Peptide-derived therapeutics for the treatment of cryptococcus neoformans infection

Abstract

The present invention relates to the treatment of Cryptococcus neoformans infection. In particular, an anti-fungal peptide therapeutic having the sequence ILSAAYSGIISLI and methods of using the peptide therapeutic to treat Cryptococcus neoformans infection.

Description

[0001] PEPTIDE-DERIVED THERAPEUTICS FOR THE TREATMENT OF CRYPTOCOCCUS NEOFORMANS INFECTION

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the treatment of Cryptococcus neoformans infection. In particular, the present invention relates to peptide-derived therapeutic for the treatment of Cryptococcus neoformans infection.

[0004] BACKGROUND

[0005] Cryptococcus neoformans is a fungal pathogen known for causing opportunistic infections, predominantly in immunocompromised individuals. This pathogen is commonly encountered in the environment, particularly in areas contaminated with bird droppings or soil harboring the fungus. Inhalation of C. neoformans spores can lead to various clinical manifestations, including pulmonary cryptococcosis and disseminated cryptococcosis.

[0006] Cryptococcus neoformans poses an international risk due to its widespread distribution and the potential for severe disease in vulnerable populations. The World Health Organization (WHO) recognizes the importance of addressing this fungal pathogen and has classified cryptococcal meningitis, the most common manifestation of disseminated C. neoformans infection, as an AIDS-defining illness. The WHO rating emphasizes the significance of cryptococcal disease in the context of HIV / AIDS and highlights the need for improved prevention, diagnosis, and treatment strategies, especially in resource-limited settings where the burden of HIV is high. International collaboration and efforts are crucial to address the global impact of Cryptococcus neoformans infection and reduce its associated morbidity and mortality.

[0007] Types of Cryptococcus neoformans infection include (1) Cutaneous cryptococcosis: Cutaneous cryptococcosis occurs when the fungus enters the skin through wounds, injuries, or sites of previous trauma. The infection may present as nodules, ulcers, abscesses, or cellulitis in the affected areas. Cutaneous cryptococcosis can occur in isolation or as part of disseminated cryptococcosis, where the infection spreads from the primary site to other organs or systems. (2) Pulmonary Cryptococcosis: Pulmonary cryptococcosis refers to the localized infection of the lungs following the inhalation of C. neoformans spores. In immunocompetent individuals, this infection may resolve spontaneously or remain restricted to the lungs without disseminating to other organs. However, in individuals with compromised immune systems, such as HIV / AIDS patients or those receiving immunosuppressive therapies, the infection can progress to disseminated cryptococcosis. (3) Disseminated Cryptococcosis: Disseminated cryptococcosis occurs when the infection spreads beyond the lungs to other sites in the body. The central nervous system (CNS) is the most affected site, leading to cryptococcal meningitis. Disseminated infection can also involve other organs, including the skin, bones, liver, and spleen. Cryptococcal meningitis is associated with significant morbidity and mortality, especially in immunocompromised individuals.

[0008] The severity of Cryptococcus neoformans infection depends on the immune status of the affected individual. In immunocompetent individuals, pulmonary cryptococcosis may present as a self-limiting respiratory illness with mild symptoms. However, in immunocompromised patients, particularly those with advanced HIV infection, disseminated cryptococcosis and cryptococcal meningitis can be severe and life-threatening. Neurological complications, such as increased intracranial pressure and neurologic deficits, are common in disseminated infection and contribute to the overall disease severity. The treatment of Cryptococcus neoformans infection primarily involves antifungal therapy. Exemplary antifungal therapies include amphotericin B, either alone or in combination with flucytosine as an induction therapy aims to rapidly control fungal growth and reduce the burden of infection; and fluconazole as a consolidation and maintenance therapy to prevent relapse and achieve long-term control of the infection. Such antifungal therapy may be associated with side effects, including nephrotoxicity with amphotericin B and drug interactions with azole antifungals. Additionally, the emergence of drug-resistant strains of C. neoformans further complicates treatment.

[0009] There exists a need for alternative C. neoformans therapies. Peptides targeting C. neoformans are being examined as potential anti-fungal therapeutics.

[0010] SUMMARY OF THE INVENTION

[0011] An object of the present invention is to provide a peptide-derived therapeutic for treatment of Cryptococcus neoformans infection. In accordance with an aspect of the present invention, there is provided a peptide comprising or consisting of the sequence ILSAAYSGIISLI (SEQ ID NO:1).

[0012] BRIEF DESCRIPTION OF THE FIGURES

[0013] FIG. 1 illustrates relative DARWIN Al scores for NeoPeptix peptide against the C. neoformans proteome. Top-ranked peptides include HSP71 and HSP71-like proteins. FIG. 2 illustrates that NeoPeptix is designed as an amphipathic peptide. Image generated using EMBOSS Pepwheel software. Aliphatic residues are marked with blue squares, hydrophilic residues are marked in red diamonds, and positively charged residues with black octagons, and hydrophobic residues in purple font.

