WO2010129267A2 - Anti-trypanosomal peptides and uses thereof - Google Patents

Abstract

The present invention provides methods of killing, inhibiting the growth, and/or inhibiting the reproduction of kinetoplastid protozoan with hydrophobic signal sequence peptides and compositions including such hydrophobic signal sequence peptides.

Description

ANTI-TRYPANOSOMAL PEPTIDES AND USES THEREOF

CONTINUING APPLICATION DATA

This application claims the benefit of U.S. Provisional Application Serial Nos. 61/172,908, filed April 27, 2009, and 61/317,895, filed March 26, 2010, each of which is incorporated by reference herein.

GOVERNMENT FUNDING

The present invention was made with government support under Grant Nos. AI039033 and 1F32AI080114-01A1, awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND

Trypanosoma brucei is the causative agent of both a veterinary wasting disease and human African trypanosomiasis, or sleeping sickness. Human African trypanosomiasis occurs in 36 countries in sub-Saharan Africa, threatening an estimated 60 million people with debilitating disease. Currently, approximately 500,000 people are infected with African trypanosomes. No vaccines are available for prevention of infection by T. brucei. Without chemotherapeutic treatment, T. brucei kills infected humans. Treatment of infected individuals is limited and there is an increase in the number of relapses from established drag treatment. Drugs currently in use are quite toxic and cause serious side effects and in some cases death. And, the development of drug resistance is of concern. Few drug candidates are currently under clinical trials, and one of the more promising compounds, DB75, has already shown a marked tendency to induce drug resistance (Lanteri et al., 2006, MoI Pharmacol; 70(5): 1585-1592). Consequently, there is a strong need for new and safer drags for the treatment of trypanosomiasis and new drags must be developed in order to prepare for possible emergence of drag resistance in the parasites. SUMMARY OF THE INVENTION

The present invention includes methods of killing a bloodstream form of a kinetoplastid protozoan of the genus Trypanosoma, the method including contacting the protozoan with a hydrophobic signal sequence peptide. The present invention includes methods of treating or preventing a trypanosomal infection in a subject, the method including administering to the subject an effective amount of a hydrophobic signal sequence peptide.

The present invention includes compositions including a hydrophobic signal sequence peptide and a pharmaceutical carrier suitable for parenteral or enteral administration to a mammal. In some aspects, the composition is pyrogen free.

The present invention includes liposomes, emulsions, and micelles including a hydrophobic signal sequence peptide.

Th present invention also includes methods of killing a bloodstream form of a kinetoplastid protozoan of the genus Trypanosoma, the method including contacting the protozoan with a composition, liposome, emulsion, or micelle including a hydrophobic signal sequence peptide.

The present invention includes methods of treating or preventing a trypanosomal infection in a subject, the method comprising administering to the subject an effective amount of a composition, liposome, emulsion, or micelle including a hydrophobic signal sequence peptide.

In some aspects of the methods, compositions, liposomes, emulsions, and micelles of the present invention, the hydrophobic signal sequence peptide may include an uncleaved signal sequence peptide.

In some aspects of the methods, compositions, liposomes, emulsions, and micelles of the present invention, the hydrophobic signal sequence peptide may be soluble in ethanol or dimethyl sulfoxide (DMSO). hi some aspects of the methods, compositions, liposomes, emulsions, and micelles of the present invention, the hydrophobic signal sequence peptide may include the signal sequence peptide of haptoglobin-related protein, paraoxonase 1, or apolipoprotein M. In some aspects of the methods, compositions, liposomes, emulsions, and micelles of the present invention, the hydrophobic signal sequence peptide may include at least nine consecutive amino acid residues of MSDLGAVISLLL WGRQLFA (SEQ ID NO:1), MAKLIALTLLGMGLALFRNHQS (SEQ ID NO:3), or MFHQIWAALLYFYGΠLNSIYQ (SEQ ID NO:5), or a derivative thereof.

In some aspects of the methods, compositions, liposomes, emulsions, and micelles of the present invention, the hydrophobic signal sequence peptide may be MSDLGAVISLLLWGRQLFA (SEQ ID NO: 1), SDLGAVISLLLWGRQLFA (SEQ ID NO:2), MAKLIATLLGMGLALFRNHQS (SEQ ID NO:3), AKLiATLLGMGLALFRNHQS (SEQ ID NO:4), MFHQIWAALLYFYGΠLNSIYQ

(SEQ ID NO:5), FHQIWAALLYFYGIILNSIYQ (SEQ ID NO:6), or SDLGAVISLLWGRQLFA (SEQ ID NO:7).

In some aspects of the methods, compositions, liposomes, emulsions, and micelles of the present invention, the trypanosome may be Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense, T. congolense, or T. vivax. hi some aspects of the methods, compositions, liposomes, emulsions, or micelles of the present invention, the composition, liposome, emulsion, or micelle including a hydrophobic signal sequence peptide may further include an RNA aptamer. In some aspects, the RNA aptamer may binds to a structurally conserved region of a trypanosome variant surface glycoprotein (VSG).

The terms "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims.

Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the killing of T. b. brucei by the Hpr-SP. hi Fig. IA, wild type 427 T.b. brucei cells were incubated with increasing concentrations of Hpr-SP (8-80 μM) added from a stock solution in DMSO. Positive controls were trypanosome lytic factor (TLF). Addition of equivolume DMSO or equimolar concentrations of a non-specific hydrophilic 19 amino acid peptide (NS 19mer; SEQ ID NO: 10) gave no trypanolysis. The inset in Fig. IA shows cells treated with Hpr-SP. Fig. IB is a cartoon of Hpr and the derived peptide Hpr-SP (SEQ ED NO:1). Hydrophobic residues are in bold. Figure 2 shows the results of lysis assays conducted with T. b. brucei expressing

SRA or procyclic form T. b. brucei.

Figure 3 presents the results of lysis assays in the presence of chloroquine, NH₄Cl or at 3⁰C.

Figure 4 shows modeling of T.b. brucei cellular membranes with liposomes. Artificial vesicles were constructed with the phosphatidylcholines corresponding in acyl chain composition to either the VSG or procyclin lipid anchors, egg phosphatidylcholine (egg PC, transition temperature < O⁰C) for VSG and distearoylphosphatidylcholine (DSPS) for procyclin. The leakage of liposomally entrapped calcein was fluorescently monitored as an indicator of Hpr-SP membrane interaction. Figure 5 monitors membrane rigidity in live T.b. brucei with the anisotropic probe TMA-DPH. The addition of Hpr-SP results in an increase in the anisotropy of TMA-DPH labeled cells, indicating membrane rigidification. Equivolmes of DMSO, the Hpr-SP solvent, results in either no change (0.167% DMSO corresponding to 8 μM Hpr- SP) or a decrease (0.4% DMSO corresponding to 20 μM Hpr-SP) in membrane rigidity. Figure 6 assayed cell viability via trypan blue exclusion. Human embryonic kidney cells were incubated with increasing concentrations of Hpr-Sp for two hours at 37⁰C. No cell death was observed with trypanolytic concentrations of Hpr-SP or equivolume addition of DMSO. Positive killing controls provided by the lytic peptide melittin. Figure 7 demonstrates toxicity and specificity of SHP-I for BSF African trypanosomes. In Fig. 7 A, SHP-I was assayed for killing activity against bloodstream form (BSF) T. b. brucei (diamond), T. b. rhodesiense (square), T. b. gambiense (triangle) and procyclic form (PCF) T. b. brucei (circle). Equivolume additions of solvent DMSO (black circle) showed no toxic effect. Fig. 7B is DIC micrographs of untreated, T. b. brucei fixed with formaldehyde and unfixed T. b. brucei incubated with 4 and 80 μM SHP-I for two hours at 37°C. Fig. 7C presents binding of Texas-red labeled SHP-I to bloodstream form (BSF), with and without peptide, and procyclic form (PCF), with and without peptide. T. b. brucei monitored by flow cytometry.

Figure 8 demonstrates that SHP-I is not toxic to mammalian cells. In Fig. 8 A, human cell lines, HEK (circles) and LNCaP (square), are not killed by SHP-I as assayed by trypan blue exclusion. Fig. 8B demonstrates that SHP-I (80 μM) does not induce morphological changes in HEK or LNCaP cells. The cytolytic peptide melittin (25 μM) was utilized as a positive control. Fig. 8C, flow cytometry indicates that Texas-red labeled SHP-I does not bind HEK or LNCaP cells. Fig. 8D, potential hemolytic activity of SHP- 1 was assayed against fresh human erythrocytes.

Figure 9 presents hydrophobicity requirements for trypanosome killing by small peptides. Fig. 9 A presents peptide amino acid sequences, N to C-terminal. SHP-I is SDLGAVISLLLWGRQLFA (SEQ ID NO:2); SHP-2 is AKLIALTLLGMGLALFRNHQS (SEQ ID NO:4); SHP-l-ΔL is SDLGAVISLLWGRQLFA (SEQ ID NO:7); SHP-I -ΔLLL is SDLGAVISWGRQLFA (SEQ ID NO:8); SHP-1-ΔLGA is SDVISLLLWGRQLFA (SEQ ID NO:9); and NSP is ERTEESWGRRFWRRGEAC (SEQ ID NO: 10). Fig. 9B demonstrates the killing of bloodstream form (BSF) T. b. brucei by SHP-I (squares) and its variants, SHP-2 (diamonds), SHP-l-ΔL (white circles), SHP-I ΔLLL (black triangles), SHP-1-ΔLGA (gray triangles) and a non-specific hydrophilic peptide (NSP, gray circles). Fig. 9C is representative traces of liposome permeabilization by 200 nM SHP-I, 200 nM SHP-2 and the deletion variants, 500 nM SHP-l-ΔL, 500 nM SHP-1-ΔLLL and 500 nM SHP-1Δ-LGA.

Figure 10 demonstrates that SHP-I acts at the surface of bloodstream form (BSF) African trypanosomes. Fig. 1OA demonstrates that killing of BSF T. b. brucei does not require cellular uptake, indicated by robust SHP-I -mediated killing at 3⁰C, a temperature non-permissive for endocytosis. Alexa 594-labeled transferrin (Tf) binds at the flagellar pocket, but is not taken up at 3⁰C (inset). In Fig. 1OB, fluorescence microscopy with Texas-red labeled SHP-I reveals diffuse labeling of the cell surface rather than accumulation within an intracellular vesicle as displayed by Alexa 594-labeled transferrin (Tf). Cell nuclei and kinetoplast are labeled with DAPI.

