemsa-stained trophozoite-stage parasites after separation via magnetic column. (4B) Representative examples of the analysis of P / -iRBCs' cytoskeleton structure of P / -iRBCs with (AmplD) or without (NT) exposure to AmplD for 30 min (5 pM) after isolation via a magnetic column. Raw images were processed to differentiate filaments from gaps and to calculate the percentage of area covered by the filaments and gaps. (4C) The area covered by filaments after treatment with AmplD. Statistical analysis: Two-Way ANOVA with random batch effect, ** p<0.01, each dot represents a cell, N=2. (4D) The mean roughness of filaments after treatment with AmplD Statistical analysis: Mann-Whitney-Wilcoxon Test: *** p<0.001.

[0073] Figure 5A-5F. LUVs model shows peptide activity is facilitated by cholesterol levels and shows distinct mechanisms of interaction with the membrane. Affinity (5A, 5B) and binding load (5C) of different AMPs tagged with NBD fluorophore (4DK5L7, AmplD, LL-37, Melittin, MSI-78, Seg5D) to LUVs. PC and PC:PS (1: 1 wt / wt) represent the elevation levels of PS in P / -IRBCs, PC: Cholesterol (9: 1 wt / wt) represents the naive RBCs’ membrane. Binding to LUVs in assemble from PC: PS: Cholesterol (4.5:4.5: 1 wt / wt / wt) and PC: PS: Cholesterol (4:4: 1 wt / wt / wt) is also presented. The values were normalized to the best-fit maximal fluorescence (Bmax) for each AMP and LUV in each batch. Statistical analysis: The Kd and Bmax values were obtained using the package "renz" for "R". The maximal fluorescence, Bmax, resulted from the interaction of each AMP to the different LUV. Statistical analysis: Two way-ANOVA followed by Tukey’s test, p<0.05 *, p<0.01 **, p< 0.001 ***. (5D) A dose-response assay between the percentage (wt / wt) of PS in PC:PS LUVs (horizontal axis) and the affinity (Kd) of NBD- AmplD to the LUVs (vertical axis). Statistical analysis: Two-way ANOVA with batch effect followed by a Tukey’s test, ** p<0.01. (5E) NBD emission blue shift of the peptides (AmplD, LL-37, Melittin, 4DK5L7, MSI-78 and Seg5D) in aqueous solution (PBS) relative to NBD emission with the interaction with LUVs. Fluorescence emission spectra of NBD-labeled peptides (AmplD, LL-37, Melittin, 4DK5L7, MSI-78 and Seg5D) in solution and the presence of LUVs. Statistical analysis: One-way ANOVA followed by a Dunnett’s test, p<0.05 *, p<0.01 ** , pcO.OOl ***. (5F) Cryo-EM representative images of LUVs incubated with or without incubation of AmplD for 1 h. Arrows are examples of nanodiscs and sheets.

[0074] Figure 6A-6C. Cholesterol protects naive RBCs in vitro from AmplD hemolytic activity. (6A) Binding analysis of the effect of cholesterol depletion from naive RBC on the binding to AmplD by IFC. On the left side, histogram plots (N=3) showing the fluorescence intensity of Rhodamine after 30 min incubation of Rho-AmplD with naive RBCs, with or without cholesterol depletion treatment (+MPC). Based on the untreated group (NT), the dashed line marks the threshold for a positive Rhodamine signal (Rho+). Representative images from the IFC analysis, BF, Rhodamine signal (Rho-AmplD), and merged images are shown on the right side. (6B) A statistical summary of the mean percentage of Rho+RBCs from (A), N=3, one-way ANOVA followed by a Tukey’s test, ** p<0.01. (6C) Naive RBCs were exposed to AmplD upon treatment / mock treatment for cholesterol depletion (MpC). The relative hemolysis was measured at 1 hpt with AmplD. Statistical analysis: t-test, p<0.01 **.

[0075] Figure 7A-7B. Minimal hydrophobicity is associated with active anti-P / AMPs. (7A) Summarizing meta-analysis using the relative hydrophobicity of the AMPs in this study (Table 1) to their Pf growth inhibition activity (as shown in Figure IB). (7B) Hierarchical clustering of AmplD, LL-37, MSI-78, Seg5D, 4DK5L7 and Melittin based on heatmap of their blue shift values after interaction with different LUVs (as shown in Figure 5E).

[0076] Figure 8A-8B. Synergistic inhibition of Plasmodium falciparum growth by AmplD in combination with artesunate using a warp-and-weft dose matrix. (8A) Inhibition curves showing increasing concentrations of AmplD at fixed artesunate concentrations. (SB) Inhibition curves showing increasing concentrations of artesunate at fixed AmplD concentrations. Data represent the mean of three independent biological replicates (N = 3). The black horizontal line indicates 50% growth inhibition.

[0077] Figure 9. Synthetic AMPs exhibit potent anti-malarial activity. Trophozoite-stage Pf parasites were treated with 5pM of each synthetic AMP, and parasite growth was quantified 24 hours post- treatment (hpt) by flow cytometry. Data represent relative parasitemia from biological replicates, normalized to untreated controls. Statistical analysis was performed using two-way ANOVA accounting for batch effects, followed by Dunnett’s post hoc test; *p < 0.05, **p < 0.01, and ***p < 0.001.

[0078] Figure 10. Cytotoxicity of the synthetic AMPs toward naive RBCs. Hemolytic activity of the synthetic AMPs was evaluated by incubating naive RBCs with a serial dilution of peptides for Ih (N = 3). The toxic AMPs Amp IL and Melittin served as positive peptide controls. Untreated cells and cells exposed to 1% Triton X-100 served as negative and positive controls, respectively. The cell viability is plotted (100%-viability), and results are presented as mean ± SEM. Statistical significance was assessed using two- way ANOVA followed by Tukey’s post hoc test; *p < 0.05, **p < 0.01, and ***p < 0.001.

[0079] Figure 11. Dose-response analysis and ICso determination of selected AMPs with significant antimalarial activity from the initial screen. Trophozoite-stage Pf parasites were treated with decreasing concentrations (25-0.39 pM) of the indicated AMPs. Parasite growth was measured 24 hpt by flow cytometry and normalized to untreated controls. Dose-response curves were used to calculate half-maximal inhibitory concentrations (ICso). Data represent SEM from three independent biological replicates (N = 3), except for peptide D4, for which one replicates were analyzed (N=l).

[0080] Figure 12. Anti-P / AMPs bind to iRBCs and localize within the intracellular parasite. A synchronized culture of trophozoite- stage Pf( % parasitemia) was incubated with Rhodamine-labeled AMPs. Peptide localization was assessed immediately following treatment using imaging flow cytometry (IFC). Representative images show bright field (BF), Rho-labeled peptide signal, nuclear DNA stained with Hoechst, and merged channels, indicating peptide accumulation within the parasite inside the iRBC.

[0081] DETAILED DESCRIPTION OF THE INVENTION

[0082] The present invention is directed to the treatment of malaria using antimicrobial peptides (AMPs) targeting malaria parasites (particularly Plasmodium falciparum (Pf ).

[0083] Finding a long-lasting solution for malaria is an endless challenge since the parasite rapidly develops resistance to all available drugs, including chloroquine, sulfadoxine / pyrimethamine and artemisinin. The present invention discloses AMPs, particularly from the D,L-KL family, as anti-P / therapeutic agents. The peptides according to the present invention are particularly advantageous, as they exhibit low hemolytic activity, high specificity for infected red blood cells and effective inhibition of Pf growth. In addition, the peptides according to the present invention are characterized by high safety profile and resistance to proteolytic degradation, rendering them particularly beneficial for safe use with sufficient lifespan in the human body to be effective against pathogens. Their general interaction with the membrane of Pf suggests they may delay the development of drug resistance, a critical trait when combating Pf.

[0084] As exemplified herein below, a mechanistic study reveals that the mode of action of peptides according to the present invention appears to involve traversing the membrane of P / -iRBCs to reach the enclosed parasites, with the highest affinity exhibited toward parasites in their later developmental stages. By combining biophysical measurements from atomic force microscopy and computer vision analysis, it was revealed that peptides according to the present invention significantly disrupt the cytoskeletal network of the infected cell. In addition, it was revealed that the cholesterol level in the plasma membrane influences the affinity of the peptides to the membrane. In particular, the reduced cholesterol levels of the P / -iRBC membrane play a significant role in the selective interaction of the peptides with infected cells.

[0085] The present invention further discloses the use of peptides described herein as a delivery system for anti-malaria drugs. The selective activity of the peptides offers an approach for targeting P / -iRBC while sparing healthy cells. For example, by conjugating a peptide according to the present invention to an anti-malaria drugs, the anti-malaria drugs can be delivered directly to the site of infection, thereby maximizing drug efficacy and minimizing off-target effects. In some particular embodiments, the present invention discloses conjugation of the peptides to small molecules. This method combines the specific targeted Mo A of the traditional anti-malaria drugs with NRM Mo A of the peptides according to the present invention. By simultaneously targeting multiple pathways, the overall therapeutic effect can be enhanced and the likelihood of resistance development can be reduced.

[0086] In some embodiments, there is provided herein a peptide of 15-25 amino acids consisting of the amino acid residues L-Lysine (K), D-Lysine (k), L-Leucine (L), and D- Leucine (1), having a net charge of (+7) to (+10) and comprising a sequence selected from L1KLL (SEQ ID NO: 53), LLKKL1 (SEQ ID NO: 54), 1KKLL (SEQ ID NO: 55), 1KL1LK (SEQ ID NO: 56), K1LLK (SEQ ID NO: 57), kLL * (wherein K* is a L-Lysine bonded by an isopeptide bond) (SEQ ID NO: 58), and kLLL (SEQ ID NO: 59).

[0087] In some embodiments, the peptide is selected from the group consisting of:

[0088] LLkLLkKllKKLlKLL (Bl) (SEQ ID NO: 12);

[0089] LLKKL1K11LKL1KK (B2) (SEQ ID NO: 13);

[0090] LKILKkLIKKLLkLL (C3) (SEQ ID NO: 33);

[0091] LLkKLIKLILKLIKK (C4) (SEQ ID NO: 34);

[0092] LLkKLIKILLKLIKK (C5) (SEQ ID NO: 35);

[0093] LKlLKkLlkKLLkLLK* (C6) (SEQ ID NO: 36);

[0094] LLKKLIKLILKLIKK (D2) (SEQ ID NO: 50);

[0095] LLKKL1K11LKL1KK1KK (D3) (SEQ ID NO: 51); and

[0096] LKlLKkLlkKLLkLLLkLL (D4) (SEQ ID NO: 52), wherein K, k, L, 1 and K* are as defined above.

[0097] In additional embodiments, there is provided herein a pharmaceutical composition comprising a peptide according to the present invention and at least one pharmaceutically acceptable excipient, for use in the treatment of malaria.