[0014] DETAILED DESCRIPTION

[0015] The present invention relates to a peptide and peptide-derived therapeutics targeting Cryptococcus neoformans and methods and uses thereof. In certain embodiments, the peptide and peptide-derived therapeutic kills and / or inhibits growth of Cryptococcus neoformans. In certain embodiments, the peptide specifically targets and kills Cryptococcus neoformans. In specific embodiments, the peptide exhibits minimal toxicity to host cells.

[0016] Peptides:

[0017] The present invention provides peptides that have anti-fungal activity. The activity may be fungistatic and / or fungicidal. In certain embodiments, the anti-fungal activity is specific for Cryptococcus neoformans. In certain embodiments, the peptide targets Ssa1 protein. In certain embodiments, the peptide comprises the sequence ILSAAYSGIISLI (SEQ ID NO:1). In specific embodiments, the peptide consists of the sequence ILSAAYSGIISLI (SEQ ID NO:1).

[0018] In certain embodiments of the present invention, the peptides comprise one or more modifications including, without limitation, glycosylations, acetylations, phosphorylations, PEG, D-amino acids, nanoparticles, solid lipid nanoparticles, esterification, N-acetylation or may be formulated with liposomes, nano-emulsions, mucoadhesive polymers, nanoparticles, solid lipid nanoparticles.

[0019] It is known in the art that peptide modifications may improve therapeutic peptide delivery by increasing stability, inhibiting enzyme activity, enhancing absorption and / or cell targeting.

[0020] Mechanisms of therapeutic peptide delivery

[0021] _ Goal _ Peptide modification / formulations _

[0022] Stomach

[0023] Increased stability PEG, D-amino acids, nanoparticles, solid lipid nanoparticles Small intestine

[0024] Increased stability cyclization, PEG, lipidation, D-amino acids, polymer matrices, nanoparticles, esterification, N- acetylation

[0025] Enzyme inhibitors soybean trypsin inhibitor, aprotinin, puromycin, bacitracin

[0026] Absorption enhancers chitosans, fatty acids, lectins, Zonula occludens toxin, cell penetrating peptides, liposomes, nanoemulsions, mucoadhesive polymers, nanoparticles, solid lipid nanoparticles Circulation

[0027] Increased stability PEG, hyper-glycosylation, liposomes, nanoparticles

[0028] Cell targeting Antibody, cell penetrating peptides

[0029] In certain embodiments of the present invention, there is provided a conjugate or fusion protein comprising the peptide of the present invention and heterologous amino acid sequence. In certain embodiments, the conjugates include a linker sequence. Linkers are known in the art and are generally classified into 3 categories according to their structures: (1) flexible linkers, (2) rigid linkers, and (3) in vivo cleavable linkers.

[0030] The peptides of the present invention can be biochemically synthesized such as by using standard solid phase techniques. These methods include exclusive solid phase synthesis, partial solid phase synthesis methods, fragment condensation, classical solution synthesis. Solid phase polypeptide synthesis procedures are well known in the art.

[0031] Recombinant techniques may also be used to generate the peptides of the present invention. Such recombinant techniques are known in the art. Accordingly, the present invention also provides a nucleic acid encoding the amino acid sequence of the peptide, and conjugates comprising the peptide. The nucleic acid can comprise DNA or RNA, and can be single or double stranded. Furthermore, the nucleic acid can comprise nucleotide analogues or derivatives (e.g. inosine or phophorothioate nucleotides and the like). The nucleic acid can encode the amino acid sequence of the peptide as part of a fusion protein comprising such sequence and a cell penetrating motif. The nucleic acid encoding the amino acid sequence of the peptide can be provided as part of a construct comprising the nucleic acid and elements that enable delivery of the nucleic acid to a cell, and / or expression of the nucleic acid in a cell. Such elements include, for example, expression vectors and transcription and / or translation sequences. Suitable vectors, transcription / translation sequences, and other elements, as well as methods of preparing such nucleic acids and constructs, are known in the art.

[0032] Accordingly, in certain embodiments, there is provided a polynucleotide encoding and expressing one or more peptide(s) of the invention. In another preferred embodiment, the polynucleotide is inserted in a vector. Preferably, said recombinant vector is an expression vector capable of expressing said polynucleotide when transfected or transformed into a host cell such as a prokaryotic or eukaryotic cell. The polynucleotide is inserted into an expression vector in proper orientation and correct reading frame for expression. In certain embodiments, the polynucleotide is operably linked to at least one transcriptional regulatory sequence and, optionally to at least one translational regulatory sequence. Recombinant vectors are known in the art and include but are not limited to plasmids and viral vectors. Viral vectors include but are not limited to oncolytic viral vectors, lentivirus and adenovirus vectors.

[0033] Pharmaceutical Compositions:

[0034] The peptides and peptide derived therapeutics of the present invention can be formulated as a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises the peptide of the present invention or one or more peptides derived therapeutics of the present invention alone or in combination with one or more other active agents and a pharmaceutically acceptable carrier.