Figure 11 shows that SHP-I interacts with membranes exhibiting fluid phase packing. Fig. 1 IA demonstrates that lateral van der Waals interactions dictate sensitivity to Hpr-SP, as indicated by the ability of 200 nM SHP-I to readily elicit calcein leakage from unilamellar liposomes composed of egg phosphatidylcholine (PC), a highly fluid, heterogeneous mixture of naturally derived lipids with T_m<0° C, whereas liposomes composed of homogenous lipid species with symmetric acyl chains, 15:0 (T_n, = 34°C) or 16:0 (T_m = 41 °C) are resistant. No permeabilization is seen against liposomes composed of symmetric 17:0 or 18:0 PC either. In Fig. 1 IB, the addition of l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (indicated by percentage) to refractory compositions l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) renders liposomes susceptible to permeabilization by SHP-I. Fig. 11C demonstrates that liposomes composed of l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) or l-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), asymmetrical PC lacking myristate, but exhibiting fluid lipid packing due to unsaturations (16:0, 18:l, T_m = -2°C and 18:0, 18:1, T_m = 6°C) are susceptible to permeabilization by SHP-I. Fig. 1 ID demonstrates that thermal fluidization of symmetrical 15:0 and 16:0 PC lipid bilayers renders liposomes susceptible to permeabilization by SHP-I. Assays were carried out at approximately 6O⁰C. Figure 12 demonstrates that SHP-I changes the mechanoelastic properties of bloodstream form (BSF) T. brucei membranes and induces dramatic changes in motility. In Fig. 12A, the rigidity of BSF T. brucei plasma membranes is increased by the addition of SHP-I as revealed by the anisotropic changes in the membrane probe l-(4-trimethylammoniumphenyl)-6-phenyl-l,3,5-hexatriene p- toluenesulfonate (TMA-DPH) (SHP-I - closed circle, DMSO only - open circles). Consistent with the lack of binding to more rigid lipid bilayers and the higher anisotropic values acquired for procyclic form (PCF) T. brucei, SHP-I has no effect on the anisotropy of PCF T. brucei cell plasma membranes (SHP-I - closed squares, DMSO only - open squares). Fig. 12B depicts the changes in motility of bloodstream form T. brucei treated with 8 M SHP-I. Fig 12C presents ilmages captured via DIC videomicroscopy illustrate the normal, hyperactive and constricted phenotype of cells treated with Hpr-SP. The normal motion of bloodstream form T. brucei displays a full rotation within 100 ms. Hyperactive cells appear to have accomplished 1.5-2 rotations within 50 ms. The constricted phenotype is commonly seen as a bent or boomerang shaped trypanosome that fails to complete a rotation.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

OF THE INVENTION

The present invention demonstrates, for the first time, the trypanocidal activity of hydrophobic signal sequence peptides and provides methods of killing, inhibiting the growth, and/or inhibiting the reproduction of kinetoplastid protozoan, including, but not limited to, kinetoplastid protozoan of the genus Trypanosoma, with hydrophobic signal sequence peptides and compositions including such hydrophobic signal sequence peptides. Protozoal pathogens a worldwide impact and cause symptomatic as well as asymptomatic infections. Unfortunately, effective treatments for the different diseases are by and large not available. This is especially true for African trypanosomiasis, a parasitic disease caused by a specific class of protozoan organisms, trypanosomes. The eukaryotic parasite Trypanosoma brucei infects mammals and causes debilitating diseases in both humans and cattle. The subspecies Trypanosoma brucei brucei (T. b. brucei ) causes the wasting disease in cattle nagana in cattle, while the two subspecies Trypanosoma brucei rhodesiense (T. b. rhodesiense) and Trypanosoma brucei gambiense (T. b. gambiense) cause sleeping sickness, also known as human African trypanosomiasis (HAT), in humans.

The methods and compositions of the present invention are applicable for a variety of protozoa, including, but not limited to, kinetoplastid protozoa. Kinetoplastids are a group of flagellate protozoa, including a number of parasites responsible for serious diseases in humans and other animals, including economically relevant livestock, as well as various forms found in soil and aquatic environments. They are included in the Euglenozoa, and are distinguished from other such forms mainly by the presence of a kinetoplast, a DNA-containing granule located within the single mitochondrion and associated with the flagellar bases. Kinetoplastids typically have complex life-cycles involving more than one host, and go through various morphological stages. The most distinctive of these is the trypomastigote stage, where the flagellum runs along the length of the cell and is connected to it by an undulating membrane. Kinetoplastid protozoa, include, for example, protozoa of the Blastocrithidia, Crithidia, Endotrypanum,

Herpetomonas, Leishmania, Leptomonas, Phytomonas, Trypanosoma, and Wallaceina genera. Diseases caused by trypanosomes include African Sleeping Sickness and South American Chagas Disease, from species of Trypanosoma, and leishmaniasis, from species of Leishmania. In some embodiments, the methods and compositions of the present invention are applicable for species of Trypanosoma, including, but are not limited to, T. avium, which causes trypanosomiasis in birds, T. boissoni, T. brucei, which causes sleeping sickness in humans and nagana in cattle, T. carassii, in freshwater teleosts, T. cruzi, which causes Chagas disease in humans, T. gambiense, T. rhodesiense, T. congolense, which causes nagana in cattle, horses, and camels, T. equinum, T. equiperdum, which causes dourine or covering sickness in horses, T. evansi, which causes one form of the disease surra in certain animals, T. lewisi, in rats, T. melophagium, T. percae in fish, T.rangeli, T. rotatorium in amphibian, T simiae, T. suis, T. theileri, T. triglae, and T. vivax.

In some embodiments, the methods and compositions of the present invention are applicable for the protozoan T. brucei, including, but not limited to, Trypanosoma brucei brucei, T. cruzi, T. brucei, Tb. gambiense, Tb. rhodesiense, T. congolense, and T. vivax. In some embodiments, the methods and compositions of the present invention are applicable for the protozoan Trypanosoma brucei brucei, Tb. gambiense, and Tb. rhodesiense, T. congolense, and T. vivax. hi some embodiments, the methods and compositions of the present invention are applicable for various protozoan Trypanosoma bracei, excluding not T. cruzi.

The present invention has identified peptides that demonstrate the ability of killing the bloodstream form (BSF) of kinetoplastid protozoans of the genus Trypanosoma. More specifically, these trypanocidal peptides are based upon the hydrophobic N-terminal signal sequences of various mammalian plasma proteins.

Additionally these peptides exhibit reduced or no toxicity towards mammalian cell lines and induce limited or no hemolysis.

Proteins have intrinsic signals that govern their transport and localization within the cell. Almost all proteins that are transported to the endoplasmic reticulum and destined either to be secreted or to be membrane components have a short signal sequence that directs the transport of a protein through the cell membrane. This signal peptide is an amino acid sequence present on the protein. It is usually at the N terminus. It is usually is cleaved off and absent in the mature protein, though in some proteins, the signal peptide uncleaved and is retained in the mature protein. Signal peptides are highly hydrophobic but with some positively charged residues.

A trypanocidal peptides of the present invention may also be referred to herein as an antitrypanosomal peptides, a signal peptide, SP, a signal sequence peptide, a hydrophobic peptide, a hydrophobic signal sequence peptide, a N-terminal signal sequence, or a hydrophobic N-terminal signal sequence. A trypanocidal peptide of the present invention may be a known hydrophobic

N-terminal signal sequence, or a fragment or derivative thereof. Derivatives include, but are not limited to, the exchange of one hydrophobic amino acid residue for another hydrophobic amino acid. Such hydrophobic amino acids include valine ("VaI" or "V"), isoleucine ("He" or "I"), leucine ("Leu" or "L"), methionine ("Met" or "M"), phenyalanine ("Phe" or "F"), tryptophan ("Trp" or "W"), cysteine ("Cys" or "C"), alanine ("Ala" or "A"), tyrosine ("Tyr" or "Y"), histidine ("His" or "H"), threonine, ("Thr" or "T"), serine ("Ser" or "S"), proline ("Pro" or "P"), glycine ("GIy" or "G"), arginine ("Arg" or "R"), and lysine ("Lys" or "K"). In some embodiments, derivatives include, but are not limited to, the exchange of one very hydrophobic amino acid residue for another very hydrophobic amino acid. Such very hydrophobic amino acids include valine ("VaI" or "V"), isoleucine ("lie" or "I"), leucine ("Leu" or "L"), methionine ("Met" or "M"), phenyalanine ("Phe" or "F"), tryptophan ("Tip" or "W"), and cysteine ("Cys" or "C"). Derivatives include, but are not limited to, the deletion of one, two, three, or more hydrophobic amino acids from the known signal sequence. Derivatives also include, but are not limited to, the deletion of one, two, three, or more very hydrophobic amino acids from the known signal sequence. Fragments may include, but are no limited to, peptides having at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive amino acids of the hydrophobic N-terminal signal peptide.

A trypanocidal peptide of the present invention may be a hydrophobic N-terminal signal sequence, with or without the N-terminal methionine residue, or a derivative or fragment thereof.

A trypanocidal peptide of the present invention may be any of a variety of lengths. For example, about 3 to about 60 amino acids in length, about 10 to about 25 amino acids in length, about 17 to about 23 amino acids, or about 20 amino acids. For example, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 amino acids in length, and/or no more than 10, no more than 15, no more than 20, no more than 25, or no more than 30 amino acids in length.

Trypanocidal peptides of the present invention may be identified by any of the assays described herein, including, but not limited to, a trypanosome lysis assay. Trypanocidal peptides of the present invention may target the cell membrane of protozoans, including, but not limited to kinetoplastid protozoans of the genus Trypanosoma, and may that increase the rigidity of lipid bilayers of the cell membrane. Trypanocidal peptides of the present invention may selectively partition into the plasma membrane of BSF trypanosomes resulting in an increase in the rigidity of the bilayer, changes in cell motility and/or subsequent cell death.

A trypanocidal peptide of the present invention may be a hydrophobic N-terminal signal sequence of a protein that is associated with high density lipoproteins (HDL). A trypanocidal peptide of the present invention may be a hydrophobic N-terminal signal sequence of an apolipoprotein, including, but not limited to a human apolipoprotein.

In some embodiments, a trypanocidal peptide of the present invention is an uncleaved, hydrophobic N-terminal signal peptide, that is, a signal peptide that is retained on the mature protein, and derivatives and fragments thereof. Examples of proteins with uncleaved signal peptides are haptoglobin-related protein (Hpr), serum paraoxonase/ arylesterase (PONl), and apolipoprotein M (Apo M), including, but not limited to, human haptoglobin-related protein, human serum paraoxonase/arylesterase (PONl), and human apolipoprotein M (Apo M). hi some embodiments, a trypanocidal peptide of the present invention includes the hydrophobic N-terminal signal peptide of haptoglobin-related protein (also referred to herein as "Hpr" or "HPR"), including, but not limited to, human haptoglobin-related protein (UniProtKB/Swiss-Prot Accession No: P00739), fragments and derivatives thereof. Hpr is an integral part of two distinct high molecular weight complexes (trypanosome lytic factor 1 (TLFl) and trypanosome lytic factor 2 (TLF2)) that are lytic for the African parasite Trypanosoma brucei brucei. See, for example, Smith et al., 1995, Science; 268(5208):284-6; Drain et al., 2001, J Biol Chem; 276(32):30254-60; and Muranjan et al., 1998, J Biol Chem; 273(7):3884-7. Human haptoglobin-related protein demonstrates an uncleaved signal peptide. For example, trypanocidal peptides of the present invention include peptides having the N-terminal amino acid residues 1-19, or 2- 19 of the human haptoglobulin-related protein (MSDLGAVISLLL WGRQLFA (SEQ ID NO:1) and SDLGAVISLLLWGRQLFA (SEQ ID NO:2), respectively), fragments, and derivatives thereof. Derivatives include, but are not limited to, the exchange of one hydrophobic amino acid residue for another hydrophobic amino acid. Derivatives may include, but are not limited to, the exchange of one very hydrophobic amino acid residue for another very hydrophobic amino acid. Derivatives may include, but are not limited to, the deletion of one, two, three, or more hydrophobic amino acids from the known signal sequence. Derivatives may also include, but are not limited to, the deletion of one, two, three, or more very hydrophobic amino acids from the known signal sequence. Fragments may include, but are no limited to, peptides having at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive amino acids of the hydrophobic N-terminal signal peptide of haptoglobin- related protein, including, but not limited to, human haptoglobin-related protein (UniProtKB/Swiss-Prot Accession No: P00739)