[0098] In additional embodiments, there is provided herein a pharmaceutical composition for use in the treatment of malaria, comprising at least one peptide of 15-25 amino acids in length comprising one or more positively-charged units and one or more hydrophobic units arranged in the peptide in an alternating order, wherein each positively-charged unit is composed of one or more consecutive positively-charged residues and each hydrophobic unit is composed of one or more consecutive hydrophobic residues, wherein the peptide has a net charge of (+7) to (+10) and relative hydrophobicity of at least 55% AcN, and wherein the peptide comprises a sequence selected from the group consisting of: LKlLKkLlkKLLkLL (AmplD) (SEQ ID NO: 1); KKkLLlLllLLLkKK (Seg5D) (SEQ ID NO: 2); LLILLkKkkKKLILL (Seg6D) (SEQ ID NO: 3);

[0099] GIGKFLKKAKKFGKAFVKILKK (MSI-78) (SEQ ID NO: 9); LLkLLkKllKKLlKLL (Bl) (SEQ ID NO: 12); LLKKL1K11LKL1KK (B2) (SEQ ID NO: 13);

[0100] LKILKkLIKKLLkLL (C3) (SEQ ID NO: 33); LLkKLIKLILKLIKK (C4) (SEQ ID NO: 34); LLkKLIKILLKLIKK (C5) (SEQ ID NO: 35); LKlLKkLlkKLLkLLK* (C6) (SEQ ID NO: 36); LLKKLIKLILKLIKK (D2) (SEQ ID NO: 50); LLKKL1K11LKL1KK1KK (D3) (SEQ ID NO: 51); and LKlLKkLlkKLLkLLLkLL (D4) (SEQ ID NO: 52), wherein K, k, L, 1 and K* are as defined above.

[0101] In some embodiments, there is provided herein a pharmaceutical composition comprising at least one peptide of 15-25 amino acids in length comprising one or more positively-charged units and one or more hydrophobic units arranged in the peptide in an alternating order, for use in the treatment of malaria, wherein: each positively-charged unit is composed of one or more consecutive positively- charged amino acid residues and each hydrophobic unit is composed of one or more consecutive hydrophobic amino acid residues; the ratio of positively-charged residues to hydrophobic residues in the peptide is in the range of 1: 1.4 to 1: 1.7; the peptide comprises positively-charged and / or hydrophobic residues in D- configuration, and the ratio of the residues in D-configuration to residues in L- configuration is in the range of 1:2 - 1:5; and the peptide is characterized by relative hydrophobicity of at least 55% AcN.

[0102] The peptides for use according to the present invention are composed of positively- charged amino acid residues (e.g., lysine residues) and hydrophobic amino acid residues in both D- and L- enantiomer configurations. In particular, the peptides for use according to the present invention are composed of positively-charged units and hydrophobic units arranged in the peptide in an alternating order, meaning that a sequence of one or more consecutive positively-charged residues (e.g., one or more consecutive lysine residues) is interposed by a sequence of one or more consecutive hydrophobic residues (e.g., consecutive leucine residues) and vice versa. The peptides for use according to the present invention are characterized by unique properties of their composition, including a defined ratio of positively-charged residues to hydrophobic residues in the peptide, a defined ratio of residues in D-configuration to residues in L-configuration, and a defined threshold of relative hydrophobicity. In particular embodiments, the peptides for use according to the present invention are composed of lysine (K) and leucine (L) residues in both D- and L- enantiomer configurations, identified as "D,L-KL peptides".

[0103] A "positively-charged unit" as described herein refers to one more consecutive positively-charged amino acid residues. In some embodiments, a positively-charged amino acid residue according to the present invention is selected from lysine and arginine residues. In some particular embodiment, the positively-charged residue is lysine residue. Consecutive positively-charged residues within a unit may be the same residues or different residues, with each possibility representing a separate embodiment. In some embodiments, a positively-charged unit (e.g., a lysine unit) is composed of 1-6 consecutive positively-charged residues, for example, 1, 2, 3, 4, 5, 6 consecutive positively-charged residues. Each possibility represents a separate embodiment. In some embodiments, a positively-charged unit (e.g., a lysine unit) is composed of up to three consecutive positively -charged residues (1, 2 or 3 consecutive positively-charged residues). In some embodiments, a peptide according to the present invention does not include lysine residues bound via isopeptide bond.

[0104] A "hydrophobic unit" (e.g., a leucine unit) as described herein refers to one or more consecutive hydrophobic amino acid residues (e.g., consecutive leucine residues). Consecutive hydrophobic residues within a unit may be the same residues or different residues, with each possibility representing a separate embodiment. In some embodiments, a hydrophobic unit is composed of 1-3 consecutive hydrophobic residues, for example 1, 2, 3 consecutive hydrophobic residues. Each possibility represents a separate embodiment. In other embodiments, a hydrophobic unit is composed of 1-9 consecutive leucine residues, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 consecutive leucine residues. Each possibility represents a separate embodiment. In some embodiments, a hydrophobic unit, for example a leucine unit, that is located at a terminus of the peptide (N- or C-terminus) is composed of up to three consecutive hydrophobic residues (1, 2 or 3 consecutive hydrophobic residues), for example up to three consecutive leucine residues (1, 2 or 3 consecutive leucine residues). Each possibility represents a separate embodiment.

[0105] Peptides according to the present invention are characterized by a net charge of between (+7) to (+10), including each value within the range, with each possibility representing a separate embodiment. The net charge of a peptide is the overall charge of the peptide molecule, determined by summing the charges of all its ionizable groups at physiological pH. These ionizable groups include the N-terminal amino group, the C- terminal carboxyl group, and the charged side chains (R-groups) of amino acids within the peptide.

[0106] In some embodiments, peptides according to the present invention are characterized by "relative hydrophobicity" of at least 55% AcN, which refers to relative hydrophobicity that is determined using the reversed-phase HPLC retention time in C18 column using a gradient of 10-90% AcN in ddHiO for 40 min. Relative hydrophobicity in the context of acetonitrile (% AcN) refers to how well a molecule or substance tends to avoid water and prefer an organic solvent like AcN. This preference is quantified by hydrophobicity scales, which assign values to amino acids, peptides, or other molecules based on their tendency to partition into a non-aqueous environment. In reversed-phase chromatography, higher %AcN in the mobile phase generally increases the hydrophobicity of the separation environment, leading to stronger retention of more hydrophobic compounds.

[0107] A peptide as described herein may include a plurality of positively-charged units (namely, at least two positively-charged units). Positively-charged units of a given peptide are not necessarily of the same length, meaning that different positively-charged units within the peptide may be composed of a different number of consecutive positively- charged residues. Similarly, a peptide as described herein may include a plurality of hydrophobic units (namely, at least two hydrophobic units, for example at least two leucine units). Hydrophobic units of a given peptide are not necessarily of the same length, meaning that different hydrophobic units within the peptide may be composed of a different number of consecutive hydrophobic residues. Each amino acid in a unit may be in D- or L-configuration. n some embodiments, a peptide for use according to the present invention is composed of at least three units, wherein the three units may be selected from: two positively-charged units interposed by a hydrophobic unit, and two hydrophobic units interposed by a positively-charged unit. Each possibility represents a separate embodiment.

[0108] A peptide according to the present invention is 15-25 amino acids in length, namely, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length. Each possibility represents a separate embodiment. In some embodiments, the peptide is 15-20 amino acids in length.

[0109] In some embodiments, the hydrophobic residues are selected from the group consisting of leucine, glycine, isoleucine, phenylalanine, alanine and valine residues. Each possibility represents a separate embodiment. In some particular embodiments, the hydrophobic residues are leucine residues. In some particular embodiments, the peptide is composed of lysine and leucine residues only.

[0110] The ratio of positively-charged residues to hydrophobic residues in peptides according to the present invention (for example, between lysine to leucine residues) is in the range of 1: 1.4 to 1: 1.7, including each value within the specified range. Each possibility represents a separate embodiment.

[0111] The ratio between residues in D-configuration to residues in L-configuration in peptides according to the present invention is in the range of 1:2 - 1:5, including each value within the specified range. Each possibility represents a separate embodiment. In some embodiments, the ratio of the residues in D-configuration to residues in L- configuration is in the range of 1:2 - 1:4, including each value within the specified range. Each possibility represents a separate embodiment.

[0112] The D-amino acids in peptides according to the present invention may be consecutive of separate. In some preferred embodiments, a peptide according to the present invention does not include more than two consecutive amino acids in D configuration.

[0113] In some embodiments, the peptide is characterized by relative hydrophobicity in the range of 55-75% AcN, for example 55-70% AcN, or 55-65% AcN, including each value within the specific ranges. Each possibility represents a separate embodiment of the present invention.

[0114] In some embodiments, a peptide for use with the present invention comprises a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12; SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52. Each possibility represents a separate embodiment of the present invention.

[0115] In some embodiments, a peptide for use with the present invention consists of a sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12; SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52. Each possibility represents a separate embodiment of the present invention. In some embodiments, the carboxy terminus of the peptide is modified with a carboxy-terminal blocking group, e.g., amide.

[0116] In some embodiments, a pharmaceutical composition according to the present invention comprising a plurality of peptides as defined herein. A "plurality" indicates at least two peptides.

[0117] In some embodiments, there is provided herein a method of treating malaria in a subject in need thereof, the method comprising administering to the subject a peptide or a conjugate as disclosed herein, or a pharmaceutical composition comprising a peptide or a conjugate as disclosed herein.

[0118] In some embodiments, there is provided herein the use of a peptide or conjugate as disclosed herein, or pharmaceutical composition comprising a peptide or a conjugate as disclosed herein, for the preparation of a medicament for the treatment of malaria.

[0119] In some embodiments, the malaria is caused by a Plasmodium species selected from P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi and P. chabaudi. Each possibility represents a separate embodiment. In some particular embodiments, the Plasmodium species is P. falciparum.

[0120] According to a further aspect, the present invention provides a conjugate comprising a peptide as described herein and an anti-malaria drug.

[0121] In some embodiments, the anti-malaria drug is a small molecule anti-malaria drug. In particular embodiments, the anti-malaria drug is selected from artemisinin or derivatives thereof (e.g., artesunate), and chloroquine or derivatives thereof (e.g., primaquine). Each possibility represents a separate embodiment.

[0122] In some embodiments, a pharmaceutical composition comprising the conjugate is provided, for use in the treatment of malaria.

[0123] In some embodiments, a method for treating malaria in a subject in need thereof is provided, comprising administering to the subject a pharmaceutical composition comprising the conjugate.

[0124] As used herein "treatment" or "treating" refers to stopping or slowing down the progression of malaria. In particular, "treating" includes inhibition of malaria parasites and their spread in an infected subject, and even complete eradication of malaria parasites from an infected subject.

[0125] As used herein "peptide" indicates a sequence of amino acids linked to one another, typically by peptide bonds. In some embodiments, a peptide according to the present invention does not include lysine residues bound via isopeptide bonds. The term "isopeptide bond" is used herein with respect to lysine residues and refers to a covalent bond between the s-amino group of a lysine residue and a carboxyl group of another amino acid. Unlike a standard peptide bond, which involves the a-amino and a-carboxyl groups, an isopeptide bond utilizes the side chain amino group of lysine. In other embodiments, a peptide according to the present invention does not include lysine residues bound via isopeptide bonds except for an isopeptide bond at the C-terminus of the peptide. Thus, in some embodiments, a peptide according to the present invention comprises a single lysine residue that is bound via isopeptide bond, at the C-terminus of the peptide.