[0035] Polynucleotides and vectors encoding the peptides of the invention may also be formulated as pharmaceutical compositions. In certain embodiments, the pharmaceutical composition comprises one or more polynucleotides or one or more vectors of the present invention alone or in combination with one or more other active agents and a pharmaceutically acceptable carrier.

[0036] The pharmaceutical composition may comprise one or more other pharmaceutically active agents or drugs. Examples of such other pharmaceutically active agents or drugs that may be suitable for use in the pharmaceutical composition include one or more other anti-fungal agents and / or one or more immunostimulatory agents. Exemplary anti-fungal agents include but are not limited to amphotericin B, flucytosine, fluconazole. Immunostimulatory agents include cytokines such as interferons, colony stimulating factors and tumor necrosis factors.

[0037] The carrier may be any of those conventionally used and is limited only by physio-chemical considerations, such as solubility and lack of reactivity with the active compound(s), and by the route of administration. The pharmaceutically acceptable carriers described herein, for example, vehicles, adjuvants, excipients, and diluents, are well-known to those skilled in the art and are readily available to the public. The pharmaceutically acceptable carrier may be one which is chemically inert to the active agent(s) and one which has no detrimental side effects or toxicity under the conditions of use. The choice of carrier will be determined in part by the active agents, as well as by the method of administration. Accordingly, there are a variety of suitable formulations of the pharmaceutical composition of the present invention. The following formulations for oral, aerosol, topical, parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, rectal, and vaginal administration are exemplary and are in no way limiting. One skilled in the art will appreciate that these routes of administering the compound of the invention are known, and, formulations appropriate for each of these routes of administration are known in the art.

[0038] In certain embodiments, one or more peptides of the present invention are conjugated, directly or indirectly, to a carrier. Appropriate carriers are known in the art and include but are not limited to proteins including but not limited to keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) and ovalbumin (OVA); virus-like particles and viruses.

[0039] Methods of Treatment

[0040] Also provided are methods of inhibiting or killing a fungus and methods of treating a fungal infection or disease caused by a fungus in a subject comprising administering to the subject in need thereof. In specific embodiments, the fungus is Cryptococcus neoformans. A worker skilled in the art would readily appreciate that Cryptococcus infection may be cutaneous, pulmonary or disseminated. Accordingly, the present invention provides methods of treating Cutaneous cryptococcosis; Pulmonary Cryptococcosis, cryptococcal meningitis and / or Disseminated Cryptococcosis.

[0041] In specific embodiments, the methods are for treatment of immunocompromised individuals.

[0042] In certain embodiments, the peptides and peptide therapeutics, alone or in combination with other therapeutics are used as an induction therapy to rapidly control fungal growth and reduce the burden of infection. Other therapeutics may include but are not limited to amphotericin B, either alone or in combination with flucytosine.

[0043] In certain embodiments, the peptides and peptide therapeutics, alone or in combination with other therapeutics are used as consolidation and maintenance therapy to prevent relapse and achieve long-term control of the infection. Other therapeutics may include but are not limited to fluconazole, an oral antifungal agent. EXAMPLE

[0044] The example below details the development of an anti-fungal peptide of an embodiment of the invention.

[0045] Molecular target and rational peptide design

[0046] Cryptococcus neoformans is a fungal pathogen that can cause severe infections, particularly in individuals with compromised immune systems, such as those with HIV / AIDS. Heat shock proteins (HSPs) are a family of proteins that play a crucial role in cellular stress responses and protein folding. HSP71 , also known as Ssa1 , is a member of the HSP70 family and is involved in C. neoformans virulence, specifically through facilitating host cell invasion (Kakeya et al., 1997; Kakeya et al., 1999). Ssa1 is localized to the cell wall of C. neoformans (Silveira et al., 2013), and accordingly, is established to be immunogenic in human infection. In C. neoformans, Ssa1 is also required to promote non-protective M2 macrophage polarization in lung monocytes upon infection (Eastman et al., 2015); mutants lacking SSA1 have reduced virulence in murine models of infection. Biochemically, Ssa1 also has an established body of literature and sufficient experimental research to support in silico development of de novo peptide inhibitors. Together, these factors support Ssa1 to be a suitable anti-fungal target for peptide design.

[0047] To develop a peptide inhibitor of C. neoformans Ssa1 protein (a.k.a. SSA1_CRYNB) an artificial intelligence platform, DARWIN Al was used. Unlike many other computationally intensive approaches to peptide design, DARWIN Al does not rely on a known crystal structure, and instead, relies on learning how proteins interact naturally and evolving novel peptides based on these principles. A key advantage of DARWIN Al is its independence from known structural information and ability to rank de novo peptides for both on- and off-target interaction probabilities. Figure 1 shows the ranked results in developing a peptide interactor of SSA1_CRYNB among the C. neoformans proteome. Top-ranked peptide targets include SSA1_CRYNB (HSP71) and a highly similar Q55VL6_CRYNB (HSP71-like) protein. Topranked proteins also included HSP75, another cell wall chaperone protein involved in fungal growth and virulence. This peptide is of the sequence ILSAAYSGIISLI (SEQ ID NO:1)(also referred to as NeoPeptix). a-helical mapping of NeoPeptix

[0048] EMBOSS pepwheel was used to visualize the formation of amino acids in the NeoPeptix peptide sequence to identify regions of hydrophobicity and hydrophilicity. Aliphatic residues are marked with blue squares, hydrophilic residues are shown in red diamonds, positively charged residues with black octagons, and hydrophobic residues in purple font. Figure 2 shows the rationally designed NeoPeptix a-helical orientation. Specifically, NeoPeptix contains rationally placed amino acids that have aliphatic characteristics with branched side groups. A key feature of NeoPeptix is a heavily hydrophilic left side of the helical peptide with abundant serine residues, creating a more amphipathic peptide for increased antimicrobial activity.