In some embodiments, a trypanocidal peptide of the present invention includes the hydrophobic N-terminal signal peptide of serum paraoxonase/ arylesterase (PONl), including, but not limited to, human serum paraoxonase/arylesterase (UniProtKB/Swiss- Prot Accession No. P27169), fragments and derivatives thereof. Serum paraoxonase/ arylesterase, like haptoglobin-related protein, demonstrates an uncleaved signal peptide. For example, trypanocidal peptides of the present invention include peptides having the N-terminal amino acid residues 1-22, or 2-22 of human paraoxonase- 1 protein (MAKLIALTLLGMGLALFRNHQS (SEQ ID NO:3) and AKLIALTLLGMGLALFRNHQS (SEQ ID NO:4), respectively), fragments, and derivatives thereof. Derivatives include, but are not limited to, the exchange of one hydrophobic amino acid residue for another hydrophobic amino acid. Derivatives may include, but are not limited to, the exchange of one very hydrophobic amino acid residue for another very hydrophobic amino acid. Derivatives may include, but are not limited to, the deletion of one, two, three, or more hydrophobic amino acids from the known signal sequence. Derivatives may also include, but are not limited to, the deletion of one, two, three, or more very hydrophobic amino acids from the known signal sequence. Fragments may include, but are no limited to, peptides having at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive amino acids of the hydrophobic N-terminal signal peptide of paraoxonase, including, but not limited to, human serum paraoxonase/ arylesterase (UniProtKB/Swiss- Prot Accession No. P27169. hi some embodiments, a trypanocidal peptide of the present invention includes the hydrophobic N-terminal signal peptide of apolipoprotein M (Apo M), including, but not limited to, human apolipoprotein M (UniProtKB/ Swiss-ProtO95445), fragments and derivatives thereof. Apolipoprotein M, like haptoglobin-related protein, and paraoxonase demonstrates an uncleaved signal peptide. For example, trypanocidal peptides of the present invention include peptides having the N-terminal amino acid residues 1-22, or 2- 22 of human apolipoprotein M (MFHQIWAALLYFYGIILNSIYQ (SEQ ID NO:5) and FHQIWAALLYFYGΠLNSIYQ (SEQ ID NO:6), respectively), fragments, and derivatives thereof. Derivatives include, but are not limited to, the exchange of one hydrophobic amino acid residue for another hydrophobic amino acid. Derivatives may include, but are not limited to, the exchange of one very hydrophobic amino acid residue for another very hydrophobic amino acid. Derivatives may include, but are not limited to, the deletion of one, two, three, or more hydrophobic amino acids from the known signal sequence. Derivatives may also include, but are not limited to, the deletion of one, two, three, or more very hydrophobic amino acids from the known signal sequence. Fragments may include, but are no limited to, peptides having at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive amino acids of the hydrophobic N-terminal signal peptide of apolipoprotein M (Apo M), but not limited to, human apolipoprotein M UniProtKB/S wiss-ProtO95445.

The present invention provides for the use of trypanocidal peptides as anti-protozoan agents and provides methods of killing, inhibiting the growth, and/or inhibiting the reproduction of a protozoan by contacting the protozoan with one or more trypanocidal peptides. As used herein, the term "inhibit" means prevent, decrease, or reverse. Such contact may be in vitro, ex vivo, and/or in vivo. As used herein in vitro is in cell culture, ex vivo is a cell that has been removed from the body of a subject, and in vivo is within the body of a subject. As used herein, the term "subject" or

"individuaP'represents an organism, including, for example, a mammal. A mammal includes, but is not limited to, a human, a non-human primate, livestock (such as, but not limited to, a cow, a horse, a goat, and a pig), a rodent, such as, but not limited to, a rat or a mouse, or a domestic pet, such as, but not limited to, a dog or a cat. The present invention provides methods of killing, inhibiting the growth, and/or inhibiting the reproduction of a protozoan in a subject by administering to the subject an effective amount of one or more trypanocidal peptides. A trypanocidal peptide of the present invention may be administered in an amount effective to inhibit replication and/or growth of the protozoan. A trypanocidal peptide of the present invention may be adrninistered in an amount effective to kill a protozoan in an infected individual.

Inhibition of the growth and reproduction of a protozoan and killing of an a protozoan may be determined by any of various known methods, including, but not limited to, the methods described in the examples herein.

The present invention provides methods of treating or preventing a protozoan infection in a subject by administering to the subject an effective amount of one or more trypanocidal peptides. Such a trypanocidal peptide may be identified by the methods described herein. As used herein "treating" or "treatment" includes therapeutic and/or prophylactic treatments. Desirable effects of treatment include preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. A trypanocidal peptide may be administered to a subject to reduce the severity of the symptoms associated with a protozoan infection. Peptides of the present invention may be taken as a prophylactic to prevent the development of a protozoan infection. A peptide of the present invention may be administered to a subject to prevent the infection of a subject with a protozoan. A peptide of the present invention may be administered to a subject prior to and/or after exposure to a protozoan.

A trypanocidal peptide may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions and methods.

By a "therapeutically effective amount" of a trypanocidal peptide is meant a sufficient amount of the compound to treat the subject at a reasonable benefit/risk ratio applicable to obtain a desired therapeutic response. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including, for example, the disorder being treated and the severity of the disorder, activity of the specific compound employed, the specific composition employed, the age, body weight, general health, sex and diet of the patient, the time of administration, route of administration, and rate of excretion of the specific compound employed, the duration of the treatment, drugs used in combination or coincidentally with the specific compound employed, and like factors well known in the medical arts.

Trypanocidal peptides of the present invention can be administered by any suitable means including, but not limited to, for example, parenteral (involving piercing the skin or mucous membrane), oral (through the digestive tract), transmucosal, rectal, nasal, topical (including, for example, transdermal, aerosol, buccal and sublingual), or vaginal. Parenteral administration may include, for example, subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrasternal, and intraarticular injections as well as various infusion techniques)

The present invention includes compositions including one or more trypanocidal peptides. Also included are compositions of one or more isolated trypanocidal peptides. As used herein, the term isolated means a preparation that is either removed from its natural environment or synthetically derived, for instance by recombinant techniques, or chemically or enzymatically synthesized. In a preferred form, an isolated trypanocidal peptides is purified and substantially free of other agents.

Compositions may be administered in any of the methods of the present invention and may be formulated in a variety of forms adapted to the chosen route of administration. The formulations may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. A composition may include a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable," as used herein, means that the compositions or components thereof so described are suitable for use in contact with human skin without undue toxicity, incompatibility, instability, allergic response, and the like. A composition may be a pharmaceutical composition.

The preparation of such compositions is well understood in the art. The formulations of this invention may include one or more accessory ingredients including, but not limited to, diluents, buffers, binders, disintegrants, surface active agents, thickeners, lubricants, preservatives, including, for example, antioxidants, and the like. Pharmaceutically acceptable includes salts, amides and esters that are well known in the art. Representative acid addition salts include, for example, hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, toluenesulfonate, methanesulfonate, citrate, maleate, fumarate, succinate, tartrate, ascorbate, glucoheptonate, lactobionate, lauryl sulfate salts, and the like. Representative alkali or alkaline earth metal salts include, for example, aluminum, calcium, lithium, magnesium, potassium, sodium, or zinc salt, an ammonium salt such as a tertiary amine or quaternary ammonium salt, and an acid salt such as a succinate, tartarate, bitartarate, dihydrochloride, salicylate, hemisuccinate, citrate, isocitrate, malate, maleate, mesylate, hydrochloride, hydrobromide, phosphate, acetate, carbamate, sulfate, nitrate, formate, lactate, gluconate, glucuronate, pyruvate, oxalacetate, fumarate, propionate, aspartate, glutamate, or benzoate salt, and the like. Pharmaceutically acceptable carriers includes, for example, non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Examples of materials that may serve as pharmaceutically acceptable carriers include, but are not limited to, sugars, such as, for example, lactose, glucose and sucrose, starches such as, for example, corn starch and potato starch, cellulose and its derivatives such as, for example, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate, powdered tragacanth, malt, gelatin, talc, excipients such as, for example, cocoa butter and suppository waxes, oils such as, for example, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil, glycols, such as, for example, propylene glycol, polyols such as, for example, glycerin, sorbitol, mannitol and polyethylene glycol, esters such as, for example, ethyl oleate and ethyl laurate, agar, buffering agents such as, for example, magnesium hydroxide and aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol and phosphate buffer solutions, as well as other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants such as, for example, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

For parenteral administration in an aqueous solution, the solution may be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. For enteral administration, the inhibitor may be administered in a tablet or capsule, which may be enteric coated, or in a formulation for controlled or sustained release. Many suitable formulations are known, including polymeric or microparticles or nanoparticles encapsulating drug to be released, ointments, gels, or solutions which can be used topically or locally to administer drug, and even patches, which provide controlled release over a prolonged period of time. These can also take the form of implants. Compositions for nasal administration may be formulated for aerosol or inhalation administration. Such compositions may include solutions in saline which may also contain, for example, benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, and/or other solubilizing or dispersing agents such as those known in the art. Compositions for rectal administration include, for example, suppositories which may contain a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquify and/or dissolve in the rectal cavity to release the drug.

For human and veterinary administration, compositions of the present invention may meet sterility, pyrogenicity, and general safety and purity standards as required by the FDA. Such compositions are considered suitable for parenteral or enteral administration to a mammal. Such compositions may be pyrogen-free.

In accordance with the present invention, trypanocidal peptides may be administered in combination with the administration of one or more previously known treatment modalities. As used herein, the term "additional therapeutic agent" represents one or more agents previously known to be effective for the treatment of a protozoan disease or other conditions. Such an additional therapeutic agent is not a trypanocidal peptide. The administration of a trypanocidal peptide may take place before, during, and/or after the administration of the other mode of therapy. The present invention includes methods of administering one or more trypanocidal peptides in combination with the administration of one or more previously known treatment modalities. The present invention includes compositions of one or more trypanocidal peptides and one or more previously known treatment modalities. hi some embodiments of the present invention, the administration of a trypanocidal peptide in combination with additional therapeutic agents may demonstrate therapeutic synergy. Likewise, the administration of two or more trypanocidal peptides may demonstrate therapeutic synergy. As used herein, a combination may demonstrate therapeutic synergy if it is therapeutically superior to one or other of the constituents used at its optimum dose (Corbett et al., 1982, Cancer Treatment Reports; 66:1187. hi some embodiments, a combination demonstrates therapeutic synergy if the efficacy of a combination is characterized as more than additive actions of each constituent.

Liposomes are currently used as drug carriers for a variety of antitumor agents, antiinflammatory agents and the like. The present invention also includes liposomes, lipid carriers, complexes, mixtures, supramolecular structures, multimolecular aggregates ans lipid-based drag delivery systems including one or more trypanocidal peptides. Such liposomes or liposome-like compositions may be in the form of a monolayer, bilayer, multimolecular aggregate, vesicle, helix, disc, tube, fiber, torus, hexagonal phase, gel phase, liquid-crystalline phase, liquid-crystalline multimolecular aggregate, micelle, reverse micelle, microemulsion, emulsion, microreservoir, oil globule, fat globule, wax globule and/or colloidal particle. Such liposomes and liposome-like compositions may further include additional agents, including, for example, additional therapeutic agents and/or targeting moieties, including, but not limited to, KNfA apatamers. Such liposomes or liposome-like compositions including one or more trypanocidal peptides may be used in any of the methods described herein. Compositions of the invention may further include one or more targeting moieties against parasite target molecules. Compositions including such targeting moieties may be used in any of the methods described herein. Targeting moieties include, but are not limited to, high-affinity nucleic acid ligands, also referred to as DNA aptamers and RNA aptamers, that bind with high affinity and high specificity to parasite target molecules. For example, a number of trypanosome-specific RNA aptamers that bind with high affinity and high specificity to the variant surface glycoprotein or to an invariant surface domain of live African trypanosomes have been identified. See, for example, Goringer et al, 20003, Int J Parasitol; 33(12):1309-17; Lorger et al., 2003, Eukaryot Cell; 2(l):84-94; Goringer et al., 2006, Handb Exp Pharmacol; (173):375-93; Homann et al., 2006, Comb Chem High Throughput Screen; 9(7):491-9; and Adler et al., 2008, Comb Chem High Throughput Screen; 11 ( 1 ) : 16-23. Any of such RNA aptamers may be used in the compositions and methods of the present invention.