[0126] The term “amino acid” refers to compounds, which have an amino group and a carboxylic acid group, preferably in a 1,2- 1,3-, or 1,4- substitution pattern on a carbon backbone, a- Amino acids are most preferred, and include the natural amino acids (which are L-amino acids except for glycine) which are found in proteins and their corresponding D-amino acids.

[0127] Some of the amino acids used in this invention are those which are available commercially or are available by routine synthetic methods. Certain residues may require special methods for incorporation into the peptide, and either sequential, divergent or convergent synthetic approaches to the peptide sequence are useful in this invention. Natural coded amino acids and their derivatives are represented by one-letter codes or three-letter codes according to IUPAC conventions. When there is no indication, the L isomer was used. The D isomers are indicated by “D” or "(D)" before the residue abbreviation or using lowercase letters.

[0128] As used herein, an "amino acid residue" means the moiety which remains after the amino acid has been conjugated to additional amino acid(s) to form a peptide, or to a moiety (such as an anti-malaria drug), typically through the alpha-amino and carboxyl of the amino acid.

[0129] In some embodiments, the carboxy terminus of the peptides described herein is modified. In some embodiments, the carboxy terminus is modified with a carboxy terminal group. In some embodiments, the carboxy terminal group is selected from the group consisting of amide, ester and alcohol group. Each possibility represents a separate embodiment of the present invention. In some particular embodiments, the carboxy terminal group is an amide group.

[0130] In some embodiments, the amino terminus is modified with an amino terminal blocking group. In some embodiments, the amino terminal blocking group is selected from the group consisting of an acetyl and alkyl. Each possibility represents a separate embodiment of the present invention.

[0131] The procedures utilized to construct peptides according to the present invention generally rely on known principles and methods of peptide synthesis, such as solid phase peptide synthesis, partial solid phase synthesis, fragment condensation and classical solution synthesis.

[0132] The peptides according to the present invention may be used in the form of pharmaceutically acceptable salts. As used herein the term “salts” refers to both salts of carboxyl groups and to acid addition salts of amino or guanido groups of the peptide molecule. The term "pharmaceutically acceptable" means suitable for administration to a subject, e.g., a human. For example, the term “pharmaceutically acceptable” can mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. Pharmaceutically acceptable salts include those salts formed with free amino groups such as salts derived from non-toxic inorganic or organic acids such as acetic acid, citric acid or oxalic acid and the like, and those salts formed with free carboxyl groups such as salts derived from non-toxic inorganic or organic bases such as sodium, calcium, potassium, ammonium, calcium, ferric or zinc, isopropylamine, triethylamine, procaine, and the like.

[0133] The term “analog” as used herein indicates the biologically active peptide which has an amino acid sequence according to the invention except for one or more amino acid changes. The term “analog" as used herein also covers analogs which may be prepared from the functional groups which occur as side chains on the residues or the N- or C- terminal groups, by means known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, i.e., they do not destroy the activity of the peptide and do not confer toxic properties on compositions containing it and have comparable or improved activity as a wild-type or unmodified peptide. The term “derivative” may be used interchangeably with “analog”. According to some embodiments, an analog according to the present invention comprises substitutions, deletions or additions of 1 to 3 amino acids. In some embodiments, an analog has at least about 80% identity to the sequence of the peptide of the invention, for example at least about 85%, at least about 90%, at least about 93%, at least about 94%, at least about 95% identity to the sequence of the peptide of the invention.

[0134] Percent (%) sequence identity with respect to a reference sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0135] As used herein, the term "about", when referring to a measurable value is meant to encompass variations of + / -10%, more preferably + / -5%, even more preferably + / - 1%, and still more preferably + / -0.1% from the specified value. Each possibility represents a separate embodiment of the present invention.

[0136] Conservative substitutions of amino acids as known to those skilled in the art are within the scope of the present invention. Conservative amino acid substitutions include replacement of one amino acid with another having the same type of functional group or side chain e.g. aliphatic, aromatic, positively charged, negatively charged.

[0137] Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

[0138] 1) Alanine (A), Serine (S), Threonine (T);

[0139] 2) Aspartic acid (D), Glutamic acid (E);

[0140] 3) Asparagine (N), Glutamine (Q);

[0141] 4) Arginine (R), Lysine (K), Histidine (H);

[0142] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0143] A peptide conjugate according to the present invention comprises a peptide according to the present invention (or a peptide analog as defined above) conjugated to a heterologous moiety, such as a carrier (e.g., a peptide or protein carrier) or moiety which improves the peptide’s solubility, stability or permeability, each possibility represents a separate embodiment of the present invention.

[0144] Water soluble, synthetic polymers, particularly polyalkylene glycols, are widely used to conjugate therapeutically active molecules such as peptides. These therapeutic conjugates have been shown to alter pharmacokinetics favorably by prolonging circulation time and decreasing clearance rates, decreasing systemic toxicity, and in several cases, displaying increased clinical efficacy. The process of covalently conjugating polyethylene glycol, PEG, to proteins is commonly known as "pegylation". Where appropriate, the abbreviation PEG is used in combination with a numeric suffix, which indicates the number of repeating ethylene glycol units. "PEG or derivatives thereof" refers to any compound including at least one polyethylene glycol moiety. PEGs exist in linear forms and branched forms comprising a multi-arm and / or grafted polyethylene glycols. The term "PEG derivative", as used herein, relates to PEG which is modified by alkylation of the terminal hydroxy group. In some embodiments, the terminal hydroxyl group is alkylated by a linear or branched C1-C6 alkyl. A PEG may further comprise a functional group. A PEG may be mono-, di-, or multifunctional polyethylene glycols. Exemplary lengths of PEG chains include 5-20 units, or 10-15 units.

[0145] The present invention further provides pharmaceutical compositions comprising a peptide or conjugate as defined herein and pharmaceutically acceptable excipients. The peptide or conjugate are present in the pharmaceutical composition in therapeutically effective amounts, namely, amounts which are sufficient to provide a therapeutic effect, an anti-malaria effect as disclosed herein.

[0146] The pharmaceutical compositions are typically formulated for systemic administration. Suitable routes of administration for the peptides, conjugates and pharmaceutical compositions disclosed herein include but are not limited to oral, rectal, buccal, nasal, intravenous, intraarticular, intramuscular, subcutaneous and intradermal. Each possibility represents a separate embodiment of the present invention.

[0147] An “excipient” as used herein refers to a non-toxic solid, semisolid or liquid filler, diluent, vehicle, solubilizing agent, encapsulating material or formulation auxiliary of any conventional type, and encompasses all of the components of the composition other than the active pharmaceutical ingredient. The carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as antioxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary.

[0148] The pharmaceutically acceptable excipient(s) useful in the composition of the present invention are selected from but not limited to a group of excipients generally known to persons skilled in the art e.g. diluents such as lactose, starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasic calcium phosphate, sucrose-based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, and bentonite; disintegrants; binders; fillers; bulking agents; organic acid(s); colorants; stabilizers; preservatives; lubricants; glidants / antiadherants; chelating agents; vehicles; hydrophilic polymers; solubility enhancing agents such as glycerin, various grades of polyethylene oxides, transcutol and glycofiirol; tonicity adjusting agents; pH adjusting agents; antioxidants; osmotic agents; viscosifying agents; wetting agents; emulsifying agents; acids; sugar alcohol; reducing sugars; non-reducing sugars and the like, used either alone or in combination thereof.

[0149] Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0150] The following examples are presented in order to more fully illustrate certain embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0151] EXAMPLES

[0152] Materials and methods

[0153] Chemicals and materials

[0154] Peptide synthesis

[0155] Peptides were synthesized by an automated solid-phase peptide synthesizer (CEM

[0156] Liberty Blue peptide synthesizer) on rink amide MB HA resin, using the Fmoc solid-phase strategy (Ben Hur et al. (2022) J. Med. Chem., 65, 9050-9062). The resin-bound peptide was washed thoroughly with DMF and DCM and dried. The addition of a Rhodamine fluorescent probe (Rho) to the N-terminus of selected peptides was performed by standard F-moc chemistry. For the addition of NBD fluorescent labeling, resin-bound peptides were treated with NBD-C1 dissolved in DMF and 5% DIEPA, leading to the formation of resin-bound N-terminal NBD peptides. The resins were washed thoroughly with DMF and then with DCM and dried under a nitrogen flow. Peptide cleavage from the resin was achieved by the addition of 95% TFA, 2.5% double distilled water, and 2.5% TIPS. The resin was filtered from the mixture, and the peptide was precipitated from the mixture using cold EtiC). Following precipitation, the supernatant was carefully decanted, and the resulting pellet, containing the crude synthesized peptide, was obtained for further cleaning (Papo, et al., 2002, J Biol Chem, 277(37), 33913-33921).

[0157] Peptide purification and purity validation

[0158] The purification of the peptides was performed by reverse-phase high-performance liquid chromatography (RP-HPLC) on an Agilent Technologies 1260 Infinity II spectrometer with a reversed-phase Vydac C4 column at a flow rate of 1.8 mL / min and monitored with an ultraviolet (UV) detector at 215 nm. Linear gradients of 10% to 90% acetonitrile in water containing 0.1% TFA were used for peptide purification for 40 min. Final products were obtained by freeze-drying the collected pure fractions. The purity of the peptides was validated with a C18 reversed-phase column (Thermo Fisher Scientific, 250 mm x 4.6 mm, 5 pm particle size) at a flow rate of 0.6 mL / min using a gradient of 10% to 90% ACN in water containing 0.1% (v / v) TFA for 40 min with UV detection at 215 nm. TOF-MS was used to determine the molecular masses of all of the peptides. The purity of all peptides examined for biological activity was >95%. To validate peptide synthesis, properties, and activity, in addition to synthesizing the peptide, the Amp ID peptide was obtained from custom peptide synthesis services, and MSI-78 peptides were obtained from a service lab.

[0159] Plasmodium falciparum parasite culture

[0160] Plasmodium falciparum parasites (NF54) were cultured in pooled donor RBCs at 4% hematocrit and incubated at 37°C in a gas mixture of 1% O2, 5% CO2 in N2 as was previously described (Camacho et al., 2023, Parasit Vectors, 1-25). Parasites were maintained in RPMI medium, 25 mg / mL HEPES, 50 pg / mL hypoxanthine, 2 mg / mL sodium bicarbonate, 20 pg / mL gentamycin and 0.5% Albumax II. Growth was monitored using methanol fixed Giemsa-stained (Merck Millipore) blood smears. Pf cultures were tested for Mycoplasma twice a month using a MycoAlert™ PLUS kit.

[0161] P culture synchronization

[0162] Ring-stage parasites were synchronized twice a week for regular maintenance and at least once prior to experiment plating using D-sorbitol (Lambros et al., 1979, Journal Parasitology, 65 (3), 418-420). Briefly, Pf media were removed from Pf cultures and the blood pellet was incubated at 37 °C with D-sorbitol 5% solution for 5 min. Immediately after, sorbitol was removed by centrifugation and the blood pellet was washed twice with pre-warmed media.