[0049] Identification of therapeutic scope

[0050] Numerous growth inhibitory peptides have been identified to date demonstrate effective towards bacteria, fungi, or both microorganisms. To better determine the therapeutic breadth and effectiveness of NeoPeptix, we carried out a primary growth inhibition screen. In the primary screening, we tested against key ESKAPE pathogens, E. coli, K. pneumoniae, A. baumannii, P. aeruginosa, S. aureus (MRSA), as well as the fungi C. neoformans and C. albicans (Table 2). The purpose of this primary screen is to identify hits to re-screen the peptides against and determine MIC.

[0051] Primary antimicrobial screening study by whole cell growth inhibition assays, using the NeoPeptix peptide at a single concentration (32 ug / mL or 20 uM), in duplicate (n=2). The inhibition of growth is measured against 5 bacteria: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus, and 2 fungi: Candida albicans and Cryptococcus neoformans. Results from the primary screen are shown in Table 3.

[0052] Hit confirmation and characterization of specific NeoPeptix inhibition of C. neoformans

[0053] Hit Confirmation of NeoPeptix by whole cell growth inhibition assays was conducted as an 8- point dose response (32- 0.25 ug / mL) to determine the Minimum Inhibitory Concentration (MIC), in duplicate (n=2). The inhibition of growth is measured against those microorganisms that showed susceptibility to the compounds tested in the Primary Screen (Table 2). Included in the Hit Confirmation were 5 bacteria: Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Staphylococcus aureus, and 2 fungi Candida albicans and Cryptococcus neoformans. In addition to determining MIC, active compounds were counter screened for cytotoxicity against a human embryonic kidney cell line, HEK293, by determining their CC50 (50% cytotoxic concentration) value. The compounds were also screened for haemolysis of human red blood cells. Results from the primary screen are shown in Table 3. To determine the therapeutic potential and toxicity of NeoPeptix peptide, cytotoxicity (Table 4) and Haemolysis (Table 5) activity are reported. Materials and Methods

[0054] In silica design of peptide inhibitors

[0055] C. neoformans Ssa1 -interacting peptides were developed in silico using the DARWIN Al platform. Briefly, peptides were developed using training data available from both Homo Sapiens and species from within the Ascomycetes and Basidiomycetes fungal divisions. Peptides were developed for on-target interaction (i.e., Ssa1) and were penalized for off-target interactions (i.e., low specificity). Targets for the top-predicted peptide (ILSAAYSGIISLI (SEQ ID NO:1); NeoPeptix) are listed in Table 1.

[0056] Peptide Synthesis

[0057] Peptides were synthesized using Solid Phase Fmoc (N-(9-fluorenyl) methoxycarbonyl) chemistry using a ResPep SL peptide synthesizer (Intavis) at a 2 pmol scale using Rink-NH2resin. All amino acid derivates and activator were purchased from p3bio systems. To allow for quantification, peptides were synthesized with a C-terminal tryptophan separated from the peptide by a 6-aminohecanoic acid (6-ahx) linker. After synthesis, peptides were released from the resin and protecting groups were cleaved using an acidic cleavage solution (95 % trifluoroacetic acid, 3 % tri-isopropylsilane, 2 % water). Cold di-ethyl ether (-20 °C) was used to precipitate and wash the peptides of residual cleavage solution. Once dried, pellets were dissolved in 1X PBS containing 4% acetic acid. Peptides were brought up to pH 7 using 5 M NaOH and quantified by A280 and molar extinction coefficients calculated by ProtParam Expasy.

[0058] Pepwheel visualization

[0059] Amino acid residue formation for NeoPeptix was visualized using the EMBOSS pepwheel tool. (EMBOSS; http: / / emboss.bioinformatics.nl / cqi-bin / emboss / pepwheel) .

[0060] Antibacterial assays

[0061] All bacteria (Table 2) were cultured in Cation-adjusted Mueller Hinton broth (CaMHB) at 37 °C overnight. A sample of each culture was then diluted 40-fold in fresh broth and incubated at 37 °C for 1.5 - 3 h. The resultant mid-log phase cultures were diluted (CFU / mL measured by OD600), then added to each well of the compound containing plates, giving a cell density of 5x 105 CFU / mL and a total volume of 50 pL. All the plates were covered and incubated at 37 °C for 18 h without shaking.