Many therapeutic agents possess a high degree of hydrophobicity which can impede their solubilization in aqueous media and thus hamper their oral or parenteral administration. Compositions of the present invention may include formulations that facilitate the solubilization and/or delivery of hydrophobic drugs. Such formulations may include any of a variety of such formulations known to the skilled artisan, including, but not limited to, amphiphilic polymers, lipid-based nanocapsules, nanoformulations, polymeric micelles, magnetic nanocarriers, nano-sized carriers that contain a hydrophobic core, polymeric vectors, lipidic vectors, emulsions, lipid emulsions, and microemulsions. Such formulations may include any of the formulations described herein, such as for example, alcohol and dimethyl sulfoxide (DMSO). The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLES

Example 1 Novel Antimicrobial Peptides Derived from Human Apolipoproteins

This example demonstrates that a small peptide derived from the human haptoglobin related protein (Hpr) exhibits potent and specific killing of African trypanosomes. The peptide acts upon the cell membrane of the trypanosome, inducing a rigidification of the bilayer lipids and subsequent cell death. The amino acid sequence of the peptide is based upon the N-terminal signal sequence of Hpr. Hpr is a secreted protein that associates with high density lipoproteins (HDL). It is unusual in that it is secreted without cleavage of its N-terminal signal sequence. Only two other proteins are known that exhibit this phenomenon, paraoxonase 1 and apolipoprotein M, both of which associate with HDL. This small 19 amino acid peptide, based upon the signal sequence of Hpr can be used as a therapeutic agent in the treatment of mammalian pathogens, particularly Trypanosoma brucei, the causative agent of African sleeping sickness. Currently, there are approximately 500,000 infected people in sub-Saharan nations. Treatment of infected individuals is limited and there is an alarming increase in the number of relapses from established drug treatment (Bran et al., 2001, Trop Medlnt Health; 6:906-914). Few new drugs are currently under clinical trials, and one of the more promising compounds, DB75, has already shown a marked tendency to induce drug resistance (Lanteri et al., 2006, MoI Pharmacol; 70:1585-1592). Based upon the ancient evolutionary origin of antimicrobial peptides that target cell membranes and their abundance in all branches of life, it is likely that drug resistance will develop slowly, if at all, to the peptide of the present invention. Haptoglobin related protein (Hpr) is a component of the trypanolytic human high density lipoprotein, termed trypanosome lytic factor (TLF). Unusual for secreted proteins, Hpr retains its N-terminal 19 amino acid signal peptide (Smith et al. 1995, Science; 268(5208):284-286). Native, delipidated Hpr has been shown to be cytotoxic to Trypanosoma brucei (Shiflett et al., 2005, J Biol Chem; 280(38):32578-32585). This example shows that trypanosome killing by Hpr is dependent upon the presence of the hydrophobic signal peptide and that a synthetic peptide corresponding to the signal sequence is sufficient for killing. Indeed, the synthetic Hpr signal peptide (Hpr-SP) specifically kills bloodstream, but not procyclic form T. brucei, is not inhibited by the serum resistance associated protein, which is responsible for evasion of TLF killing in T. b. rhodesiense (Hager and Hajduk, 1997, Nature; 385(6619):823-826), and Hpr-SP appears to act at the surface of the trypanosome. hi vitro studies with model liposomes suggests that bloodstream form T. brucei are uniquely susceptible to killing by the peptide due to the acyl chain composition of their cellular membrane. Due to the VSG coat, bloodstream form T. brucei have a high content of relatively short myristoyl acyl chains (14 carbon, saturated) (Ferguson et al., 1985, J Biol Chem; 260(27):14547-14555; and Ferguson et al., 1985, J Biol Chem; 260(8):4963-4968), whereas the signal peptide resistant procyclic form T. brucei (the developmental stage found within the insect vector) contain a larger content of palmitoyl (16 carbon, saturated) and stearoyl (18 carbon, saturated) chains (Treumann et al., 1997, JM?/ Biol; 269(4): 529-547), providing for greater van der Waals interactions within the bilayer, possibly inhibiting signal peptide penetration. Data show that the Hpr-SP is not toxic towards mammalian cells, thus it may provide a novel therapeutic for treatment of African trypanosomiasis.

Materials and Methods

Peptides. Synthetic peptides corresponding to the 19 amino acid N-terminal signal peptide of Hpr (MSDLGAVISLLLWGRQLFA) (SEQ ID NO:1) were purchased from Bio-Synthesis, Inc. (Lewisville, TX). Non-specific, hydrophilic peptides (ERTEESWGRRFWRRGEAC) (SEQ ID NO: 10) were predicted from the N-terminus of the alternatively edited protein- 1 (AEP-I) from mitochondria of T. b. brucei (Ochsenreiter and Hajduk, 2006, EMBO; 7(11):1128-1133) and purchased from Alpha Diagnostic (San Antonio, TX).

Lipids. All lipids were purchased from Avanti Polar Lipids (Alabaster, AL). These include phosphatidylcholine from egg (#840051) and 1,2-distearoyl-sn-glycero- 3-phosphocholine (DSPC) (#850365).

Trypanosoma lysis assays. Trypanosomes were cultured in the appropriate media and incubated at a cell density of 3 X 10⁶ cells/ml with the addition of peptide from a 10 mg/ml stock in DMSO, or control reagents for 2 hours (h) at 37°C. Lysis of bloodstream form trypanosomes was evaluated by phase-contrast microscopy. Procyclic form trypanosomes were stained with 0.1 % trypan blue. Trypan blue staining was also used for some bloodstream form assays to eliminate any possible discrepancy between the procyclic and bloodstream form assays. No difference was observed with the presence of trypan blue in bloodstream form lysis assays.

Mammalian cell viability assays. Human embryonic kidney cells (HEK, ATCC # CRL- 1573) were cultured in Eagle's Minimum Essential Medium supplemented with 10 % fetal bovine serum. Cell viability after treatment with Hpr-SP or the relevant controls was determined via trypan blue exclusion. Briefly, cells were plated at approximately 50-60% confluency into 96-well poly-lysine coated microtiter plates. Serial dilutions of Hpr-SP from a 10 mg/ml stock in DMSO were added and the cells were incubated for 2 hours at 37°C. Positive killing controls were serial dilutions of melittin, a membrane permeabilizing peptide from honey bee venom.

Anisotropy assays. Bloodstream form trypanosomes were washed and resuspended at 3 X 10⁶ cells/ml in phosphate buffered saline. The anisotropic probe trimethylammonium-l,6-diphenyl-l,3,5-hexatriene (TMA-DPH) was added to a concentration of 0.2 μM and allowed to intercalate into the cell membrane for 15 minutes (min) at ambient temperature (25⁰C). The anisotropic value was acquired with a Perkin Elmer Life Sciences fluorescence spectrophotometer LS-55 with excitation at 358 run and emission read at 430 nm, both with 10 nm slit widths. Values expressed were determined immediately after the addition of Hpr-SP or DMSO only and are the average of three independent assays (except for no addition and 0.167 % DMSO). The anisotropy values did not change over the course of an hour.

Liposome permeabilization assays. Liposomes were constructed by hydration of dried lipid films with 10 mM Hepes and 30 mM calcein to a final lipid concentration of 10 mg/ml. Resulting multilamellar liposomes were extruded through a 0.1 μm polycarbonate filter at temperatures above the transition point of the particular lipid mixture. Untrapped calcein was removed by gel filtration (Sephacryl S-300 HR). Permeabilization of liposomes was monitored fluorimetrically with a Perkin Elmer LS-55 luminescent spectrophotometer. Assays were performed by diluting liposomes 1 : 1000 into the appropriate buffer and monitoring fluorescence when excited by 484 nm light (5 nm slit width) and reading the emission intensity at 513 nm (10 nm slit widths). The percentage calcein release was calculated relative to the 100 % fluorescence intensity, achieved by the addition of 0.01 % Triton X-100.

Results Hpr-SP is sufficient for trypanolysis. Based upon the necessity of the Hpr-SP for killing activity by Hpr, the trypanolytic activity of a synthetic peptide corresponding to the 19-amino acids of Hpr-SP was investigated. Addition of Hpr-SP to bloodstream form trypanosomes efficiently killed cells in a dose dependent fashion. The Hpr-SP solubilizing agent DMSO did not exhibit toxicity at concentrations equal to the highest dosage of Hpr-SP tested. The morphology of Hpr-SP killed trypanosomes is dramatic and distinct from cells killed by TLF. Excessive fraying of the cellular membrane, and what appears to be leakage of cytoplasmic contents is apparent. These data suggest that the mechanism of Hpr-SP trypanosome killing is quite different than that employed by TLF. Fig. IA shows the killing of T. b. brucei by the Hpr-SP. Wild type 427 T.b. brucei cells were incubated with increasing concentrations of Hpr-SP (8-80 μM) added from a stock solution in DMSO. Positive controls were trypanosome lytic factor (TLF). Addition of equivolume DMSO or equimolar concentrations of a non-specific hydrophilic 19 amino acid peptide (NS 19mer; SEQ ID NO: 10) gave no trypanolysis. The inset in Fig. IA shows cells treated with Hpr-SP. Fig. IB presents a cartoon of Hpr and the derived peptide Hpr-SP (SEQ ID NO:!). Hydrophobic residues are in bold. Hpr-SP kills SRA-expressing T. brucei and exhibits specificity for bloodstream form trypanosomes. The trypanolytic activity of Hpr-SP against procyclic trypanosomes (the insect stage form which dramatically down regulates endocytic activity) was assayed. Surprisingly, only very minor trypanolytic activity was found against these cells (> 20 % at 80 μM Hpr-SP). This discrepancy may be accounted for by the different compositions of the bloodstream and procyclic form trypanosome membranes. Because the Hpr-SP is derived from TLF, we asked whether SRA, the protein responsible for inhibiting TLF killing in the human pathogenic trypanosome strain T. b. rhodesiense, provided protection against Hpr-SP mediated killing. SRA expressing trypanosomes were killed with identical activity as non-SRA expressing T. b. brucei. The morphology of Hpr-SP killed trypanosomes is dramatic and distinct from cells killed by TLF. Excessive fraying of the cellular membrane, and what appears to be leakage of cytoplasmic contents is apparent. These data suggest that the mechanism of Hpr-SP trypanosome killing is quite different than that employed by TLF. In Fig. 2, lysis assays were conducted with T. b. brucei expressing SRA or procyclic form T. b. brucei. SRA protects human pathogenic T. b. rhodesiense from the lytic activity of TLF. However, SRA offers no protection against Hpr-SP. Procylic form T. b. brucei are inefficiently killed by Hpr-Sp. As discussed below, it may be the case that a higher content of stearoyl acyl chains in the trypanosome membrane protects against the lytic effect of the small highly hydrophobic Hpr-Sp.