[0163] Growth assay

[0164] Growth assays of malaria parasites were based on a previously described method (Camacho et al., 2023, supra). Flow cytometry analysis was performed using a ZE flow cytometer (Bio-Rad) equipped with 355 nm and 488 nm lasers to detect infected cells based on their signal from the Hoechst-stained DNA (Camacho et al., 2023; Dekel et al., 2017, Methods, 112, 157-166). Gating for infected RBCs was based on a naive RBCs culture. To identify potent antimalarial peptides, a culture of 2% synchronized trophozoite P. falciparum-infected erythrocytes, 4% hematocrit, was incubated with 12.5 pM of different peptides. The culture was collected 24 hours post treatment (hpt) at the rings stage. Growth was monitored using methanol-fixed Giemsa- stained blood smears (Dekel et al., 2017, supra) and flow cytometry (Camacho et al., 2023; Dekel et al., 2017). To identify the IC50 values of potent antimalarial peptides, cultures of 2% synchronized trophozoite P. falciparum-infected erythrocytes, 4% hematocrit, were treated with increasing drug concentrations (0.19-25 pM). To compare the effect of Amp ID on rings versus trophozoite stage parasites, parasites were synchronized and incubated at ring and trophozoite stages with 5 pM of Amp ID for 3 h. Cells were then washed and measured for the relative growth after invasion, 48 hpt and 24 hpt for rings and trophozoites respectively using flow cytometry. Hemolytic activity assay

[0165] The hemolytic activity assay was carried out as previously described (Wani et al., 2021, ACS Infect Dis, 7(6), 1702-1712) with the following alterations: Briefly, naive RBCs were suspended in sterile phosphate-buffered saline (PBS, pH 7.4) at 4% hematocrit. The naive RBCs were exposed to the different peptides at different concentrations (ranging from 0.19-25 pM) and were incubated as at 37 °C under a gas mixture of 1% O2, 5% CO2 in N2. At 1 hpt and 24 hpt the supernatant was taken out and transferred to a 96-well plate. The sample supernatant absorbance was measured at 450 nm using a microplate auto reader (SynersyMx, Biotek). Untreated naive RBCs were used as a negative control, and cells treated with 1% Triton X-100 were used as a positive control. The percentage of hemolysis was calculated as [(sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance)] x 100 (Grieco et al., 2013, Biochim Biophys Acta Biomembr, 1828 (2), 652-660).

[0166] Binding selectivity and binding load analysis

[0167] The binding affinity of NBD-AMPs was tracked using flow cytometry to compare the percentage of P / -iRBCs positive to NBD against the percentage of naive RBCs positive to NBD fluorescent signal. In order to classify the RBC population as naive or Pf- iRBCs, DNA-Hoechst staining was used (Camacho et al., 2023, supra). In addition, an untreated RBC sample was used as a reference to classify naive and P / -infected RBCs as NBD positive or negative controls.

[0168] Subcellular localization of AMPs

[0169] For subcellular localization of NBD-AMPs, synchronized trophozoite-stage parasites (2% parasitemia) were incubated with NBD-AMPs for 8 h. Parasites were stained with Hoechst for visualization of the nucleus. For subcellular localization of Rho- labeled AMPs, trophozoite stage parasites were isolated using a magnet column (Dekel et al., 2021, Nat Commun , 12(1), 1-19) and incubated with labeled AMPs for 30 min. Parasites were stained using Hoechst (2 pM) and Rhodamine 123 (IpM) for visualization of the nucleus and the plasma membrane (Love et al., 2012, Cell Host & Microbe, 12 (6), 815-823), respectively. Cells were imaged using a multispectral image flow cytometry (IFC) (ImageStreamX mark II imaging flow-cytometer: Amnis Corp, Seattle, WA, Part of Cytek) as described before (Alfandari et al, 2022, Front Cell Infect Microbiol, 11). Data were analyzed using the manufacturer's image analysis software (IDEAS 6.3; Amnis Corp). Cells were gated for focused cells, using the Gradient RMS feature and contrast, as previously described (Fendi et al., 2016, Biochem Biophys Res Commun, 478(1), 168— 173). Cropped cells were further eliminated by plotting the cell area of the BF image against the Centroid X feature (the number of pixels in the horizontal axis from the left corner of the image to the center of the cell mask). Then, single cells were gated, using the area and aspect ratio features. Cells positive for either NBD or Rhodamine were gated by using the intensity (the sum of the background-subtracted pixel values within the masked area of the image) and the max pixel (the largest value of the background- subtracted pixel). The gate was set according to untreated cells. To quantify whether the Rho-AmplD is located within the nucleus, a mask was created for the DNA staining (delineating the parasite nucleus) and calculated the peptide intensity within it. An additional mask was created for the rest of the parasite and RBC and calculated the peptide intensity within it. Finally, the ratio between the intensity of the peptide within the nucleus and outside of it was calculated.

[0170] Atomic force microscopy (AFM)

[0171] Synchronized trophozoites (2% parasitemia) were treated with the peptide Amp ID (2.5 p M) for 2 h. Then, using AFM, the effect of Amp ID was quantified on the height and Young's modulus of naive or P / -infected RBCs (Dekel et al., 2021, supra). Cells were identified as naive or infected based on the presence or absence of hemozoin pigment vacuole (Wongtanachai et al., 2012, Southeast Asian J Trop Med Public Health., 43(1): 1-9; Jackson et al., 2004, Mol Microbiol, 54(1), 109-122) in the bright field channel. Data was collected as follows: P / -iRBCs in RPMI media were placed on a freshly cleaved mica surface for 5 min and rinsed with RPMI medium. AFM imaging was performed by a JPK Nanowizard III AFM microscope (Bruker Nano GmbH, Berlin, Germany) in QI mode. Measurements were conducted with a qp-BioAC-CI probe (Nanosensors, Neuchatel, Switzerland), with a spring constant - of 0.06 N / m. In the QI mode, forcedistance curves are recorded at each pixel. These are used to acquire topographic images simultaneously with nano-mechanical data. Force curves from the center of each cell were used to calculate the elastic modulus by applying the contact mechanics Hertzian model (using JPK data processing software version 6.1.86) with a Poisson ratio of 0.5 and a conical tip shape with 22 deg half cone angle. For the elastic modulus measurements on the RBCs, images of 50x50 pm2were captured in 60x60 pixel resolution. Images at higher resolution (20x20 pm2at 100x100 pixel resolution) were also recorded to follow more precisely the differences in cell structure. The force applied on each pixel was 120-140 pN and the approach speed was 30 pm / s. Image analysis was performed using Gwyddion (Necas et al., 2012, Central European Journal of Physics., pp 181-188): an open-source software for SPM data analysis. JPK-SPM data processing software and MountainsSPIP® Academic (Digital Surfe version 10.0.10510).

[0172] Sample preparation for cytoskeleton AFM imaging

[0173] Freshly cleaved mica surfaces were incubated with 0.1% polyLysine solution for 2 h and then rinsed with PBS. This was followed by the deposition of 100 pl P / -RBCs in PBS (106cells per ml) which was incubated for 10 min after which the cells were shear washed with 60-ml 5P8-10 buffer (5 mM Na2HPO4 / NaH2PO4, 10 mM NaCl, pH 8.0) using a syringe at a -20° angle from the surface plane. The cytoskeleton-exposed samples were dried with nitrogen flow and kept in a desiccator overnight (or longer).

[0174] AFM cytoskeleton imaging

[0175] Initial AFM imaging was carried out by using a MultiMode AFM with Nanoscope V electronics (Bruker AXS SAS, Santa Barbara, CA) controlled with Nanoscope 9.2 software (Build R2Srl.130547). Scans were made in PeakForce Tapping mode using a PNP-TRS, Pyrex-Nitride-probes with Silicone Nitride sensor (NanoWorld). To locate and sample enough cells with exposed cytoskeleton for proper statistical analysis images were collected with a fast-scan AFM system, using photothermal off-resonance tapping and small cantilevers, which fit on the base of the commercial MultiMode AFM system (Nievergelt et al., 2018, Nature Nanotechnology , 696-701). Images were collected with custom software. Scans were made with a silicon tip on a silicon nitride cantilever- (Bruker FASTSCAN-C). All cytoskeleton images were analyzed using Gwyddion 2.62 software (Necas et al., 2012, supra). Traditional computer vision and image analysis

[0176] Two independent samples were supplied. The first sample constituted a set of 13 and 15 AFM images of untreated P / -iRBCs and P / -iRBCs treated with AmplD, respectively. The second sample was composed of 14 and 10 AFM images of untreated P / -iRBCs and P / -iRBCs treated with AmplD, respectively. All images were acquired by scanning the center of a cell with an exposed cytoskeleton. Each image captures a 4 pm x 4 pm area with a resolution of 512 x 512 pixels.

[0177] The computational pipeline was applied on two samples, independently, and composed of two parts.

[0178] In the first part, the cytoskeleton backbone was segmented in each image. To achieve satisfactory segmentation, a few steps were applied. First, Gaussian blurring was applied for each image with a kernel size of 5x5 to reduce noise and make binarization more robust. Next, each image was binarized to yield the segmented backbone. For each image a unique threshold value for binarization was calculated. To determine a unique threshold value, a pixel value distribution was obtained for each image. Then, for a shape distribution of a valley between two maxima, the threshold value was chosen as the local minima in between the two maxima. Alternatively, for a shape of distribution of maxima between two minima a different approach was applied. The distribution was clustered to yield 8 cluster centers, and then the first 3 cluster centers were averaged to yield a threshold for a good backbone segmentation. This enables calculating the total size of the filaments (backbone) in each image. Then, each image was inverted, so the backbone had a value of 0 and all other image features had value of 1. Contours were calculated for all image features that were not the backbone for visualization purposes. In addition, connected component analysis was applied to label the image features. This enables the calculation of the image features’ area.

[0179] In the second part of the pipeline, each image (4 pm x 4 pm, 512x512 pixels) was sliced into 8 images of (0.5 pm x 0.5 pm, 64x64 pixels). Then, the mean roughness (Ra) was calculated for the sliced images. Slicing was applied to reduce the parasite crystals' effect on the image roughness and make the statistics more robust and specific to the cytoskeleton network. All pipeline calculations were fully automated.

[0180] All code was written in Python 3.9.13 (Van Rossum, Centrum Voor Wiskunde En Informatica Python Reference Manual). OpenCV library (4.9.0, opencv.org) was applied for traditional computer vision together with Scikit-learn and Scipy (1.9.1) libraries (Virtanen et al., 2020, Nat Methods, 17(3), 261-272).