[0062] Inhibition of bacterial growth was determined measuring absorbance at 600 nm (OD6oo), using a Tecan M1000 Pro monochromator plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate as references. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (bacteria without inhibitors) on the same plate. The MIC was determined as the lowest concentration at which the growth was fully inhibited, defined by an inhibition > 80%. In addition, the maximal percentage of growth inhibition is reported as DMax, indicating any compounds with partial activity. Hits were classified by MIC < 16 pg / mL or MIC < 10 pM in either replicate (n=2 on different plates).

[0063] Antifungal assays

[0064] Fungi strains (Table 2) were cultured for 3 days on Yeast Extract-Peptone Dextrose (YPD) agar at 30 °C. A yeast suspension of 1 x 106to 5 x 106CFU / mL (as determined by OD530) was prepared from five colonies. The suspension was subsequently diluted and added to each well of the compound-containing plates giving a final cell density of fungi suspension of 2.5 x103CFU / mL and a total volume of 50 pL. All plates were covered and incubated at 35 °C for 36 h without shaking.

[0065] Growth inhibition of C. albicans was determined measuring absorbance at 630 nm (OD63o), while the growth inhibition of C. neoformans was determined measuring the difference in absorbance between 600 and 570 nm (OD6oo-57o), after the addition of resazurin (0.001% final concentration) and incubation at 35 °C for 2 h. The absorbance was measured using a Biotek Multiflo Synergy HTX plate reader. In both cases, the percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control (fungi without inhibitors) on the same plate. The MIC was determined as the lowest concentration at which the growth was fully inhibited, defined by an inhibition > 80% for C. albicans and an inhibition > 70% for C. neoformans. Due to a higher variance in growth and inhibition, a lower threshold was applied to the data for C. neoformans. In addition, the maximal percentage of growth inhibition is reported as DMax, indicating any compounds with marginal activity. Hits were classified by MIC < 16 pg / mL or MIC < 10 pM in either replicate (n=2 on different plates).

[0066] Cytotoxicity Assay

[0067] HEK293 cells were counted manually in a Neubauer haemocytometer and then plated in the 384-well plates containing the compounds to give a density of 5000 cells / well in a final volume of 50 pL. DMEM supplemented with 10% FBS was used as growth media and the cells were incubated together with the compounds for 20 h at 37 °C in 5% CO2. Cytotoxicity (or cell viability) was measured by fluorescence, ex: 560 / 10 nm, em: 590 / 10 nm (F560 / 590), after addition of 5 pL of 25 pg / mL resazurin (2.3 pg / mL final concentration) and after incubation for further 3 h at 37 °C in 5% CO2. The fluorescence intensity was measured using a Tecan M1000 Pro monochromator plate reader, using automatic gain calculation. CC5o (concentration at 50% cytotoxicity) were calculated by curve fitting the inhibition values vs. log(concentration) using a sigmoidal dose-response function, with variable fitting values for bottom, top and slope. In addition, the maximal percentage of cytotoxicity is reported as DMax, indicating any compounds with partial cytotoxicity. The curve fitting was implemented using Pipeline Pilot's dose-response component, resulting in similar values to curve fitting tools such as GraphPad's Prism and IDBS's XIFit. Any value with > indicate sample with no activity (low DMax value) or samples with CC50 values above the maximum tested concentration (higher DMax value). Cytotoxic samples were classified by CC5o 32 pg / mL or CC5o < 10 pM in either replicate (n=2 on different plates). In addition, samples were flagged as partial cytotoxic if DMax > 50%, even with CC5o > the maximum tested concentration.

[0068] Haemolysis Assay

[0069] To determine haemolysis activity of NeoPeptix, Human whole blood was washed three times with 3 volumes of 0.9% NaCI (saline) and then resuspended in saline to a concentration of 0.5 x 108cells / mL, as determined by manual cell count in a Neubauer haemocytometer. The washed cells were then added to the 384- well compound-containing plates for a final volume of 50 pL. After a 10 min shake on a plate shaker the plates were then incubated for 1 h at 37 °C. After incubation, the plates were centrifuged at 10OOxg for 10 min to pellet cells and debris, 25 pL of the supernatant was then transferred to a polystyrene 384-well assay plate.

[0070] Haemolysis of NeoPeptix was determined by measuring the supernatant absorbance at 405 mm (OD405). The absorbance was measured using a Tecan M1000 Pro monochromator plate reader. HC10 and HC5o (concentration at 10% and 50% haemolysis, respectively) were calculated by curve fitting the inhibition values vs. log(concentration) using a sigmoidal doseresponse function with variable fitting values for top, bottom and slope. In addition, the maximal percentage of haemolysis is reported as DMax, indicating any compounds with partial haemolysis. The curve fitting was implemented using Pipeline Pilot's dose-response component, resulting in similar values to curve fitting tools such as GraphPad's Prism and IDBS's XIFit. Any value with > indicate sample with no activity (low DMax value) or samples with HC-io values above the maximum tested concentration (higher DMax value). Haemolysis samples were classified by HC10 < 32 pg / mL or HC10 < 10 pM in either replicate (n=2 on different plates). In addition, samples were flagged as partial haemolytic if DMax > 50%, even with HC-io > the maximum tested concentration. Antibiotic, Cytotoxic and Haemolytic Standards Preparation and Quality Control

