WO2005086590A2 - Muscle-relaxing proteins and uses thereof - Google Patents

Abstract

The invention provides a 12kDa protein, herein designated muscle-relaxing protein (MRP), obtainable from the lysate or from the growth medium of Leishmania major organisms and capable of causing reversible muscle relaxation, and useful for treatment or prevention of a cardiac disease or condition such as angina pectoris, cardiac arrythmia, heart failure, or myocardial infarction, hypertension, a respiratory such as asthma, emphysema, pneumonia or bronchitis, and for the prevention of premature labor.

Description

MUSCLE-RELAXING PROTEINS AND USES THEREOF

FIELD OF THE INVENTION The present invention relates to new proteins isolated from Leishmania major organisms, which are capable of causing reversible smooth muscle relaxation and can be used for several pharmaceutical purposes.

BACKGROUND OF THE INVENTION Leishmania, a genus of parasitic flagellate protozoa (order Kinetoplastida, suborder Trypanosomatidae), comprise parasites of worldwide distribution, several species of which are pathogenic to humans. Because all species are morphologically indistinguishable, the organisms have usually been assigned to species and subspecies according to their geographic origin, the clinical syndrome they produce, and their ecologic or other characteristics. In some classifications, leishmanias are placed in four complexes comprising species and subspecies: L. donovani, L. tropica, L. mexicana, and L. vivannia or L. brasiliensis. All the parasites develop in and are transmitted by female sand flies that in the Old World belong to the genus Phlebotomas (Diptera: Nematocera: Psychodidae). The organisms have two morphologic stages in their life cycle: a flagellated, extracellular promastigote stage in the gut of the invertebrate, i.e. sand fly host, and an intracellular amastigote stage within the reticuloendothelial cells of a mammalian (e.g. human). Survival in the sand fly requires a complex system of vector-parasite interactions including these that allow persistence in the gut. Leishmania parasites that are ingested with blood are initially enclosed with the blood within the peritrophic membrane, a semi-permeable sac, produced by the gut epithelium. At a later stage a great number of the free parasites attach to the epithelial cells of the gut by some flagellar-surface protein. Another factor in the attachment of one class of parasites to the gut epithelial cells is the lipophosphoglycan (LPG), the most abundant promastigote surface membrane glycoconjugate. This, however, does not explain why the majority of free promastigotes are not excreted. Blood feeding female sand flies may acquire Leishmania parasites and later, after a complex growth cycle in the gut, transmit them by bite to another host. Leishmania major that causes rural cutaneous leishmaniasis is transmitted by the sand fly Phlebotomus papatasi.

SUMMARY OF THE INVENTION Based on the observation that in Phlebotomus papatasi infected with L. major, the midgut is distended and has lost peristalsis, we presumed that the parasites produce a substance that affects sand fly gut. Therefore, the effect of L. major proteins on sand fly gut, and later on Guinea pig ileum and uterus, rat cardiomyocytes and mammalian heart cells and smooth muscle was tested. It has now been found, in accordance with the present invention, that L. major parasites in their vector P. papatasi, express and secrete at least one protein that causes reversible muscle relaxation and distention of the insect gut, and that these proteins can be obtained from the lysates or from the culture medium of L. major organisms. The protein causes reversible inhibition of the sand fly hind gut peristalsis, but has no effect on skeletal muscles. The present invention thus relates to one or more proteins obtainable from the lysates or from the culture medium of Leishmania major organisms, which are capable of causing reversible muscle relaxation, herein designated muscle-relaxing ρrotein(s) (MRP). The present invention further provides a pharmaceutical composition comprising a MRP protein and a pharmaceutically acceptable carrier, particularly for treatment of angina pectoris, cardiac arrythmia, hypertension, heart failure, or prevention of myocardial infarction, treatment of asthma, emphysema, pneumonia or bronchitis, and for the prevention of premature labor. In another aspect, the present invention relates to a method for treatment or prevention of a cardiac disease or condition which comprises administering to a subject in need an effective amount of a muscle-relaxing protein of the invention. In one embodiment, the cardiac disease or condition is angina pectoris. In another embodiment, the cardiac disease or condition is cardiac arrhythmia. In a further embodiment, the cardiac disease or condition is heart failure. In yet another embodiment, the cardiac disease or condition is myocardial infarction. In another aspect, the present invention relates to a method for treatment of hypertension which comprises administering to a subject in need an effective amount of a muscle-relaxing protein of the invention. In a further aspect, the present invention relates to a method for treatment or prevention of a respiratory disease or condition which comprises administering to a subject in need an effective amount of a muscle-relaxing protein of the invention. In one embodiment, the respiratory disease or condition is asthma. In another embodiment, the respiratory disease or condition is emphysema. In yet another embodiment, the respirator}' disease or condition is pneumonia. In a further embodiment, the respiratory disease or condition is bronchitis. In yet another aspect, the present invention relates to a method for the prevention of premature labor which comprises administering to a female subject in need an effective amount of a muscle-relaxing protein of the invention.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 is a graph showing the inhibition of Phlebotomus papatasi hindgut contractions with L. major lysate proteins (0.05-0.07mg/ml). All hindguts were observed initially for 5 min. Hindguts in the "Return" group were treated with L. major- ysatQ proteins for 20 min, rinsed in ABS, and immediately returned to fresh