[0181] Large Unilamellar Vesicles (LUV) preparation

[0182] LUV preparation was conducted as previously described (Rotem et al., 2016, Biochemistry, 55(7), 1049-1057). Briefly, Large Unilamellar Vesicles (LUVs) from PC, PC / PS (1: 1, wt / wt), PC / PS (3: 1, (wt / wt) and PC / Cholesterol (9: 1, wt / wt), which mimic the outer membranal leaflets of cholesterol depletion post-parasite invasion, additional negative charge on the outer membrane of P / -iRBCs and uninfected (naive) red blood cells, receptively. Lipids were dissolved in chloroform / DCM (3: 1) and evaporated using nitrogen stream, followed by 3-24 h of vacuum pumping or lyophilization. The lipid film was then hydrated with PBS solution (osmol=300 mOsmo / kg) to reach the desired concentration, and the solution was gently vortexed. The resulting MLV (multilamellar vesicles) suspensions were sonicated for 15 min to disperse larger aggregates. The vesicles were subsequently downsized by extrusion through 1pm, 400 nm, 200 nm, and 100 nm polycarbonate membranes. Extrusion was performed 11 times through each membrane. The liposomes' size was measured using Malvern NanoSight NS300, Malvern, UK (NT A) (Figure S4C). Samples were diluted with PBS. Optimal dilution for each sample was found by pre-testing the sample until an ideal particle-per-frame value (30-100 particles / frame) was obtained. For each measurement, five 1 min videos were captured at 25°C, with at least 300 pL displacement between each video. The number of completed tracks in NTA measurements was always greater than the proposed minimum of 1000 to minimize data skewing based on single large particles.

[0183] AMP binding affinity to LUV assay

[0184] AMPs binding affinity to LUV assay was conducted as previously described (Saar- Dover et al., 2013, Methods in Molecular Biology, 1033, 173-183). Briefly, binding equilibrium (Bmax) was determined by the varying lipid / peptide ratios, and the affinity constant was calculated from the relationship between the equilibrium emission level and lipid concentration using a steady-state affinity model. The affinity constants (Ka) were determined using a steady-state affinity model with non-linear least squares (NLLSQ) analysis. The NLLSQ fitting was done using the following equation: where X is the lipid concentration, Y(x) is the fluorescence emission, Bmax is the maximal difference in the emission of peptide before and after the addition of lipids (it represents the maximum amount of peptides bound to a lipid), and Kais the affinity constant. The accessibility of the peptides to proteolytic cleavage in the membrane-bound state was measured using Proteinase K (125 pg / mL), which was added to each well to validate the insertion of the peptides into the lipid vesicles. In a control experiment, a labeled peptide was first added to the solution containing the enzyme, followed by the addition of LU Vs.

[0185] NBD fluorescence measurements

[0186] The fluorescence of an NBD-labeled peptide increases upon transfer from a solution to a hydrophobic environment (Saar-Dover et al., 2013, supra). NBD-labeled peptides were diluted in PBS to a concentration of 0.1 pM, either alone or in the presence of LUV s. LUVs were employed to minimize differential light scattering effects, maintaining a high lipid / peptide molar ratio (2000: 1) to ensure negligible spectral contributions from free peptides. The fluorescence was recorded with excitation set at 470 nm and an emission spectrum from 510 nm to 610 nm. Under these conditions, most of the peptide was bound to the vesicles.

[0187] Cryo-EM sample preparation and imaging

[0188] A sample solution of 3 pL was applied to the carbon side of lacey carbon 200 mesh copper grid (Electron Microscopy Sciences, USA), which was glow discharged for 120s at 0.5 mbar and 18 W with Evactron CombiClean System (XEI Scientific, USA). Samples were blotted for Is from both sides at 4°C and 100% humidity and plunged frozen into the liquid ethane cooled by liquid nitrogen using Vitrobot Mark IV (Thermo Fisher Scientific, USA). Cryo-EM images were collected using a Tales Arctica TEM, equipped with a Falcon 4i direct detector (Thermo Fisher Scientific, USA) and operated at 200 kV. Image acquisition was performed using Tomography 5.16 and Velox 3.8 software (Thermo Fisher Scientific, USA) at a nominal magnification of 17.500x (pixel size 1.64 nm, defocus 10-15pm), 45.000x (pixel size 0.62 nm, defocus 5-8 pm) or 120.000x (pixel size 0.118 nm, defocus 3-5pm) with a total dose not exceeding 100 e- / A2. Hemolytic activity under cholesterol depletion

[0189] Naive RBCs were subjected to treatment / mock treatment for cholesterol depletion using 3.12 M of methyl-beta-cyclodextrin (MpC) for 30 min. Then, MpC was washed and the cells were subjected to 5 pM Amp ID for 1 h (AmplD+MpC group). The nontreated group (NT) was used as a control, as well as Amp ID (5 pM) treated RBCs (Amp ID group), a MpC treated cells (MpC group) and a positive control group exposed to 1% Triton X-100. was collected and analyzed as explained above under the "Hemolytic activity assay" section, with the following modification: The relative hemolysis of the AmplD+MBC group was normalized to the MpC group, while the relative hemolysis of the Amp ID group was normalized to the NT group.

[0190] Statistical analysis

[0191] All statistical analyses were carried out on the mean of at least three independent biological replicates using "R" version > 4, as indicated in each figure legend. The mean of each set of biological repeats is represented in all cases. Multiple comparisons were performed where needed, as indicated in the respective figure legends, and p-values were calculated to show statistical significance.

[0192] Example 1 - Antimicrobial peptides (AMPs) inhibit malarial growth

[0193] AMPs comprised of lysine (K) and leucine (L) residues in both D and L enantiomer configurations, identified as "D,L-KL peptides", were previously shown to have therapeutic potential against bacteria without hemolytic and cytotoxic effect (Papo et al., 2002, The Journal of Biological Chemistry, 277(37), 33913-33921; Ben-Hur et al., 2022, J Med Chem, 65(13), 9050-9062; Segev-Zarko et al., 2015, Biochem J, 468(2), 259-270). Yet, AMPs are not intuitively a straightforward choice for combating malaria. This is mainly due to the asexual blood stage, in which the intracellular Pf parasite is enclosed within its host RBC and lacks direct contact with the extracellular environment. Hence, there is uncertainty about whether such peptides could penetrate the host cell and access the encapsulated parasite.

[0194] The membrane of P / -infected RBCs differs greatly from naive cells (Paulitschke et al., 1993, J Lab Clin Med., 122(5):581-9; Kutner et al., 1983, J Cell Physiol, 114(2), 245— 251; Mauritz et al., 2009, PLoS Comput Biol., 5(4):el000339). The present experiment tested the efficacy of synthetic de novo designed AMPs (Table 1) in the context of Pf growth. In particular, their effect on P / (NF54 strain) growth in culture was tested.

[0195] Table 1 - Peptide identification and properties isopeptide bond formation. All the peptides were amidated on at their C-terminus.bThe RP-HPLC retention time in the C18 column using a gradient of 10-90% ACN in water for 40 min.cHydrophobic moment (pH) The peptides identified as "AmplD", "Seg5D", and "Seg6D" are D,L-KL peptides (Papo et al., 2002, supra', Ben-Hur et al., 2022, supra', Segev-Zarko et al., 2015, supra', Saar-Dover et al., 2013, Methods in Molecular Biology, 1033, 173-183), which differ in the distribution of their positively charged lysine residues. AmplD has an even distribution of lysine residues compared to Seg6D and Seg5D, with lysine residues grouped in the center or at the margins of the molecule, respectively (Figure 1A).

[0196] The peptide identified as "4DK5L7" (Shprung et al. 2021, Biochemistry, 60(39), 2943-2955; Kapach et al., 2020, Front Microbiol, 11: 189) is a variant representing a shorter version of the D,L-KL peptides, 12 residues compared to the 15 residues in the other KL peptides on the list (Table 1).

[0197] The peptides identified as "AmplEP", "AmpEP9", and "AmpEPIO" (Wani et al., 2021, ACS Infect Dis, 7(6), 1702-1712; Wani et al., 2022, J Med Chem, 65(6), 5085- 5094) are KL peptides with lysine residues that form a peptide bond with their s-amino group, termed an isopeptide bond.

[0198] The study further included AMPs such as the human AMP "LL-37" (Chennupati et al., 2009 , Am J Rhinol Allergy , 23(1), 46-51; Lofton et al., 2013, PLoS One, 8(7): e68875; Overhage et al., 2008, Infect Immun, 76(9), 4176-4182; Oren et al., 1999, Biochem J., 341(Pt 3): 501-513), "MSI-78" (Hallock et al., 2003, Biophys J, 84(5), 3052-3060) (Figure 1A) (commercially known as pexiganan), "MSIEP" (Wani et al., 2021, supra), which is an analog to MSI-78 with isopeptide lysine residues, and the toxic AMP Melittin as a control.

[0199] All peptides in this study shared common characteristics, including the presence of hydrophobic amino acids and a net positive charge ranging from +5 to +10 ("Net ch." in Table 1). Their molecular weights fell within a similar range (approximately spanning from 1450 g / mol to 4490 g / mol). However, the hydrophobic moment (pH), which affects the insertion of the AMP to the membrane (Fink et al., 2024, ACS Pharmacol Transl Sci, 7(6): 1795-1806), varied among the peptides. All peptides were designed and structured using ChemDraw, synthesized on rink-amide resin using a peptide synthesizer, purified, and assessed for purity via RP-HPLC. All the peptides were characterized using MALDL TOF mass spectrometry. To identify peptides with anti-P / activity, the AMPs (12.5 pM) were incubated with trophozoite- stage synchronized Pf cultures for 24 h. The growth inhibition effect of each AMP was measured using flow cytometry as described above.

[0200] The assay with the peptides Amp ID, Seg5D, Seg6D, 4DK5L7, AmplEP, AmpEP9, AmpEPIO, LL-37, MSI-78, MSIEP and Melittin showed that AmplD and Seg5D (D,L- KL family, more particularly D.L-KeLg family), and MSI-78 (a synthetic analog of a natural peptide) significantly inhibited Pf growth leading to a significant reduction in parasitemia levels (Figure IB) while exhibiting low hemolytic activity (Figures 1B-1C). The peptide Seg6D was partially active while exhibiting low hemolytic activity (Figures 1B-1C). The human AMP LL-37 exhibited potent anti-P / activity but also high hemolytic activity, leading to the destruction of naive RBCs (Figures 1B-1C), as previously described (Love et al., 2012, supra). 4DK5L7 was found to be inactive (Figure IB).

[0201] The analogs of AmplD and MSI-78 with isopeptide lysine residues (AmplEP AmpEP9, AmpEPIO and MSIEP) displayed low relative activity (Figure IB). These data suggest that incorporating lysine residues in their isopeptide bond form resulted in loss of anti-P / activity. Thus, the specific chemical and biophysical properties of the peptides rather than the amino acid sequence itself appear to be crucial factors contributing to the potency of anti-P / activity in AMPs.

[0202] A dose-response assay was then carried out to evaluate the potency of AmplD, MSI-78 and Seg5D on Pf growth. The human LL-37 peptide was also evaluated in this dose-response assay. The toxic peptide Melittin was used as a positive control. Half- maximal inhibitory concentration (IC50) values were obtained after treating synchronized trophozoite- stage Pf cultures for 24 h. The results are shown in Figure ID. With the exception of Melittin (IC50 =0.921 pM), AmplD exhibited the highest potency (IC50 = 2.5 pM), followed by LL-37 (IC50 = 9.0 pM), Seg5D (IC50 = 9.0 pM) and MSI-78 (IC50 =10.2 pM).