[0071] Colistin and Vancomycin were used as positive bacterial inhibitor standards for Gram-negative and Gram-positive bacteria, respectively. Fluconazole was used as a positive fungal inhibitor standard for C. albicans and C. neoformans. Tamoxifen was used as a positive cytotoxicity standard. Melittin was used as a positive haemolytic standard. Each antibiotic standard was provided in 4 concentrations, with 2 above and 2 below its MIC or CC5o value and plated into the first 8 wells of column 23 of the 384-well NBS plates. Tamoxifen and melittin was used in 8 concentrations in 2-fold serial dilutions with 50 pg / mL highest concentration. The quality control (QC) of the assays was determined by Z’-Factor, calculated from the Negative (media only) and Positive Controls (bacterial, fungal or cell culture without inhibitor), and the Standards (Table 7). Plates with a Z’-Factor of > 0.4 and Standards active at the highest and inactive at the lowest concentration, were accepted for further data analysis.

[0072] Table 1. Top-ranked NeoPeptix targets from within the C. neoformans proteome.

[0073] Targets identified and ranked with the NuvoBio DARWIN Al peptide discovery platform.

[0074] Peptide Target Relative score Target description

[0075] NeoPeptix F5HB71 1.000 HSP71 CRYNB Transcriptional coregulator SSA1

[0076] NeoPeptix Q55VL6 0.979 Q55VL6_CRYNB Hsp71 -like protein

[0077] NeoPeptix Q55YF7 0.894 Q55YF7 CRYNB Glycoside hydrolase family 31 N-terminal domain-containing protein

[0078] NeoPeptix P0CS29 0.833 ATG18 CRYNB Autophagy-related protein 18

[0079] NeoPeptix Q560D4 0.773 Q560D4_CRYNB Hsp75-like protein

[0080] NeoPeptix F5HGB9 0.692 F5HGB9 CRYNB FMN hydroxy acid dehydrogenase domain-containing protein

[0081] NeoPeptix F5HE90 0.659 F5HE90 CRYNB Endoplasmic reticulum chaperone BiP

[0082] NeoPeptix P0CM41 0.626 ATG9 CRYNB Autophagy-related protein 9

[0083] NeoPeptix F5H9D5 0.604 F5H9D5_CRYNB Endoplasmic reticulum chaperone BiP

[0084] NeoPeptix Q55YC9 0.553 Q55YC9_CRYNB PUM-HD domain-containing protein

[0085] NeoPeptix F5HAN7 0.459 F5HAN7_CRYNB Chaperone DnaK

[0086] NeoPeptix F5HC93 0.421 F5HC93_CRYNB RNA helicase

[0087] NeoPeptix F5H9M6 0.331 F5H9M6_CRYNB L-lactate dehydrogenase

[0088] NeoPeptix F5HB25 0.293 F5HB25_CRYNB L-lactate dehydrogenase (Cytochrome)

[0089] NeoPeptix F5H8V1 0.293 F5H8V1_CRYNB L-mandelate dehydrogenase

[0090] NeoPeptix F5HA22 0.289 F5HA22 CRYNB PUM-HD domain-containing protein

[0091] NeoPeptix Q55J68 0.288 Q55J68 CRYNB L-lactate dehydrogenase (Cytochrome)

[0092] NeoPeptix Q55QM3 0.275 Q55QM3 CRYNB Uncharacterized protein

[0093] NeoPeptix F5H964 0.272 F5H964 CRYNB Solute carrier family 25 (Mitochondrial dicarboxylate transporter), member 10

[0094] NeoPeptix F5HI19 0.246 F5HI19 CRYNB L-mandelate dehydrogenase

[0095] NeoPeptix F5HAE9 0.230 F5HAE9 CRYNB Uncharacterized protein

[0096] NeoPeptix F5HGR4 0.167 F5HGR4 CRYNB Fanconi-associated nuclease

[0097] NeoPeptix Q55HI0 0.163 Q55HI0 CRYNB Solute carrier family 25 (Mitochondrial citrate transporter), member 1

[0098] NeoPeptix Q55VR2 0.148 Q55VR2_CRYNB Solute carrier family 25, member 34 / 35

[0099] NeoPeptix F5HEX1 0.145 F5HEX1_CRYNB Alpha-glucosidase

[0100] NeoPeptix F5HIC0 0.141 F5HIC0_CRYNB Uncharacterized protein

[0101] NeoPeptix F5HCU9 0.139 F5HCU9_CRYNB Heat shock 70kDa protein 4 Table 2. Microbial strains and cell lines.