Hpr-SP acts at the cytoplasmic membrane of bloodstream form trypanosomes. A diagnostic feature of TLF killing is the necessity for trafficking to the lysosome of the target trypanosome, where the acidic environment activates a membrane disruptive activity by TLF. Therefore we asked if compounds that inhibit the acidification of the lysosome (and are known inhibitors of TLF) block the killing activity of Hpr-SP. Thus, lysis assays were performed with Hpr-SP in the presence of 10 mM NH₄Cl or 50 μM chloroquine. No inhibitory effect by these pH neutralizing agents was found, suggesting that either Hpr-Sp is endocytosed but does not require acidic conditions or that the peptide acts at the surface of the target trypanosome. hi order to answer this question, lysis assays were performed at 3⁰C, a temperature that effectively halts endocytosis. Robust trypanolysis was observed, equivalent to activity observed at 37⁰C, indicating that the Hpr-SP does not require cellular uptake to exert its toxic effects.

In Fig. 3, the site of action was probed by conducting lysis assays in the presence of chloroquine, NH₄Cl, or at 3⁰C. Inhibiting the acidification of the lysosomes with either chloroquine NH₄Cl, protects against TLF killing. No inhibition of Hpr-Sp was observed, suggesting that Hpr-SP does not require targeting tp the intracellular compartment. Endocytosis was inhibited by incubating T. b. brucei at 3⁰C. The Hpr-SP readily killed cells at the non-permissive temperature, confirming the cell surface as the site of action of Hpr-Sp. Hpr-SP exhibits specificity of permeabilizing activity against different model membranes. In order to test the effect of membrane composition on the specificity displayed by Hpr-SP, a model liposome system was utilized in which we fluorescently monitored the leakage of dye from the liposome interior. Liposomes composed entirely of egg PC₃ a heterogeneous mixture with respect to the length and degree of unsaturations, are readily permeabilized by nanomolar concentrations of Hpr-SP. Permeabilizing activity was robust at pH 6.8, consistent with the ability of Hpr-SP to target trypanosomal membranes in the extracellular medium. However, when Hpr-SP permeabilizing activity against liposomes composed entirely of DSPC, 18- carbon saturated lipids, no permeabilizing activity at neutral or acidic pH was found. This suggests that either the Hpr-SP is incapable of intercalating into the hydrophobic regions of the DSPC bilayer or that intercalation of the peptide is incapable of producing sufficient destabilization, and thus permeability increase, of the DSCP bilayer.

Fig. 4 shows modeling of T.b. brucei cellular membranes with liposomes. Artificial vesicles were constructed with the phosphatidylcholines corresponding in acyl chain composition to either the VSG or procyclin lipid anchors, egg phosphatidylcholine (eggPC, transition temperature < 0⁰C) for VSG and distearoylphosphatidylcholine (DSPS) for procyclin. The leakage of liposomally entrapped calcein was fluorescently monitored as an indicator of Hpr-SP membrane interaction.

Hpr-SP induces a rigidifϊcation of the bilayer lipids in T. brucei. In order to understand the effect of Hpr-SP on the cell membrane of target trypanosomes the lipid bilayer fluidity of trypanosomes treated with Hpr-SP was analyzed via anisotropy determinations. The addition of lytic concentrations of Hpr-SP induced a rigidity increase, i.e. a constriction of rotational and lateral motion of the lipid components, in the cell membrane. These data are particularly important as bloodstream form trypanosomes rely upon a fluid membrane to recycle their VSG coat and thus avoid killing mediated by host antibodies.

In Fig. 5, membrane rigidity in live T.b. brucei was monitored with the anisotropic probe TMA-DPH. The addition of Hpr-SP results in an increase in the anisotropy of TMA-DPH labeled cells, indicating membrane rigidification. Equivolmes of DMSO, the Hpr-SP solvent, results in either no change (0.167% DMSO corresponding to 8 μM Hpr-SP) or a decrease (0.4% DMSO corresponding to 20 μM Hpr-SP) in membrane rigidity. Modulation of the membrane fluidity may contribute to the cytotoxity of Hpr-SP.

Hpr-SP is not toxic towards mammalian cells. The potential toxicity of Hpr-SP towards human cells was assayed by determining the effect of exogenous Hpr-SP on the viability of human embryonic kidney cells. No toxicity was observed towards these cells by Hpr-SP or equivolume addition of DMSO, the solubilizing agent. These data are not unexpected as Hpr-SP is an endogenously derived molecule that is associated with hepatic cell membranes. hi Fig. 6, cell viability was assayed via trypan blue exclusion. Human embryonic kidney cells were incubated with increasing concentrations of Hpr-Sp for two hours at 37⁰C. No cell death was observed with trypanolytic concentrations of Hpr-SP or equivolume addition of DMSO. Positive killing controls provided by the lytic peptide melittin. Lack of cytotoxicity against mammalian cells suggest that Hpr-SP provide a novel therapeutic strategy for treating African trypanosomiasis. hi conclusion, this example demonstrates that the N-terminal signal sequence of Hpr is sufficient for trypanosome killing; that Hpr-SP exhibits specificity for BSF form T.b. brucei and evades the protective effect of SRA; that Hpr-SP acts at the surface of target trypanosomes, inducing a rigidification of the lipid bilayer; and that the lack of toxicity towards mammalian cells support Hpr-SP as a therapeutic agent. Discussion

Previous studies had revealed that human Hpr, a protein encoded by a gene that evolved during primate evolution, was toxic to trypanosomes and when assembled in TLF, with another primate specific protein, ApoL-1, had a high specific activity for killing trypanosomes (Shifted: et al, 2005, J Biol Chem; 280(38):32578-32585). Studies to characterize the mechanism of Hpr killing revealed that Hpr was unusual in that it contained an unprocessed N-terminal signal peptide (Hpr-SP). Deletion of the Hpr-SP inactivated recombinant Hpr (Vanhollebeke et al., 2007, PNAS; 104(10):4118-4123). The 19 amino acid Hpr-SP was synthesized and tested for trypanosome killing activity. Hpr-SP damage to the trypanosomes is striking. Cells rapidly fragment and the rate of killing is independent of temperature. On the other hand, TLF killing of trypanosomes requires endocytosis and lysosomal trafficking, has a characteristic lag phase of ~20 min and killing activity is completely blocked at temperatures below 4°C. When human embryonic kidney cells were incubated at the same concentration of Hpr-SP no toxicity was observed, indicating the Hpr-SP selectively interacts with trypanosomes.

These results show that Hpr-SP is highly toxic to trypanosomes, at low μM concentrations, and acts rapidly at the cell surface. In addition, mammalian cells are resistant to Hpr-SP. The rigidification increase seen upon addition of Hpr-SP to bloodstream form trypanosomes may be directly involved in the cytotoxicity of the peptide. T. brucei rapidly turn over a dense protein surface coat through endocytosis at a specific site at the posterior of the cell (Engstler et al., 2007, Cell; 131(3):505-515). This turnover facilitates evasion from the host immune system. The fluidity of the cell membrane has been directly implicated in this process as the proteins are swept backwards towards the endocytic site by outside physical forces, i.e. the flow of the extracellular environment. In this regard it may even be the case that the peptide will facilitate immune destruction of trypanosomes by blocking the turnover of the cellular protein coat. Example 2

Plasma Membrane of Bloodstream Form African Trypanosomes Confers Susceptibility and Specificity to Killing by Hydrophobic Peptides

The developmental stage of T. brucei found within the mammalian host, the bloodstream form (BSF), is a highly motile cell (Rodriguez et al, 2009, PNAS; 106(46): 19322- 19327; and Oberholzer et al., 2010, PLoS Pathog; 6(l):el000739), exhibits rapid rates of endocytosis (Engstler et al., 2004, JCe// Sci; 117(Pt 7):1105-1115) and free diffusion of densely packed GPI-anchored proteins (Bulow et al., 1988, Biochem; 27(7):2384-2388). Endocytosis is restricted to the flagellar pocket, a small, specialized membrane structure at the posterior of the cell (Field and Carrington, 2009, Nature Rev; 7(11):775-786). This morphology requires that surface associated cargo destined for endocytosis be laterally sorted in the plane of the membrane to the flagellar pocket. Perhaps the most striking example of this activity is the sorting of immunoglobulin bound variant surface glycoproteins (VSG) to the flagellar pocket by physical forces generated from the flow of extracellular medium over the surface of the trypanosome (Engstler et al. Al., 2007, Cell; 131(3):505-515). The nature of the trypanosome plasma membrane that facilitates rapid lateral sorting is not known. In the case of VSG, surface flow may benefit from the use of myristate, a relatively short acyl chain, as the membrane-anchoring moiety (Ferguson et al., 1985, J Biol Chem;

260(27): 14547-14555; and Ferguson et al., (1985), J Biol Chem, 260(8):4963-4968). The procyclic form (PCF) T. brucei, found within the insect midgut, does not exhibit rapid rates of endocytosis (Langreth and Balber, 1975, J Protozool; 22(l):40-53; and Morgan et al., 2001, JCe// Sci; 114(Pt 14):2605-2615) and the GPI-anchored procyclin surface proteins are anchored with longer palmitoyl and stearoyl acyl chains (Field et al., 1991, EMBO; 10(10):2731-2739).

The structure of the plasma membrane is also important for avoiding lysis by host defense factors such as complement or antimicrobial peptides. The terminal components of the complement membrane attack complex are sterically hindered from assembling by the dense surface coat of VSG. Human defensins, antimicrobial peptides with a distinct tertiary structure, are relatively inefficient at killing BSF T. brucei (McGwire et al., 2003, JInfectDis; 188(1):146-152) and it may be the case that the VSG coat inhibits interaction with the cell membrane. Interestingly BSF and PCF T. brucei exhibit differential susceptibility to a number of antimicrobial peptides (McGwire et al., 2003, J Infect Dis; 188(1):146-152). This distinction may be attributed to the different surface density of GPI-anchored proteins between the two forms, VSG being present at roughly an order of magnitude greater than procylin, thereby providing greater steric hinderance. Alternatively, or in addition to the difference in surface protein density, the differences in phospholipid and sterol composition between the two developmental forms may play a role.

The present example describes the trypanocidal activity of a small hydrophobic peptide (SHP-I), derived from the signal sequence of a human apolipoprotein, haptoglobin related protein (Hpr), which circumvents steric hinderance from the VSG coat and interacts with the plasma membrane of BSF T. brucei. This example shows that this non-cationic peptide specifically targets fluid membranes. The SHP-I rapidly binds BSF cells rather than PCF or mammalian cells. Consistently, BSF T. brucei are readily killed by SHP-I and no lysis or killing is observed with PCF T. brucei, mammalian cells or human erythrocytes.

Methods

Peptides. Synthetic peptides corresponding to the N-terminal signal peptide of human apolipoproteins haptoglobin related protein ("SHP-I," SDLGAVISLLLWGRQLFA (SEQ ED NO:2)), paraoxonase-1 ("SHP-2," (AKLIATLLGMGLALFRNHQS (SEQ ID NO:4)) and all derivatives ("SHP-l-ΔL," SDLGAVISLLWGRQLFA (SEQ ID NO:7); "SHP-1-ΔLLL,"

SDLGAVISWGRQLFA (SEQ ID NO:8); and "SHP-1-ΔLGA," SDVISLLLWGRQLFA (SEQ ID NO:9) were purchased from Bio-Synthesis, Inc. (Lewisville, TX). A non-specific, hydrophilic peptide (ERTEESWGRRFWRRGEAC (SEQ ID NO: 10)) predicted from the N-terminus of the alternatively edited protein- 1 from mitochondria of T. b. brucei, was purchased from Alpha Diagnostic (San Antonio, TX) (Ochsenreiter and Hajduk, 2006, EMBO; 7(11):1128-1133).