[0203] Next, the anti-P / activity of AmplD was tested on specific parasitic stages - ring stage and trophozoite stage. In order to isolate the effect on each specific stage and avoid transition between these stages that occurs in a 24h exposure to AMPs, AmplD was applied on either ring or trophozoite- stage parasites for a short 3h pulse (5 pM), followed by a wash. The results are shown in Figure IE. Noticeably, trophozoite- stage parasites exhibited higher sensitivity to Amp ID, and their relative growth was reduced compared to the ring-stage parasites.

[0204] Overall, the two D,L-KL AMPs, AmplD and Seg5D, demonstrated significant anti- Pf activity combined with negligible damage to naive healthy human RBCs, which suggests a selective activity against P / -iRBCs.

[0205] Example 2 - Novel AMPs that inhibit malarial growth

[0206] The following peptides were designed and synthesized:

[0207] "AmplD+LN", also referred to herein as "Bl" (SEO ID NO: 12):

[0208] LLKLLKKLLKKLLKLL

[0209] The peptide is a derivative of AmplD which includes an added leucine residue at the N-terminus. The peptide is characterized by a net positive charge of +7, MW of 1917 g / mol, and pH (hydrophobic moment) of 0.874.

[0210] "Amp4D", also referred to herein as "B2" (SEO ID NO: 13):

[0211] LLKKLLKLLLKLLKK

[0212] The peptide is a further D,L-KL peptide (in particular, D,L-KeL9 peptide) characterized by even distribution of K and L residues. The peptide is characterized by a net positive charge of +7, MW of 1805 g / mol, and pH (hydrophobic moment) of 0.782.

[0213] Underlined amino acids are D-enantiomers. The peptides were amidated at their C- terminus

[0214] A dose-response assay testing anti-P / activity of the novel peptides was carried out as described in Example 1. The results are shown in Figure IF. The two peptides were highly potent - Amp4D provided IC50 = 2.833 pM, and AmplD+LN provided IC50 = 5.322 pM.

[0215] Example 3 - Anti-Pf AMPs selectively bind to / 7-iRBCs

[0216] The present experiment investigated the binding ability of AmplD, Seg5D and MSI-78 to either P / -iRBCs throughout the parasite blood stages (rings, trophozoites, schizonts) or naive RBCs. LL-37 was used as a control more prone to cause hemolysis and less active against Pf compared to the three other tested AMPs.

[0217] In order to track the binding of the peptides, the small fluorophore nitrobenzoxadiazole (NBD), which emits a fluorescent signal in a membranal environment (Amaro et al., 2016, Physical Chemistry Chemical Physics, 18(10), 7042- 7054), was conjugated to each peptide at the N-terminus (Fendi et al., 2016, supra). Importantly, since each AMP molecule incorporates a single NBD molecule, the signal intensity is a reliable proxy for the quantity of peptides bound to the cells (Papo et al., 2003, Journal of Biological Chemistry, 278(23), 21018-21023), that is, the binding load (Figure 2C).

[0218] Synchronized trophozoite- stage parasites were treated with either NBD-AmplD, NBD-Seg5D, NBD-MSI-78 or NBD-LL-37. The binding of the fluorescent peptides to P / -iRBCs or naive RBCs was monitored in time points throughout 24h using flowcytometry as illustrated in Figure 2A.

[0219] Remarkably, despite P / -iRBCs representing only about -2% of the cells in the sample (i.e., 2% parasitemia), NBD-AmplD exhibits an exclusive binding to the infected host cells and not to the naive RBCs within the same sample, under both IC50 and IC25 concentrations (Figure 2B, top and bottom panels, respectively). Furthermore, within the population of the infected cells, it was found that the interaction of the NBD-AmplD to the host membrane undergoes changes during the parasitic developmental blood stages, reaching its peak binding at the late trophozoite- schizont stage (Figure 2B). In contrast, NBD-LL-37 was more prone to bind naive human RBCs unselectively (Figure 2B), indicating it has weaker binding preference between infected or naive RBCs. The two additional active anti-P / AMPs, the D,L-KL AMP NBD-Seg5D and NBD-MSI-78 demonstrated preferred binding affinity to P / -iRBCs in a similar trend to NBD-AmplD (Figure 2B, right panel).

[0220] The flow cytometry binding data was then analyzed using the signal intensity of NBD, as it was reported to be correlated with AMPs activity (Saar-Dover et al., 2013, supra). The analysis revealed that up to 8h the signal intensity of NBD-AmplD was significantly higher for P / -iRBCs compared to the signal derived from control naive RBCs (Figure 2C). The intensity of the peptide binding signal reached peak intensity four hours post treatment. These results correlate with the observed increased growth inhibition of trophozoite- stage parasite after exposure to Amp ID (Figure IE). In contrast to the binding load results of Amp ID, the difference in the binding load of NBD-LL-37 was less pronounced between P / -iRBCs and naive RBCs (Figure 2C), correlating with its lack of binding selectivity (Figure 2B). To determine the subcellular localization of the set of NBD-AMPs within Pf- iRBCs, imaging flow cytometry (IFC) was utilized (Shmuel-Galia et al., 2017, Journal of Biological Chemistry, 292(32), 13415-13427; Cohen et al., 2023, ACS Omega, 8(20), 17856-17868). The anti-P / active peptides AmplD, LL-37, Seg5D and MSI-78 were tested. Based on the observed peak binding, the NBD-AMPs were incubated with synchronized trophozoite- stage parasites for eight hours. The peptide 4DK5L7 was used as inactive peptide control. In line with the binding selectively results of AmplD, a robust fluorescent signal was detected in P / -iRBCs treated with NBD-AmplD both at IC50 and IC25 concentrations (Figure 2D, top and bottom panels, respectively), but not in naive RBCs (Figure 2D, top panel). Interestingly, the signal from NBD-AmplD was observed to be co-localized with the nucleus of Pf similarly to the other active peptides Seg5D and MSI-78 (Figure 2D). In contrast, a signal from the inactive peptide 4DK5L7 could not be detected (Figure 2D). These results suggest the three active peptides can penetrate the plasma membrane of the infected host RBC and access the encapsulated parasitic cell. Opposingly, the detected signal for peptide NBD-LL-37 mainly appeared outside of the cells, within small extracellular structures that are positive for DNA signal (Figure 2D), which might suggest that those structures are post-egress merozoites.

[0221] To confirm the potential nuclear subcellular localization of AmplD within Pf- iRBCs, AmplD was labeled using a Rhodamine fluorophore (Rho-AmplD). The difference between Rhodamine and NBD lies in the fact Rhodamine’ s emits a fluorescent- signal irrespective of membrane binding. Indeed, the accumulation and diffusion of Rho- AmplD towards the parasitic nucleus within P / -iRBCs could be visualized using imaging flow-cytometry (Figure 2E). In addition, on average the signal intensity of Rho-AmplD near the nucleus is stronger compared to regions external to the nucleus (Figure 2F).

[0222] Together, these results suggest that AmplD can exclusively penetrate the multiple membranes of P / -iRBCs and ultimately reach the interior of the parasitic cell. The binding rate and intensity peaked during the late developmental blood stages, namely, the late trophozoites-schizonts (Figures 2B-C), suggesting that it may be driven by membranal alterations occurring in the infected cells at this stage of development (Flammersfeld et al., 2018, International Journal of Medical Microbiology, 308(1), 129-141. Example 4 - AmplD selectively disrupts the membranal and cytoskeletal structure of / 7-iRBCs

[0223] To elucidate how AmplD affects the biophysical properties of P / -iRBCs, atomic force microscopy (AFM) was used to measure the structure and stiffness of the cells (Dekel et al., 2021, supra). AmplD was incubated with a synchronized trophozoite- stage Pf culture. The alterations in the cell stiffness of Amp ID-treated and untreated naive RBCs and of P / -iRBCs was analyzed. P / -iRBCs were identified based on the presence or absence of hemozoin pigment vacuole (Wongtanachai et al., 2012, supra', Jackson et al., 2004, supra) in bright field (BF) and by areas with higher Young's modulus containing parasites (Figure 3A).

[0224] Although Pf is hidden within the host RBCs, AmplD (2.5 pM) induced exclusive structural changes in the membrane of P / -iRBCs, 2 hpt. While the mean sampled height values from treated P / -iRBCs (+AmplD) were lower compared to untreated (NT) Pf- iRBCs, naive RBCs in the culture did not show a similar response, exhibiting no significant change in height after treatment (Figure 3B).

[0225] To further investigate the differences in cell structure, the resolution of the atomic force microscopy (AFM) scans was increased by approximately four times (Figure 3C). Under the tested experimental conditions, naive RBCs and P / -iRBCs adhere to the surface with a round, dome-like morphology (Figures 3A and 3C). Noticeably, upon exposure to AmplD, P / -iRBCs lost their round dome structure characteristic, and the membrane of the host RBC appears to deflate and collapse (Figures 3A and 3C). In contrast, untreated P / -iRBCs maintain their round, dome-like morphology (Figures 3A and 3C). These observed differences in cell topography may indicate alterations in the P / -iRBCs' cell membrane. AmplD treatment could compromise the infected cell's ability to maintain its inflated, dome-like shape, leading to a collapsed morphology around the parasite. In agreement with the change in the height of P / -iRBCs (Figure 3B), the mean Young’s modulus of treated P / -iRBCs was reduced compared to untreated P / -iRBCs (Figure 3D).

[0226] Having established that the effect of AmplD is restricted to P / -iRBCs, further experiments tested the influence of AmplD on the structure of P / -iRBCs' cytoskeleton. RBCs mechanical properties are strongly influenced by the underlying cytoskeleton network (Dekel et al., 2021, supra; Millholland et al., 2011, Molecular and Cellular Proteomics, 10(12): Ml 11.010678; Li et al., 2007, Proc Natl Acad Sci USA, 104(12):4937-42; Sinha et al., 2015, Sci Rep, 5, 9768). Trophozoite-stage parasites were isolated using a magnetic column (Figure 4A) and treated with Amp ID for 30 min. To expose the cytoskeleton of P / -iRBCs treated or untreated, cells adsorbed onto poly-L- ly sine-modified mica were washed with a hypotonic solution and then scanned in a dry state using atomic force microscopy (AFM) (Figure 4A). Figures of 4pm x 4um were scanned in the center of each exposed cell cytoskeleton and used as an input for the computer vision and image analysis pipeline. Notably, two significant structural differences in the phenotype of the cytoskeleton were observed between the untreated Pf- iRBCs and P / -iRBCs treated with Amp ID. First, each image was segmented to identify the backbone area (Figure 4B) and the filaments coverage area was calculated in each image (Figure 4C). It was found that the untreated P / -iRBCs have a statistically significant higher percentage of area covered by filaments than the treated P / -iRBCs. This may indicate that the disrupted skeleton of the treated P / -iRBC cells was washed away in the preparation procedure. This leads to a lower coverage of filaments in the scanned area. Secondly, the mean roughness (Ra - profile height deviations from the mean line) was calculated on 0.5 pm x 0.5 pm areas covered with a cytoskeleton network (Figure 4D). The mean roughness was found to be larger in the untreated P / -iRBCs in comparison to the treated P / -iRBCs, indicating that the filaments lost their structural height characteristics due to the treatment.