[0102] ID Batch Abbrev. Organism Strain Description

[0103] GN_001 2 Ec Escherichia coli ATCC 25922 FDA control strain

[0104] GN_003 2 Kp Klebsiella pneumoniae ATCC 700603 MDR

[0105] GN_034 2 Ab Acinetobacter baumannii ATCC 19606 Type strain

[0106] GN_042 2 Pa Pseudomonas aeruginosa ATCC 27853 Quality control strain

[0107] GN_020 2 Sa Staphylococcus aureus ATCC 43300 MRSA

[0108] FG_001 1 Ca Candida albicans ATCC 90028 CLSI reference

[0109] FG_002 1 Cn Cryptococci neoformans ATCC 208821 H99, Type strain

[0110] MA_007 2 Hk Homo sapiens ATCC CRL-1573 HEK 293

[0111] HA 150 - Hm Homo sapiens ARCBS 5400 00150 Whole blood

[0112] Table 3. Primary hit screen results.

[0113] Peptide OrgID OrgCode Organism Strain (ug / mL) DMax

[0114] ATCC 700603;

[0115] NeoPeptix GN_003 Kp MDR Klebsiella pneumoniae MDR >32.0 11.80

[0116] ATCC

[0117] NeoPeptix GN_034 Ab 19606 Acinetobacter baumannii 19606 >32.0 20.50

[0118] ATCC

[0119] NeoPeptix GN_034 Ab 19606 Acinetobacter baumannii 19606 >32.0 13.10

[0120] ATCC 43300;

[0121] NeoPeptix GP_020 Sa MRSa Staphylococcus aureus MRSA >32.0 8.30

[0122] ATCC

[0123] NeoPeptix GN_001 Ec Escherichia coli 25922 >32.0 4.30

[0124] ATCC

[0125] NeoPeptix GN_001 Ec Escherichia coli 25922 >32.0 0.90

[0126] ATCC 700603;

[0127] NeoPeptix GN_003 Kp MDR Klebsiella pneumoniae MDR >32.0 10.00

[0128] ATCC

[0129] NeoPeptix FG_001 Ca Candida albicans 90028 >32.0 -42.00

[0130] ATCC

[0131] NeoPeptix GN_042 Pa Pseudomonas aeruginosa 27853 >32.0 17.80

[0132] ATCC 208821 ;

[0133] NeoPeptix FG_002 Cn H99 Cryptococcus neoformans H99 >32.0 -8.00

[0134] ATCC

[0135] NeoPeptix FG_001 Ca Candida albicans 90028 >32.0 -5.70

[0136] ATCC

[0137] NeoPeptix GN_042 Pa Pseudomonas aeruginosa 27853 >32.0 19.40

[0138] ATCC 43300;

[0139] NeoPeptix GP_020 Sa MRSa Staphylococcus aureus MRSA >32.0 10.10

[0140] ATCC 208821 ;

[0141] NeoPeptix FG_002 Cn H99 Cryptococcus neoformans H99 <=32.0 86.70 Table 4. Hit Confirmation of NeoPeptix by whole cell growth inhibition assays.

[0142] OrgID OrgCode Organism Strain (ug / mL) Max

[0143] GP_020 Sa MRSa Staphylococcus aureus ATCC 43300; MRSA >32.0 15.20

[0144] GN_003 Kp MDR Klebsiella pneumoniae ATCC 700603; MDR >32.0 23.00

[0145] GN_042 Pa Pseudomonas aeruginosa ATCC 27853 >32.0 3.10

[0146] FG_002 Cn H99 Cryptococcus neoformans ATCC 208821 ; H99 <=0.250 103.10

[0147] GP_020 Sa MRSa Staphylococcus aureus ATCC 43300; MRSA >32.0 31.60

[0148] GN_034 Ab 19606 Acinetobacter baumannii ATCC 19606 >32.0 25.00

[0149] GN_001 Ec Escherichia coli ATCC 25922 >32.0 37.70

[0150] FG 001 Ca Candida albicans ATCC 90028 >32.0 8.00

[0151] Table 5. Cytotox activity of NeoPeptix

[0152] CC50

[0153] OrgID OrgCode Organism Strain (uq / mL) ^Max

[0154] HEK293; ATCC

[0155] MA_007 HEK293 Homo sapiens CRL1573 >32.0 17.20

[0156] HEK293; ATCC

[0157] MA 007 HEK293 Homo sapiens CRL1573 >32.0 20.90

[0158] Table 6. Haemolysis activity of NeoPeptix

[0159] OrgID OrgCode Organism Strain (ug / mL) ( ^u^g / m0 L) DMax

[0160] HA_150 RBC Homo sapiens Red blood cell >32.0 >32.0 -5.20

[0161] HA 150 RBC Homo sapiens Red blood cell >32.0 >32.0 -0.40

[0162] Table 7. Standards used on growth inhibition assays.

[0163] Stock Cone.