Lipids. All lipids were purchased from Avanti Polar Lipids (Alabaster, AL). These include phosphatidylcholine from egg (egg PC, #840051), l,2-distearoyl-OT-glycero-3- phosphocholine (DSPC) (#850365), 1 ,2-diheptadecanoyl-,y«-glycero-3 -phosphocholine (#850360), l₅2-dipalmitoyl-OT-glycero-3 -phosphocholine (DPPC) (#850355), 1,2- dipentadecanoyl-sn-glycero-S-phosphocholine (#850350), 1,2-dimyristoyl-^-glycero- 3 -phosphocholine (DMPC) (#850345), l-pahnitoyl-2-oleoyl-OT-glycero-3-phosphocholine (POPC) (#850457) and (#850467). Trypanosome killing assays. Bloodstream form T. b. brucei Lister 427 (MiTat

1.2), T. b. gambiense type 1 (Eliane strain) and T. b. rhodesiense KETRI2482 were used in these studies. Trypanosome killing assays were performed as described previously (Hajduk et al, 1989, J Biol Chem; 264(9):5210-5217; Shiflett et al., 2005, J Biol Chem; 280(38):32578-32585; and Widener et al., 2007, PLoS Pathog; 3(9): 1250-1261). Cultured trypanosomes (except for T. b. gambiense which were incubated at 1 x 10⁶ cells/ml) were incubated in HMI-9 media (BSF) or SM media (PCF) at a density of 1 x 10⁷ cells/ml containing either 10% heat inactivated fetal bovine serum or bovine serum albumin and peptide or control reagents for two hours at 37°C. Killing of BSF trypanosomes was evaluated by phase-contrast microscopy. Procyclic form trypanosomes were stained with 0.1% trypan blue. Trypan blue staining was also used for some BSF assays to eliminate any possible discrepancy between the PCF and BSF assays. Trypan blue was added at the endpoint of each assay to avoid any toxic effects. No difference was observed with the presence of trypan blue in BSF killing assays.

Mammalian cell viability and hemolysis assays. Human embryonic kidney cells (HEK) (ATCC CRL-1573) and LNCaP prostate cancer cells (ATCC CRL-1740) were utilized for cell viability assays. Human embryonic kidney cells were cultured hi Dulbecco's Modified Eagles Medium, high glucose (Thermo Scientific, cat # SH30243.01) with 10% fetal bovine serum at 37° C and 5% CO₂. Prostate cancer cells were cultured in RPMI-1640 medium (Invitrogen, cat # Al 0491-01) with 10% fetal bovine serum at 37⁰C and 5 % CO₂. hi both cases, assays were performed by aliquoting cells into 96 well plates at approximately 60% confluency and allowing the cells to adhere for two hours. Cells were then incubated with serial dilutions of SHP-I, or the relevant control, in the corresponding media for two hours at 37⁰C. Cell viability was determined by the ability of live cells to exclude trypan blue. The cells were incubated with 0.1% trypan blue for 10 minutes and examined microscopically for cytoplasmic staining. The potential for SHP-I to induce hemolysis was assayed by monitoring hemoglobin (Hb) release from freshly collected human erythrocytes that had been washed and incubated with SHP-I in PBS. The Hb concentration of the supernatant was determined by the absorbance at 412 nm and compared to 100% hemolysis acquired by hypotonic lysis. The membrane permeabilizing peptide, melittin, was used a positive control for killing or hemolysis.

Microscopy. All images were acquired with an Axio Observer Zl equipped with an Axiocam MRm controlled by the Axiovision 4.6 software or a Zeiss Imager Al . Fluorescent microscopy with Texas Red-labelled SHP-I was performed by incubating 3 x 10⁶ cells/ml with 8 μM TR-Hpr-SP in media for 10 min before fixing with 1 % paraformaldehyde for 1 min, air drying on glass slides and covering with DAPI containing antifade reagent ProlongGold (Molecular Probes). Videos were acquired with live cells at a density of 3 x 10⁶ cells/ml incubated with the indicated concentration of SHP-I at 37⁰C. Movies were recorded at 63 magnification at 50 msec acquisition times for normal and constricted motion trypanosomes and 25 msec acquisition times for hyperactivated trypanosomes. Visualized trypanosomes presented in supplementary movies were centered in the videos by digital tracking with Final Cut software (Apple) and videos were looped to facilitate comparisons.

Flow cytometry. Peptide binding to cells was monitored by flow cytometry. Binding assays were performed with 3 x 10⁶ cells/ml in PBS at 25⁰C. Texas red-labelled SHP-I was added to a final concentration of 0.8 μM and 50,000 cells were immediately counted with the CyAn ADP flow cytometer (Dako, available on the worldwide web at _ dako.com). Data were analyzed with FlowJo software (Treestar, available on the worldwide web at treestar.com). Calcein release assays. Permeabilization of unilamellar liposomes was assayed as described previously (Harrington, et al., 2009, J Biol Chem; 284(20): 13505-12). Briefly, liposomes were diluted 1:1000 into 50 mM Tris, pH 7.0 and calcein fluorescence was monitored at 513 ran when excited at 484 nm. The percent calcein release was calculated relative to the 100% fluorescence intensity, achieved by the addition of 0.01% Triton X-100.

Anisotropy assays. The membrane fluidity of live T. b. brucei was probed by measuring the fluorescence depolarization of l-(4-trimethylammoniumphenyl)- 6-phenyl-l,3,5-hexatriene p-toluenesulfonate (TMA-DPH) (Invitrogen, cat. # T204). Cells were washed twice with, and resuspended in PBS, or Voorheis's modified PBS (supplemented with 10 mM glucose, 79 mM sucrose) in the case of procyclic cells, at a density of 3 x 10⁶ cells/ml. The anisotropic probe, TMA-DPH, was added to a final concentration of 0.5 μM and allowed to intercalate into the cell membrane for 15 min. Anisotropic values were acquired via the software function of the Perkin Elmer Life Sciences LS55. Samples were excited at 358 nm and emission was read at 430 nm, both with 10 nm slit widths. Data was corrected for light scattering with an unlabeled sample of cells and anisotropy was calculated according to the equation, r = (I_w -

+ 2GI_Y11), where r is the anisotropy value, I_w is the emission intensity acquired with the excitation and emission polarizing filters set vertically, G is the instrument correction factor and Iv_n is the emission intensity acquired with the excitation polarizing filter set vertically and the emission polarizing filter set horizontally. All assays were conducted at ambient temperature, -25⁰C.

Results

Small hydrophobic peptides specifically kill BSF African trypanosomes - Previously our laboratory has reported that delipidated native human Hpr, purified from HDL, kills BSF T. b. brucei (Shiflett et al., 2005, J Biol Chem; 280(38):32578-32585; and Smith et al. 1995, Science; 268(5208):284-286). The lack of killing activity from a recombinant form of Hpr that lacks the N-terminal signal peptide (VanhoUebeke et al., 2007, PNAS; 104(10):4118-4123) led us to investigate the potential trypanocidal activity of a synthetic peptide corresponding to the signal sequence. Addition of SHP-I to BSF T. b. brucei killed cells in a dose dependent fashion (Fig. 7A). Equi volume amounts of DMSO did not exhibit toxicity. The human pathogenic subspecies T. b. rhodesiense and T. b. gambiense are also sensitive to killing by SHP-I (Fig. 7A). Cells treated with lower concentrations of SHP-I, ie. 4 M, retain their elongated and twisted shape, and cell membranes appear completely intact, however cells are motionless and unable to exclude trypan blue (Fig. 7B). At higher concentrations of SHP-I, 40-80 M, BSF trypanosomes retain their overall shape, however cellular membranes appear to have been stripped off or collapsed into the cytoplasm of the cells (Fig. 7B). Membrane degradation occurs subsequent to cell death, as revealed by video DIC microscopy. SHP-I exhibits specificity for BSF African trypanosomes. The specificity of SHP was initially examined by conducting killing assays with PCF T. b. brucei. No killing of PCF cells was observed (Fig. 7A). Flow cytometry was utilized to determine if the lack of PCF killing was due to a failure of SHP-I to bind the PCF trypanosome (Fig. 7C). Texas-red SHP-I rapidly binds BSF T. brucei as indicated by the labeling of the entire population of cells immediately after the addition of peptide. No labeling of PCF cells was observed immediately after addition of peptide indicating a dramatic difference in the affinity of SHP-I for BSF and PCF trypanosomes.

The analysis of the spectrum of SHP-I toxicity was broadened by testing the peptide against two human cell lines. Incubating human embryonic kidney cells (HEK) or prostate cancer cells (LNCaP) with relatively high concentrations of SHP-I, 80-160 M, for 2 h at 37°C did not result in cell death as evaluated by changes in morphology or the ability to exclude trypan blue (Figs. 8A and 8B). Consistent with a lack of toxicity, flow cytometry indicated that these cells are not bound by SHP-I (Fig. 8C). Additionally no hemolysis of freshly collected human erythrocytes incubated with 240 M SHP-I, the highest concentration tested, was detected (Fig. 8D).

SHP-I killing of BSF trypanosomes requires a hydrophobic stretch of amino acids. The sequence requirements for trypanocidal activity by SHP-I (SEQ ID NO:2) were analyzed by conducting killing assays with synthetic peptides that mimicked, lessened or ablated the core hydrophobic region of SHP-I (Fig. 9A). A peptide exhibiting similar hydrophobicity and length was derived from the signal sequence of the human apolipoprotein paraoxonase-1 (SHP-2) (SEQ ID NO:4) (Sorenson, 1999, Arterioscler Thromb Vase Biol; 19(9):2214-2225). Trypanocidal activity was observed against BSF T. brucei equivalent to SHP-I (Fig. 9B). Conversely, a non-specific hydrophilic peptide of equal length to SHP-I exhibited no toxicity (Fig. 9B). The size of the hydrophobic core of SHP-I was decreased by a single leucine deletion from the C-terminal leucine triplicate (SEQ ID NO:7). Decreasing the length and hydrophobicity by a single amino acid resulted in a five-fold decrease in trypanocidal activity (Fig. 9B). Further deletions of the C-terminal leucine triplicate (SEQ ID NO: 8) or the N-terminal leucine-glycine- alanine (SEQ ID NO:9) resulted in a complete lose of trypanosome killing activity (Fig. 9B).

The effect of altering peptide hydrophobicity on trypanosome killing suggested that membrane interaction might play a mechanistic role in SHP-I 's trypanotoxicity. hi an initial attempt to address this possibility, a model liposome system was utilized in which the release of internally trapped fluorophore, calcein, was monitored as an indicator of membrane interaction. The SHP-I elicits calcein release from unilamellar liposomes composed of phosphatidylcholine (PC) from egg at nanomolar concentrations (Fig 9C). Variant SHP peptides performed in the calcein release assay in a manner consistent with their ability to kill BSF trypanosomes, with a lack of liposome permeabilization by the deletion variants. The SHP-2 elicited calcein release, albeit reduced from SHP-I, while deleting a single leucine, the leucine truplicate or the leucine-glycine-alanine stretch resulted in a loss of permeabilizing activity at concentrations of 500 nM, the highest concentration tested (Fig. 9C).