[0227] Overall, these results indicate that Amp ID reshapes the mechanical properties of P / -iRBCs both at the membrane and cytoskeleton levels. In addition, the exclusive interaction of Amp ID with P / -iRBCs suggests its selective MoA is likely dependent on specific membrane components.

[0228] Example 5- Membranal cholesterol level determines the activity of AmplD

[0229] The present experiment sought to identify the key feature in the membrane of Pf- iRBCs that enhances the peptide interaction. AMPs' activity is positively correlated with reduced cholesterol and increasing negative charge in the pathogens' membrane (Shai et al., 2002, Biopolymers - Peptide Science Section, 66(4), 236-248; Vale et al., 2014, Front Pharmacol, 5(DEC): 275). Additionally, Pf dramatically changes the host RBCs' membrane, causing a reduction in cholesterol levels as well as exposure of negatively charged PS lipids (Brown et al., 2020, Sci Rep, 10(1), 1-15; Maguire et al., 1991, Parasitology, 102(2), 179-186; Kaushik et al., 2012, Malar J, 11, 256). It was therefore questioned whether the cholesterol and the phosphatidylserine (PS) modifications in the host membrane of the P / -iRBCs' play a role in the selective binding of Amp ID.

[0230] To address that and investigate the nature of the interactions between Amp ID and the host membrane of P / -iRBCs, LUVs were utilized as a membranal model (Papo et al., 2005, Journal of Biological Chemistry, 280(11), 10378-10387; Sam-Yellowe et al., 1991, Exp Parasitol, 171, 161-171). NBD was used as a fluorescent probe to monitor the binding of NBD-AMPs to the lipidic bilayer of the LUVs (Saar-Dover et al., 2013, supra). In addition, the change in NBD’s emission spectra towards the blue edge of the spectrum, blue shift, was monitored upon binding to LUVs, to better understand its localization inside the hydrophobic lipidic core (Amaro et al., 2016, supra', Gazit and Shai, 1993, Biochemistry, 32(46), 12363-12371; Ashkenazi et al., 2013, Blood, 121(12):2244-52; Hiratsukasg and Katol, 1987, J Biol Chem., 262(13):6318-22).

[0231] To model the membranal lipid composition of naive RBCs a mixture of PC: cholesterol (Makovitzki et al., 2006, Proc Natl Acad Sci U S A, 103(43), 15997- 16002) was used and the membranal changes post-invasion were examined using: i. LUVs comprised of only phosphatidylcholine (PC) to represent the impact of cholesterol depletion post-parasite invasion (Hernandez-Castaneda et al., 2021, Front Immunol, 12: 643746) and ii. a mix of PC and negatively charged PS (PC:PS) to simulate the effect of additional negative charge on the outer membrane of P / -iRBCs due to the exposure of negatively charged PS post-parasite invasion. The peptide's affinity for phospholipid membranes was assessed through titration of NBD-labeled peptide with LUVs (Figure 5A, 5B). Then, based on the emitted signal from the interaction of NBD- AMPs with the LUVs, the affinity of the AMPs to different LUVs (Kd) was measured (Figures 5A, 5B, 5D). In addition, the maximal fluorescence value (Bmax) was used as a proxy to the binding load of the AMPs 4DK5L7, AmplD, LL-37, Melittin, MSI-78, and Seg5D to the LUVs (Figure 5C).

[0232] It was found that the presence of cholesterol in PC:Cholesterol LUVs significantly reduced the binding affinity (increased Kd) of AmplD, compared to LUVs with the absence of cholesterol (PC and PC:PS LUVs) (Figure 5A). These results were similar for other peptides with anti-P / activity, Seg5D and MSI-78 (Figure 5A). Notably, Melittin and LL-37, which have shown relatively higher hemolytic activity were less affected by the presence of cholesterol (Figure 5A). Surprisingly, although Amp ID and other AMPs are positively charged, the additional negative charge in PC:PS LUVs did not improve the binding affinity (Figure 5D). This indicates that the presence of cholesterol rather than the absence of PS, as in the naive RBCs, prevents the activity of AMPs and promotes the selective binding of anti-P / AMPs. Surprisingly, in LUVs with a combination of PC, PS and Cholesterol, the addition of PS increases AMPs affinity compared to PC: Choi LUVs (Figures 5A, 5B). However, the affinity remained lower compared to the Pf postinvasion LUV model, PC (Figures 5A, 5B).

[0233] Binding load is a good predictor of AMP activity (Saar-Dover et al., 2013, supra), possibly due to lower levels of peptide oligomerization (Fink et al., 2024, supra', Shai et al., 1999, Biochim Biophys Acta Biomembr, 1462(1-2), 55-70. Therefore, the maximal binding load of six NBD labeled peptides (4DK5L7, AmplD, LL-37, Melittin, MSI-78, and Seg5D) to LUVs was analyzed by using the maximal emission, Bmax, as an approximation. Interestingly, a significant change in Bmax level was detected when AMPs were introduced to PC: Cholesterol LUVs (Figure 5C). The peptides AmplD, Melittin and LL-37 showed higher Bmax levels than 4DK5L7 Seg5D and MSI-78 (Figure 5C). These results suggest that AMPs may need to achieve a minimal binding load when encountering P / -iRBCs' membrane to manifest as an active anti-P / AMP.

[0234] The subcellular localization of AMPs labeled at their N-terminal with the fluorescent probe NBD was further investigated within the membrane milieu. Fluorescence emission spectra of these NBD-labeled peptides were recorded in PBS and in the presence of various combinations of LUVs to measure the insertion of AMPs into the lipidic environment (Figure 5E). The altered spectra indicates the relocation of the NBD group into a more hydrophobic environment, assessed by the degree of blue shift. Greater blue shift suggests deeper peptide insertion into the membrane, implying a higher propensity for channel formation due to interactions with the membrane's hydrophobic core (Shai et al., 1999, supra). However, smaller or no shifts indicate peptides remaining on the surface. Overall, in AmplD a blue shift of range -3.8 to -5.5 nm was observed, LL- 37 and Melittin showed a stronger shift of -7.1 range to -9.3 nm and -5.0 to -15.4 nm respectively (Figure 5E, left panel). Additionally, 4DK5L7, MSI-78 and Seg5D showed a blue shift range -3.8 to -8.2 nm, -3.6 to -6.8 nm, 0 to -6.3 nm, respectively, except the blue shift occurred with the interaction of Seg5D peptide with PC:PS that showed a blue shift of -13.6 nm (Figure 5E, right panel). These spectral changes indicate for the NBD moiety's relocation to a more hydrophobic environment, consistent with its positioning within the membrane. Thus, the observed differences in blue shifts among the peptides indicate distinct mechanisms of interaction with the membrane.

[0235] Lastly, the interaction of Amp ID on PC LUVs was investigated by visualizing its effect. PC LUVs were exposed to Amp ID (at a ratio of 100: 1 Lipids:peptide) and imaged using cryo-EM. The cryo-EM images revealed that Amp ID disrupts a fraction of the PC vesicles, resulting in the formation of small fragments (Figure 5F). Additionally, the addition of Amp ID induced lipid aggregation and deformation to the LUVs, causing a formation of structures resembling nanodiscs and sheets that were not observed in untreated PC LUVs (Figure 5F). Certain amphiphilic proteins can assemble into lipid- protein nanodiscs. In these assemblies, proteins wrap around lipid bilayer discs and reduce line tension at the bilayer edge. These phenomena are shown also with 1- 1- palmitoyl-2-oleoyl-glycero-3 -phosphocholine (POPC) LUVs with interaction HNP1 and LL-37 peptides (Drab et al., 2020, Biophys J, 119(12), 2440-2450; Denisov et al., 2004, J Am Chem Soc, 126(11), 3477-3487; Xu et al., 2013, Cell Death Dis, 4(6): e683).

[0236] Together, the data indicate that the activity of anti-P / AMPs strongly depends on the reduced levels of cholesterol within the host's RBCs during Pf infection. Both the binding affinity and Bmax levels of the LUVs membranal model could be used to evaluate anti-P / activity and cytotoxicity of AMPs.

[0237] Example 6 - Cholesterol depletion from naive RBCs increases their susceptibility to the lytic activity of AmplD

[0238] Based on the LUV models, the present experiment tested whether the high level of cholesterol in the plasma membrane protects naive RBCs from the hemolytic effect of AmplD. Initially, the cholesterol levels in naive RBCs were reduced using methyl-beta- cyclodextrin (MpC) (Ahiya et al., 2022, Microbiol Spectr, 10(1): e0015822). The treated RBCs were then exposed to 5pM Rho-AmplD, a non-hemolytic concentration, and recorded their interaction using IFC (Figures 6A-6B).

[0239] Noticeably, only upon cholesterol depletion, Rho-AmplD was able to bind to the RBCs in sufficient quantity to reach detectable fluorescent signals in the cells (Figures 6A-6B). Moreover, post-cholesterol depletion from the naive cells, AmplD exhibited significant hemolytic activity (Figure 6C) under a non-hemolytic concentration. These results further support the importance of cholesterol in the preferred interaction of the D,L-KL peptide with P / -iRBCs under the LUV model data (Figure 4).

[0240] Example 7 - Meta-analysis of anti-P / activity

[0241] A meta-analysis of the anti-P / activity data suggests that a critical hydrophobicity threshold exists for achieving anti-P / activity (Figure 7A). In addition, the data suggest that a minimal length of 15 amino acids is needed. For example, 4DK5L7, a truncated version of Amp ID containing only 12 aa, lacked anti-P / activity, although it was previously found to be active against other pathogens (Kapach et al., 2020, Front Microbiol., 11: 189).

[0242] The above described data demonstrate how Amp ID binds and crosses the membrane of P / -iRBCs, deforms it biophysically and induces irregularity in their cytoskeleton. These results provide evidence of the NRM MoA used by Amp ID and strengthen the possibility it can be used as an anti-P / drug without inducing drug resistance. Additionally, the data identified that cholesterol reduction in RBCs post Pf invasion is a key component in mediating D,L-KL peptides' exclusive interaction with P / -iRBCs. The observed sensitivity of Amp ID to cholesterol but not to PS level demonstrates its ability to interact selectively with P / -iRBC and with naive RBCs or with RBCs with higher exposure of PS due to Pf infection. Thus, heightened AmplD distinct advantage in the context of Pf infection.

[0243] The AmplD signal co-localized with the nucleus signal of Pf This finding is particularly intriguing given that the parasite vacuole membrane (PMV) contains higher cholesterol levels than the P / -iRBCs membrane. Typically, increased cholesterol within the membrane is associated with greater rigidity and stability which would theoretically reduce the efficacy of AMPs. This unexpected result suggests that AmplD may possess unique properties or MoA that enable it to effectively target and interact with the PMV despite these biophysical challenges.