[0164] Sample Name Sample ID Full MW (mg / mL) Solvent Source

[0165] Colistin - sulphate MCC_000094:03 1400.63 10 DMSO Sigma; C4461

[0166] Vancomycin - HCI MCC_000095:39 1485.71 10 DMSO Sigma: 861987

[0167] Fluconazole MCC_008383:01 306.27 2.56 DMSO Sigma; F8929

[0168] Tamoxifen MCC_000096:06 371.5 10 DMSO Sigma; T5648

[0169] Melittin MCC 008868:05 2846.46 10 Water Sigma; M2272 References

[0170] Eastman, A.J.; He, X.; Qiu, Y.; Davis, M.J.; Vedula, P.; Lyons, D.M.; Park, Y.-D.; Hardison, S.E.; Malachowski, A.N.; Osterholzer, J. J.; et al. Cryptococcal Heat Shock Protein 70 Homolog Ssa1 Contributes to Pulmonary Expansion of Cryptococ- cus neoformans during the Afferent Phase of the Immune Response by Promoting Macrophage M2 Polarization. J. Immunol. 2015, 194, 5999-6010.

[0171] Kakeya, H.; Udono, H.; Ikuno, N.; Yamamoto, Y.; Mitsutake, K.; Miyazaki, T.; Tomono, K.; Koga, H.; Tashiro, T.; Nakayama, E.; et al. A 77-kilodalton protein of Cryptococcus neoformans, a member of the heat shock protein 70 family, is a major antigen detected in the sera of mice with pulmonary cryptococcosis. Infect. Immun. 1997, 65, 1653-1658.

[0172] Kakeya, H.; Udono, H.; Maesaki, S.; Sasaki, E.; Kawamura, S.; Hossain, M.A.; Yamamoto, Y.; Sawai, T.; Fukuda, M.; Mitsutake, K.; et al. Heat shock protein 70 (hsp70) as a major target of the antibody response in patients with pulmonary cryptococcosis. Clin. Exp. Immunol. 1999, 115, 485-490.

[0173] Silveira, C.P.; Piffer, A.C.; Kmetzsch, L.; Fonseca, F.L.; Soares, D.A.; Staats, C.C.; Rodrigues, M.L.; Schrank, A.; Vainstein, M.H. The heat shock protein (Hsp) 70 of Cryptococcus neoformans is associated with the fungal cell surface and influences the interaction between yeast and host cells. Fungal Genet. Biol. 2013, 60, 53-63.

Claims

Claims1 . A peptide comprising the sequence ILSAAYSGIISLI.

2. A peptide consisting of the sequence ILSAAYSGIISLI.

3. The peptide of claim 1 or 2, wherein said peptide has anti-fungal activity, optionally anti- Cryptococcus neoformans activity.

4. A polynucleotide encoding the peptide of claim 1 or 2.

5. A vector comprising the polynucleotide of claim 4.

6. An anti-fungal therapeutic comprising the peptide of claim 1 or 2.

7. A pharmaceutical composition comprising (a) the peptide of any one of claims 1 to 3, the polynucleotide of claim 5, the vector of claim 6 or the anti-fungal therapeutic of claim 6 and (b) a pharmaceutically acceptable carrier.

8. A method of treating a fungal infection or disease caused by a fungus in a subject comprising administering to the subject in need thereof, the peptide of any one of claims 1 to 3, the polynucleotide of claim 5, the vector of claim 6 or the anti-fungal therapeutic of claim 6 or the pharmaceutical composition of claim 7 alone or in combination with one or more other therapeutics.

9. The method of claim 8, wherein the fungal infection or disease is caused by Cryptococcus neoformans.

10. A method of inhibiting or killing Cryptococcus neoformans comprising administering to the subject in need thereof, the peptide of any one of claims 1 to 3, the polynucleotide of claim 5, the vector of claim 6 or the anti-fungal therapeutic of claim 6 or the pharmaceutical composition of claim 7 alone or in combination with one or more other therapeutics.11 . Use of the peptide of any one of claims 1 to 3, the polynucleotide of claim 5, the vector of claim 6 or the anti-fungal therapeutic of claim 6 or the pharmaceutical composition of claim 7 alone or in combination with one or more other therapeutics to treat a fungal infection or disease caused by a fungus in a subject.

12. The use according to claim 11 , wherein the fungal infection or disease is caused by Cryptococcus neoformans.

13. Use of the peptide of any one of claims 1 to 3, the polynucleotide of claim 5, the vector of claim 6 or the anti-fungal therapeutic of claim 6 or the pharmaceutical composition of claim 7 alone or in combination with one or more other therapeutics to inhibit or kill Cryptococcus neoformans.

14. Use of the peptide of any one of claims 1 to 3, the polynucleotide of claim 5, the vector of claim 6 or the anti-fungal therapeutic of claim 6 or the pharmaceutical composition of claim 7 alone or in combination with one or more other therapeutics in the manufacture of a medicament to treat a fungal infection or disease caused by a fungus in a subject.

15. The use according to claim 14, wherein the fungal infection or disease is caused by Cryptococcus neoformans.

16. Use of the peptide of any one of claims 1 to 3, the polynucleotide of claim 5, the vector of claim 6 or the anti-fungal therapeutic of claim 6 or the pharmaceutical composition of claim 7 alone or in combination with one or more other therapeutics in the manufacture of a medicament to inhibit or kill Cryptococcus neoformans.

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