These data indicate that, the amino acid sequence of SHP-I is not strictly required but the necessary characteristic for trypanosome killing activity is a significantly high degree of hydrophobicity, and the ability to interact with lipid bilayers is consistent with the ability to kill BSF trypanosomes.

SHP acts at the surface of BSF trypanosomes. Multiple trypanocidal molecules have been identified which exert their toxic effect after localization within an intracellular vesicle rather than at the cell surface (Hager et al., 1994, JCeIl Biol; 126(1):155-167; and Delgado et al., 2009, Cell Death Differ; 16(3):406-416). Therefore, whether SHP-I requires internalization by the target trypanosome was addressed. Killing assays were performed with SHP-I at 3 C, a temperature that halts endocytosis (Hager et al, 1994, J Cell Biol; 126(1):155-167). Robust trypanocidal activity (Fig. 10A) was observed, equivalent to killing assays performed at 37 C, suggesting that SHP-I does not require cellular uptake to exert its toxic effects. Consistent with these data, Texas

Red-labeled SHP-I uniformly binds the surface of BSF trypanosomes at 37 C rather than accumulating in intracellular vesicles (Fig. 10B).

SHP-I -membrane interaction is dependent on lipid bilayer fluidity. Based upon the physiological necessity of membrane surface flow in BSF T. brucei, the role lipid bilayer fluidity plays in dictating sensitivity to SHP-I was addressed. Liposomes composed of highly fluid compositions such as egg phosphatidylcholine (PC) (Tm < 0 C) (Koynova and Caffrey, 1998, Biochim Biophys Acta; 1376(1):91-145), are readily permeabilized by SHP-I (Fig. HA). However, homogenous liposomes composed entirely of symmetric PC with saturated chains of 15 to 18 carbon atoms (Tm 34 C and 55 C respectively) (Koynova and Caffrey, 1998, Biochim Biophys Acta; 1376(1):91-145) are not permeabilized by SHP-I (Fig. 1 IA). Fluidizing refractory lipid compositions by incorporating increasing mole percent DMPC (symmetric 14:0, Tm = 23.6 C) (25) led to a DMPC-concentration-dependent increase in susceptibility to SHP-I (Fig. HB). To determine whether SHP-I has a specific affinity for membrane myristate or susceptibility is due to a general physical property imparted by the short acyl chain, liposomes composed of PC with different acyl chain moieties and transition temperatures (Tm) were assayed. The presence of an unsaturation in 16- or 18-carbon acyl chains results in a large decrease in bilayer transition temperature relative to their unsaturated counterparts. Liposomes composed of POPC₅ Tm = -2°C (25), or SOPC, 18:0-18:1 PC, Tm = 6°C (25), are susceptible to permeabilization by SHP-I indicating that myristate is not a requirement for membrane interaction (Fig. 11C). The role of membrane fluidity in mediating membrane sensitivity to SHP-I was further investigated by conducting permeabilization assays with the refractory lipid compositions at temperatures above their Tm. Fluidizing lipid bilayers composed of 1,2-dipentadecanoyl-sn-glycero- 3-phosphocholine (symmetric 15:0, Tm = 34°C) or DPPC (symmetric 16:0, Tm = 41.3°C) (Koynova and Caffrey, 1998, Biochim Biophys Acta; 1376(1):91-145) by bringing liposomal suspensions above their respective Tm resulted in sensitivity to permeabilization by SHP-I (Fig. 1 ID). These data indicate that it is not affinity for a specific bilayer moiety but the general physical property of membrane fluidity that confers susceptibility to SHP- 1.

SHP-I induces rigidification of BSF trypanosome cell membranes. Based upon the role of lipid bilayer fluidity in SHP-I membrane interaction and the restriction of liposomal acyl chain motion by E. coli LarnB signal sequence peptides (Jones and Gierasch, (1994) Biophys J; 67(4): 1534- 1545), the effect SHP-I has upon the fluidity of BSF T. brucei membranes was assayed. The surface membrane probe TMA-DPH, a cationic lipophilic molecule that rapidly partitions into the outer leaflet of the cellular lipid bilayer, was utilized. Addition of SHP-I increases the rigidity of BSF cell membranes as indicated by an increase in the anisotropy of the surface membrane probe TMA-DPH (Fig. 12A). The difference in membrane composition of PCF trypanosomes is apparent in the higher anisotropic values, i.e. greater rigidity, acquired for PCF cells. Consistent with the lack of SHP-I binding to PCF cells and data from model liposomes indicating that a high degree of fluidity is required for SHP-I intercalation, the anisotropy of PCF trypanosomes is unchanged by the addition of peptide. In contrast to BSF cells, PCF trypanosomes are significantly less motile and do not exhibit high turnover rates of surface proteins or endocytic activity.

SHP-I induces dramatic changes in cell motility. During the course of our investigations on BSF T. brucei killing, it readily became apparent that treatment of cells with SHP-I resulted in motility changes. In order to assess the effect of SHP-I on the motility of trypanosomes BSF cells were analyzed by DIC video microscopy. Addition of 8 M SHP-I resulted in a progression of motility changes over 30 min (Fig 12B). Untreated BSF trypanosomes exhibit a characteristic corkscrew motion (Fig. 12C). Within a minute of SHP-I addition, a significant fraction of cells exhibit hyperactive motility (Fig. 12C). Hyperactivated cells are still present at 10-20 minutes, however, a portion of cells display highly constricted motion (Fig. 12C). The constricted motility phenotype is increasingly apparent at 20 minutes, with approximately 50 % of the cells displaying constriction, the other 50 % remaining hyperactivated in their motion. During the time course dead cells are increasingly apparent. This phenomenon is more pronounced at 80 M SHP-I with all of the cells exhibiting hyperactivity until slowing down and dying within 10 min.

Discussion

This example demonstrates that a small peptide that rapidly intercalates into the plasma membrane of BSF T. brucei and induces cell death. The peptide is highly specific for the developmental form found within a mammalian host. Specificity is mediated at the level of binding, or more likely intercalation into the acyl chain region of the target membrane. Indeed, studies with model membrane systems indicate that the peptide is sensitive to the acyl chain composition of lipid bilayers. In particular the membrane fluidity that is imparted by a given acyl chain composition dictates the ability of this peptide to interact with membranes. Killing of BSF T. brucei has been demonstrated for a variety of bona fide antimicrobial peptides (McGwire et al., 2003, J Infect Dis; 188(1):146-152; and Haines et al., 2009, PLoS Negl Trop Dis; 3(2):e373), such as the cathelicidins and their derivatives novispirin and ovispirin, as well as unusual candidates such as neuropeptides (Delgado et al., 2009, Cell Death Differ, 16(3):406-416). In the case of cathelicidins it has been shown that these peptides permeabilize the plasma membrane and cells assume a rounded and crumpled morphology upon death (McGwire et al., 2003, J Infect Dis; 188(1):146-152). At relatively high concentrations of SBDP-I, massive disruption of the cellular membrane is observed, however, as revealed by video microscopy, this occurs subsequent to cell death. Treatment with lower concentrations of SHP-I, i.e. 4 M, results in dead T. brucei that exhibit a normal morphology and DIC microscopy indicates that the cellular membrane remains intact. The lack of osmotic swelling suggests that permeabilization of the plasma membrane is not the mechanism of killing. Furthermore, the observed changes in BSF T. brucei cell membrane rigidity by SHP-I suggest a novel mechanism of toxicity. Bloodstream form African trypanosomes are highly dynamic cells with respect to motility (Rodriguez et al., 2009, PNAS; 106(46):19322-19327; and Oberholzer et al, 2010, PLoSPathog; 6(l):el000739), endocytosis and vesicle trafficking (Engstler et al., 2004, JCeIl Sci; 117(Pt 7): 1105-1115) and lateral flow of surface molecules (Bulow et al., 1988, Biochem; 27(7):2384-2388; and Engstler et al. Al., 2007, Cell; 131 (3):505-515). All of these activities require movement of membrane components such as phospholipids, and in the case of endocytosis and vesicle trafficking, remodeling of the lipid bilayer. Decreasing the physical property of fluidity in the bulk membrane such that these activities are burdened may contribute to a general poisoning of the cell by SHP-I. Alternatively, or in synergy with membrane rigidification, SHP-I may act through non-specific alterations of integral membrane protein stability/activity (Lee, 2004, Biochim Biophys Acta; 1666(l-2):62-87). For example, distortion of the lateral pressure profile, a mechanism that has been attributed to general anesthetics (Cantor, 1998, Toxicol Lett; 100-101 :451-458), may lead to increased lateral pressure which in turn may physically inhibit transmembrane channels (Kamaraju and Sukharev, 2008, Biochem; 47(40):10540-10550). Additionally, peptides corresponding to the E. coli LamB signal peptide have been shown to effect the oligomerization of integral transmembrane proteins (Benach et al., 2003, J Biol Chem; 278(6):3628-3638). Such surface activity is consistent with the SHP-I site of action being the plasma membrane as suggested by killing assays performed at 3 C (Fig. 10A) and labeling of the surface of BSF T. brucei by Texas-red-SHP-1 (Fig. 10B). In either case, the fluidity of the BSF trypanosome cell membrane appears to not only be modified by the SHP-I, but also specifically targeted.

The studies of this example, with model liposomes, indicate that the fluidity of the target lipid bilayer determines the ability of SHP-I to bind and permeabilize membranes. These data are consistent with previous studies showing that modulation of the surface pressure of lipid monolayers dictates the ability of signal peptides to intercalate into the acyl chain region (Briggs et al., 1986, Science; 233(4760):206-208). Membranes exhibiting gel phase lipid order are refractory to permeabilization by SHP-I. When these refractory liposomes are brought above their transition temperatures, thus exhibiting liquid crystalline order, they are rendered susceptible to permeabilization by SHP-I . These data indicate that the lateral van der Waals forces dictate the ability of SHP-I to intercalate into target lipid environments. The immediate labeling of the entire population of BSF T. brucei by Texas-red labeled SHP indicates a very rapid rate of bilayer intercalation (Fig. 7C). The consequences of SHP-I intercalation into BSF trypanosome membranes are immediately apparent on the cellular level. Cells exhibit hypermotility immediately after introduction of SHP-I subsequently becoming constricted in their motion before dying. The cause for these changes in motility can only be speculated upon. Spermatozoa are examples of flagellated cells that undergo changes in membrane fluidity concurrent with the onset of hypermotility (Visconti et al, 2002, J Reprod Immunol; 53(l-2):133-150). Hyperactivation is associated with efflux of cholesterol and an increase in membrane fluidity. In this regard Tyler et al. have recently demonstrated that T. brucei flagellar membranes are enriched in cholesterol and exhibit a higher degree of order than the pellicle of the cell (Tyler et al., 2009, JCeIl Sci; 122(Pt 6):859-866). It is intriguing to imagine that SHP-I induces a redistribution of cholesterol from the flagellum to the pellicular fraction of the cell membrane. This would result in an overall increase in cell membrane rigidity coupled with a localized decrease of the flagellar rigidity, and thus alleviation of physical strain against flagellar motors and acceleration of flagellar beating. The observed constricted motility at later time points following treatment with SHP-I may be the result of metabolic depletion.