[0244] A cluster analysis of the blue shift showed closer hierarchical clustering of AMPs with hemolytic activity, namely LL-37 and Melittin, compared to AMPs without hemolytic activity, namely AmplD, Seg5D, MSI-78, and 4DK5L7 (Figure 7B). Example 8 - AmplD in combination with artesunate

[0245] Trophozoite- stage Pf parasites were treated with an 8x8 concentration matrix combining serial dilutions of AmplD and the antimalarial drug artesunate. Parasite growth inhibition was measured 24 hpt using FACS and normalized to untreated controls. The results are summarized in Figures 8A-8B. Advantageously, AmplD and artesunate showed a synergistic inhibition of the malaria parasite. AmplD enhanced the efficacy of the conventional antimalarial drug artesunate, potentially allowing dose reduction of the latter while maintaining or improving therapeutic outcomes. Example 9 - Design and screening of additional synthetic AMPs for inhibiting the malaria parasite

[0246] A series of peptides with targeted structural modifications compared to AmplD and MSI-78 were designed (Table 2). These peptides were tested together with the peptides set forth as SEQ ID NO: 12 and SEQ ID NO: 13 that are described in Example 2.

[0247] Table 2 - Peptide identification and properties

[0248]

[0249] aUnderlined amino acids are D-enantiomers. Boldface amino acids are bound via isopeptide bond ("EP"). All the peptides were amidated on at their C-terminus.bThe reversed-phase HPLC retention time in the C18 column using a gradient of 10-90% ACN in water for 40 min.

[0250] Amp ID analogs identified as peptides B2-B4, C2-C5 , C8-C19 and D2 in Table 2 contained six lysines and nine leucines in an amphipathic configuration with a high propensity to adopt an a-helical structure. These analogs included incorporating 3 - 5 D-amino acids at defined positions for every 1-5 amino acids.

[0251] Additional analogs contained a reduced (B 14 and B5) or increased (Bl, C6, C7, D3 and D4) number of amino acids compared to AmplD, to modulate hydrophobicity.

[0252] Also, an analog containing partial substitution of lysine residues (2 out of 5) with histidine residues was designed (R4 in Table 2), to introduce pH-dependent charge.

[0253] Additionally, as shown in Example 1, one or more isopeptide bonds in the middle of the peptide sequence disrupted antimalarial activity. In the present example, AmplD analogs with a mix of D-amino acids and lysines bound via isopeptide bonds where designed (B7-B11), and also an analog in which an isopeptide bond is located at the peptide's terminus (C6, which is mentioned above with respect to the peptide's length compared to AmplD).

[0254] Makovitzki et al. (2006) Proc Natl Acad Sci U S A., 103(43): 15997-6002 report ultrashort lipopeptides containing palmitic acid moieties, having antibacterial and antifungal activities. Peptides identified as B6 and B12-B13 in Table 2 comprise a lipid moiety of palmitic acid ("C16") and four amino acid residues (B12-B13 include only lysine residues), containing D-amino acids and isopeptide bonds.

[0255] Several modified forms of the peptide MSI-78 were also evaluated. These variants (M1-M5 in Table 2) incorporated distinct structural modifications, including charge segregation, sequence scrambling of the segregated residues, substitution of phenylalanine with glycine to modulate hydrophobicity, L-to-D amino acid inversion to alter chirality, and other structural rearrangements.

[0256] To assess anti-plasmodial activity of the peptides, they were initially screened against synchronized trophozoite- stage Pf (NF54 strain) cultures at a concentration of 5pM, using flow cytometry. Among all tested peptides, 10 peptides significantly inhibited Pf growth, leading to a substantial reduction in parasitemia levels (Figure 9), while demonstrating low hemolytic activity (Figure 10): AmplD, Bl, B2, C3, C4, C5, C6, D2, D3 and D4.

[0257] The results suggest that shortening the peptide sequence below 15 amino acids disrupts antimalarial activity. In addition, the results support the finding that incorporating lysine residues via their s-amino group to form isopeptide bonds in the middle of the peptide disrupts antimalarial activity.

[0258] A dose-response analysis was subsequently performed to determine the potency of the active peptides (Figure 11). The half-maximal inhibitory concentration (ICso) values were determined following 24 h treatment of synchronized trophozoite- stage Pf cultures. Peptides C4, D2, and D4 demonstrated the highest anti-plasmodial potency, with ICso values of 2.06, 1.93, and 1.91 pM, respectively, followed by peptide B2 (ICso = 2.42 pM).

[0259] B2 and C4 were further tested for their binding to iRBC and localization within the intracellular parasite, in comparison to Amp ID, which was already demonstrated to bind iRBC and localize within the intracellular parasite. The results are summarized in Figure 12, indicating peptide accumulation within the parasite inside the iRBC.

[0260] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed functions may take a variety of alternative forms without departing from the spirit and scope of the present invention as described by the claims, which follow.

Claims

1. CLAIMS1. A peptide of 15-25 amino acids consisting of the amino acid residues L-Lysine (K), D-Lysine (k), L-Leucine (L), and D-Leucine (1), having a net charge of (+7) to (+10) and comprising a sequence selected from kLLL, LLKKL1, 1KL1LK, L1KLL, 1KKLL, K1LLK and kLL * (wherein K* is a L-Lysine bonded by an isopeptide bond).

2. The peptide of claim 1, wherein the peptide consists of 15-20 amino acids, of which 3-5 residues are in D-configuration.

3. The peptide of claim 1 or claim 2, comprising a sequence selected from the group consisting of:LKlLKkLlkKLLkLLLkLL (D4) (SEQ ID NO: 52);LLKKL1KL1LKL1KK (D2) (SEQ ID NO: 50);LLkKLIKLILKLIKK (C4) (SEQ ID NO: 34);LLKKL1K11LKL1KK (B2) (SEQ ID NO: 13);LLkLLkKllKKLlKLL (Bl) (SEQ ID NO: 12);LKILKkLIKKLLkLL (C3) (SEQ ID NO: 33);LLkKLIKILLKLIKK (C5) (SEQ ID NO: 35);LKlLKkLlkKLLkLLK* (C6) (SEQ ID NO: 36); and LLKKL1K11LKL1KK1KK (D3) (SEQ ID NO: 51), wherein K, k, L, 1 and K* are as defined in claim 1.

4. The peptide of claim 1, selected from the peptides set forth as SEQ ID NO: 52, SEQ ID NO: 50, SEQ ID NO: 34, SEQ ID NO: 13, SEQ ID NO: 12, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 36, and SEQ ID NO: 51.

5. The peptide of any one of the preceding claims, characterized by relative hydrophobicity of at least 55% AcN.

6. The peptide of claim 5, selected from the peptides set forth as SEQ ID NO: 52, SEQ ID NO: 50, SEQ ID NO: 34, SEQ ID NO: 13 and SEQ ID NO: 12.

7. A pharmaceutical composition comprising a peptide according to any one of the preceding claims and at least one pharmaceutically acceptable excipient.

8. The pharmaceutical composition of claim 7, for use in the treatment of malaria.

9. The pharmaceutical composition of claim 8, wherein the malaria is caused by a Plasmodium species selected from P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi and P. chabaudi.

10. The pharmaceutical composition of any one of claims 7-9, comprising a plurality of peptides according to any one of claims 1-6.

11. The pharmaceutical composition of any one of claims 7-10, for use in the treatment of malaria in combination with at least one anti-malaria drug.

12. The pharmaceutical composition of claim 11, wherein the anti-malaria drug is selected from artemisinin or derivatives thereof, and chloroquine or derivatives thereof.

13. The pharmaceutical composition of claim 12, wherein the anti-malaria drug is artesunate.

14. The pharmaceutical composition of claim 12, wherein the anti-malaria drug is primaquine.

15. A conjugate comprising a peptide according to any one of claims 1-6 and a heterologous moiety.

16. The conjugate of claim 15, wherein the heterologous moiety is an anti-malaria drug.

17. The conjugate of claim 16, wherein the anti-malaria drug is selected from artemisinin or derivatives thereof, and chloroquine or derivatives thereof.

18. The conjugate of claim 17, wherein the anti-malaria drug is artesunate.

19. The conjugate of claim 17, wherein the anti-malaria drug is primaquine.

20. A pharmaceutical composition for use in the treatment of malaria, comprising a conjugate according to any one of claims 15-19.

21. A pharmaceutical composition for use in the treatment of malaria, comprising at leastone peptide of 15-25 amino acids in length comprising one or more positively- charged units and one or more hydrophobic units arranged in the peptide in an alternating order, wherein each positively-charged unit is composed of one or more consecutive positively-charged residues and each hydrophobic unit is composed of one or more consecutive hydrophobic residues, wherein the peptide has a net charge of (+7) to (+10), and wherein the peptide comprises a sequence selected from the group consisting of:LKlLKkLlkKLLkLL (AmplD) (SEQ ID NO: 1);KKkLLlLllLLLkKK (Seg5D) (SEQ ID NO: 2);LLILLkKkkKKLILL (Seg6D) (SEQ ID NO: 3);GIGKFLKKAKKFGKAFVKILKK (MSI-78) (SEQ ID NO: 9);LLkLLkKllKKLlKLL (Bl) (SEQ ID NO: 12);LLKKL1K11LKL1KK (B2) (SEQ ID NO: 13);LKILKkLIKKLLkLL (C3) (SEQ ID NO: 33);LLkKLIKLILKLIKK (C4) (SEQ ID NO: 34);LLkKLIKILLKLIKK (C5) (SEQ ID NO: 35);LKlLKkLlkKLLkLL^* (C6) (SEQ ID NO: 36);LLKKLIKLILKLIKK (D2) (SEQ ID NO: 50);LLKKL1K11LKL1KK1KK (D3) (SEQ ID NO: 51); and LKlLKkLlkKLLkLLLkLL (D4) (SEQ ID NO: 52), wherein K, k, L, 1 and K* are as defined in claim 1.

22. The pharmaceutical composition for use according to claim 21, wherein the peptide is 15-20 amino acid in length.

23. The pharmaceutical composition for use according to claim 21, wherein the peptide is selected from the peptides set forth as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 50, SEQ ID NO: 51, and SEQ ID NO: 52.

24. The pharmaceutical composition for use according to any one of claims 21-23, wherein the peptide is selected from the peptides set forth as: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 9.

25. The pharmaceutical composition for use according to any one of claims 21-24, comprising a plurality of peptides as defined in claim 21.

26. The pharmaceutical composition for use according to any one of claims 21-25, wherein the malaria is caused by a Plasmodium species selected from P. falciparum, P. malariae, P. ovale, P. vivax, P. knowlesi and P. chabaudi.

27. The pharmaceutical composition for use according to any one of claims 21-26, for use in combination with at least one anti-malaria drug.

28. The pharmaceutical composition of claim 27, wherein the anti-malaria drug is selected from artemisinin or derivatives thereof, and chloroquine or derivatives thereof.

29. The pharmaceutical composition of claim 28, wherein the anti-malaria drug is artesunate.

30. The pharmaceutical composition of claim 28, wherein the anti-malaria drug is primaquine.

31. A conjugate comprising a peptide as defined in claim 21 and an anti-malaria drug.

32. The conjugate of claim 31, wherein the anti-malaria drug is selected from artemisinin or derivatives thereof, and chloroquine or derivatives thereof.

33. The conjugate of claim 32, wherein the anti-malaria drug is artesunate.

34. The conjugate of claim 32, wherein the anti-malaria drug is primaquine.

35. A pharmaceutical composition comprising the conjugate of any one of claims 31-34, for use in the treatment of malaria.

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