Small hydrophobic peptides target a fundamental physiological characteristic of T. brucei, namely the fluid physical property of the cell membrane. It is not likely that BSF trypanosomes will quickly develop a strategy to circumvent the cytotoxicity of molecules that alter the mechanoelastic properties of the cell membrane. This example demonstrates that the BSF trypanosome cell membrane is an attractive target for the development of novel therapeutics. Example 3 Killing T. congolense, T. vivax, and the Metacyclic Forms of T. brucei

African trypanosomes are eukaryotic parasites that cause sleeping sickness in humans and a wasting disease known as Nagana in cattle. As shown in the previous examples, a small trypanocidal peptide has been derived from the human trypanosome lytic factor (TLF), a subset of high density lipoproteins that confers immunity to veterinary pathogenic trypanosomes. As described in Examples 1 and 2, the peptide kills both the veterinary pathogen Trypanosoma brucei brucei and the human pathogenic subspecies responsible for African sleeping sickness, T. b. rhodesiense and T. b. gambiense. Screening of the peptide against a number of eukaryotic pathogens, human cell lines and the procyclic form trypanosome, the developmental stage within the insect vector midgut, indicates that the peptide uniquely targets the developmental stage of trypanosome found within the mammalian host. The lack of toxicity against mammalian cell lines suggests a therapeutic potential for treatment of human and animal trypanosomiasis. This Example will test the peptides against the major cattle pathogens T. congolense and T. vivax. Additionally, the activity of the peptide will be tested against metacyclic trypanosomes, the developmental form within the tsetse fly salivary gland and thus the first developmental stage encountered by a potential human or animal host. Trypanosome lytic factor uniquely contains two trypanolytic proteins, haptoglobin related protein (Hpr) and apolipoprotein L-I (Shiflett et al., 2005, J Biol Chem; 280(38):32578-32585). Haptoglobin related protein is unusual in that it is secreted from hepatocytes without cleavage of its hydrophobic N-terminal signal peptide (Hpr-SP) (Smith et al., 1995, Science; 268(5208): 284-286). Previous work established that human Hpr, when purified in a delipidated form, kills bloodstream form T. b. brucei (Shiflett et al., 2005, J Biol Chem; 280(38): 32578-32585). The lack of trypanolytic activity from recombinant Hpr (Vanhollebeke et al. 2006, PNAS; 104(10), 4118-4123) led us to investigate the potential activity of a synthetic 19-amino acid peptide corresponding in sequence to the Hpr-SP. The previous examples have established that Hpr-SP is toxic to all bloodstream form T. brucei and that the specificity of Hpr-SP is mediated by the high presence of myristate, a short 14 carbon, saturated acyl chain, in the cell membrane. The unusually high content of myristate is due to the glycerophosphoinositol- anchoring of the trypanosome's variable surface glycoproteins. The high content of myristate lends the cell membrane a high degree of fluidity that facilitates both the rapid uptake of bound host antibodies and cell surface bound nutrients. The Hpr-SP induces a rigidification of the cell membrane that results in dramatic motility changes and rapid onset of cell death (Examples 1 and 2).

In the case on of the T. brucei subspecies, the membrane composition is particularly well characterized; The VSG proteins in bloodstream form trypanosomes are membrane anchored via myristate (Ferguson et al., 1985, J Biol Chem;

260(27):14547-14555), whereas the procyclic forms utilize longer saturated chains, stearate and palmitate (Field et al., 1991, EMBO; 10(10): 2731-2739) which provide increased lateral van der Waals forces and presumably protect the membrane from intercalation of Hpr-SP. Example 2 shows that cells refractory to Hpr-SP killing are not bound by the peptide.

With this example, the cattle pathogens 71 congolense and T. vivax will be tested for susceptibility to binding and killing by hydrophobic signal sequence peptides, including, but not limited to, SEQ ED NO: 1-7. There are some indications that these species possess different membrane compositions than the T. brucei subspecies. For instance probing the surface with antibodies against surface lectins suggests a less dense VSG coat, and therefore less myristate within the cell membrane, hi vitro killing assays will be carried out against T. congolense and T. vivax. Assays will be performed by incubating trypanosomes with various concentrations of peptide in HMI-9 media for two hours at 37°C. Purified human TLF will serve as a positive killing control. Additionally, the binding, or lack thereof, of hydrophobic signal sequence peptides to these cell lines will be determined utilizing Texas-red labeled Hpr-SP and analysis by flow cytometry or fluorescence microscopy.

The metacyclic form of T. brucei resides within the salivary glands of the tstese fly vector. This developmental stage is primed for encountering a potential mammalian host through the bite of the fly. Consistently, the metacyclic form expresses GPI-anchored VSG rather than the procyclic acid repeats characteristic of the developmental stages within the mid- and hind gut of the tsetse fly. Thus, the metacyclic forms will likely be susceptible to killing by Hpr-SP. Metacyclic T. brucei will be collected from tsetse fly salivary glands, washed into PBS supplemented with 30 % media and subject to the previously described assays. These assays will indicate the possibility of utilizing hydrophobic signal sequence peptides, such as Hpr-SP, as a molecule capable of preventing initial infection of potential animal hosts from interaction with the trypanosome containing insect vector. The studies of this example will greatly inform our knowledge of the therapeutic potential of Hpr-SP and the membrane physiology of different Trypanosoma sp. and developmental forms.

Example 4

Mouse Infection Studies

Peptides of the present invention may be tested in animal models, including, but not limited to, a T. b. rhodesiense mouse model, for antitrypanosomal activity. Pre-toxicity screens in non infected mice using up to 100 mg/kg may be undertaken. Further, guidelines of the World Health Organization (WHO) parasite drug discovery initiative may be followed for the identification and development of new drug candidates (reviewed, for example, by Nwaka and Hudson, 2006, Nat Rev Drug Discov; 5:941-95, Nwaka and Ridley, 2003, Nat Rev Drug Discov; 2:919-928, and Nwaka et al., 2009, PLoS Negl Trop Dis; 3(8):e440).

The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Sequence Listing Free Text

SEQ ID NO : 1 N-terminal amino acid residues 1 - 19 of the human haptoglobulin- related protein

SEQ ID NO:2 N-terminal amino acid residues 2-19 of the human haptoglobulin- related protein

SEQ ED NO: 3 N-terminal amino acid residues 1-22 of the human paraoxonase-1 protein

SEQ ID NO:4 N-terminal amino acid residues 2-22 of the human paraoxonase-1 protein

SEQ ID NO: 5 N-terminal amino acid residues 1-22 of the human apolipoprotein

M (Apo M)

SEQ ID NO: 6 N-terminal amino acid residues 2-22 of the human apolipoprotein M (Apo M)

SEQ ID NO: 7 Derivative of N-terminal amino acid residues 2-19 of the human haptoglobulin-related protein, with single leucine deletion

SEQ ID NO: 8 Derivative of N-terminal amino acid residues 2-19 of the human haptoglobulin-related protein, with deletion of C-terminal leucine triplicate

SEQ ID NO: 9 Derivative of N-terminal amino acid residues 2-19 of the human haptoglobulin-related protein, with deletion of the N-terminal leucine-glycine-alanine residues SEQ ID NO: 10 Non-specific, hydrophilic peptide predicted from the N-terminus of the alternatively edited protein- 1 from mitochondria of T. b. brucei

Claims

What is claimed is:

1. A method of killing a bloodstream form of a kinetoplastid protozoan of the genus Trypanosoma, the method comprising contacting the protozoan with a hydrophobic signal sequence peptide.

2. A method of treating or preventing a trypanosomal infection in a subject, the method comprising administering to the subject an effective amount of a hydrophobic signal sequence peptide.

3. The method of claim 1 or 2, wherein the hydrophobic signal sequence peptide is an uncleaved signal sequence peptide.

4. The method of claim 1 or 2, wherein the hydrophobic signal sequence peptide is soluble in ethanol or dimethyl sulfoxide (DMSO).

5. The method of claim 1 or 2, wherein the hydrophobic signal sequence peptide is the signal sequence peptide of haptoglobin-related protein, paraoxonase 1, or apolipoprotein M.

6. The method of claim 1 or 2, wherein the hydrophobic signal sequence peptide comprises at least nine consecutive amino acid residues of MSDLGAVISLLLWGRQLFA (SEQ ED NO: 1), MAKLIALTLLGMGLALFRNHQS (SEQ ID NO:3), or MFHQIWAALLYFYGIILNSIYQ (SEQ ID NO:5), or a derivative thereof.

7. The method of claim 1 or 2, wherein the hydrophobic signal sequence peptide is selected from the group consisting of MSDLGAVISLLLWGRQLFA (SEQ BD NO:1), SDLGAVISLLLWGRQLFA (SEQ ID NO:2), MAKLIATLLGMGLALFRNHQS (SEQ ID NO:3), AKLIATLLGMGLALFRNHQS (SEQ DD NO:4), MSDLGAVISLLWGRQLFA (SEQ ID NO:5), SDLGAVISLLWGRQLFA (SEQ ID NO:6), or SDLGAVISLLWGRQLFA (SEQ ID NO:7).

8. The method of any one of claims 1-7, wherein the trypanosome is selected from the group consisting of Trypanosoma brucei brucei, T.b. gambiense, and T.b. rhodesiense, T. congolense, and T. vivax.

9. A composition comprising a hydrophobic signal sequence peptide and a pharmaceutical carrier suitable for parenteral or enteral administration to a mammal.

10. A liposome, emulsion, or micelle comprising a hydrophobic signal sequence peptide.

11. The composition of claim 9 or the liposome, emulsion, or micelle of claim 10, wherein the hydrophobic signal sequence peptide is an uncleaved signal sequence peptide.

12. The composition of claim 9 or the liposome, emulsion, or micelle of claim 10, wherein the hydrophobic signal sequence peptide is soluble in ethanol or dimethyl sulfoxide (DMSO).

13. The composition of claim 9 or the liposome, emulsion, or micelle of claim

10, wherein the hydrophobic signal sequence peptide is the signal sequence peptide of haptoglobin-related protein, paraoxonase 1 , or apolipoprotein M.

14. The composition of claim 9 or the liposome, emulsion, or micelle of claim

10, wherein the hydrophobic signal sequence peptide comprises at least nine consecutive amino acid residues of MSDLGAVISLLLWGRQLFA (SEQ ID NO:1), MAKXIALTLLGMGLALFRNHQS (SEQ ID NO:3), or MFHQIWAALLYFYGΠLNSIYQ (SEQ ID NO:5), or a derivative thereof.

15. The composition of claim 9 or the liposome, emulsion, or micelle of claim 10, wherein the hydrophobic signal sequence peptide is selected from the group consisting of MSDLGAVISLLLWGRQLFA (SEQ ED NO:1),

SDLGAVISLLLWGRQLFA (SEQ ID NO:2), MAKLIATLLGMGLALFRNHQS (SEQ ID NO:3), AKLIATLLGMGLALFRNHQS (SEQ ID NO:4), MSDLGAVISLLWGRQLFA (SEQ ID NO:5), SDLGAVISLLWGRQLFA (SEQ ID NO:6), or SDLGAVISLLWGRQLFA (SEQ ID NO:7).

16. The composition, liposome, emulsion, or micelle of any one of claims 9-15, further comprising an RNA aptamer that binds to a structurally conserved region of a trypanosome variant surface glycoprotein (VSG).

17. A method of killing a bloodstream form of a kinetoplastid protozoan of the genus Trypanosoma, the method comprising contacting the protozoan with a composition, liposome, emulsion, or micelle of any one of claims 9-16.

18. A method of treating or preventing a trypanosomal infection in a subject, the method comprising administering to the subject an effective amount of a composition, liposome, emulsion, or micelle of any one of claims 9-16.

19. The method of claim 17 or 18, wherein the trypanosome is selected from the group consisting of Trypanosoma brucei brucei, Tb. gambiense, and Tb. rhodesiense, T congolense, and T. vivax.