ABS. Each point represents 36 repeats. Fig. 2 shows dose-response inhibition of P. pαpαtαsi hindgut contractions by different quantities of L. major lysate proteins (1.2 μg, 2.4 μg, 4.8 μg, 9.6 μg). Each experiment was repeated 5 times. Fig. 3 shows the specific MRP activity of proteins from nine trypanosomatid parasites in the bioassay of sand fly hindgut contractions. Fig. 4 shows the effect of 0.034 mg/ml L. m^tjor lysate and 0.042 mg/ml

Crithidia fasciculate lysate proteins on spontaneous contractions of a neonatal rat ventriculocyte monolayer. Proteins were added at time point 0 and contractions counted for 10 min. Cell cultures were then incubated for 30 min and contractions counted again for 10 min. Cultures were then washed with saline and incubated for further 30 min. Contractions were counted every minute for 10 min after incubation. Figs. 5A-5D depict contractions of strips of Guinea pig ileum. (5A) Spontaneous contractions, without treatment. (5B) LMuscle relaxing effect of L. major- ysatQ proteins on ileum strips pre-contracted with nicotine, wherein: 1 - nicotine application; 2 - Tyrode's solution wash; 3 - application of 0.88 mg/ml L. major lysate proteins; 4 - reapplication of nicotine; 5 — wash. (5C) Muscle-relaxing effect of L. major lysate proteins on ileum strips pre-contracted with acetylcholine, wherein: 1 - acetylcholine application; 2 - Tyrode's solution wash; 3 - application of 0.88 mg/ml L. major lysate proteins; 4 - reapplication of acetylcholine; 5 - wash; 5D) cumulative muscle-relaxing effect of L. major-Y s&te proteins on ileum strips repeatedly stimulated with 40V, wherein: 1 - electric stimulation; 2 - 0.22 mg/ml L. wα/ør-lysate proteins; 3 - 0.43 mg/ml L. major lysate proteins; 4 - 0.82 mg/ml L. mα/ør-lysate proteins. Fig. 6 depicts the muscle-relaxing dosage effect of L. major lysate proteins on Guinea pig uterine muscle pre-contracted with oxytocin, wherein: 1 - 0.17 mg/ml L. major lysate proteins; 2 - 0.31 mg/ml . major lysate proteins; 3 - Tyrode's solution wash; 4 - oxytocin application. Fig. 7 is a graph depicting size exclusion chrornatography analysis of MRP, using a Fractogel EMD BioSEC column. Arrow denotes retention time of MRP (134.9 min) and protein markers of known molecular mass. Fig. 8 is a graph depicting reversed phase high performance liquid chromatography (RP-HPLC) of MRP Fraction 6, using an HPLC Vydac C4 column. Elution of the muscle-relaxing activity is denoted by the black arrow (48.3593). Numbers above peaks denote exact run-time.

DETAILED DESCRIPTION OF THE INVENTION According to the present invention, a small protein was initially isolated from L. major lysates and found to inhibit sand fly hindgut contractions in a standard bioassay. Muscle-relaxing activity was detected in parasite proteins from day 3-5 in vitro. This timing is consistent with the cycle in the sand fly where, by day 4, normally the blood meal has been digested and voided, and parasites that are not attached by their flagella remain vulnerable in the midgut. Further experiments according to the invention demonstrated that this protein also causes a reversible relaxation of mammalian heart cells and smooth muscles of different organs. In the experiments, crude protein preparation of lysed L. major organisms completely inhibited the spontaneous contractions of isolated neonatal rat ventricular heart cells at a concentration of 0.034 mg protein/ml medium. Segments of Guinea pig gut (ileum), electrically stimulated with 40 V, showed a dose-dependent reduction in the strength (29.7% to 75.65%) and in the frequency of contraction, when treated with 0.22 to 0.82 mg cell proteins /ml medium. Similarly, Guinea pig gut (ileum) segments treated with cell lysate proteins (0.9 mg/ml medium) and then exposed to nicotine, showed a rapid 35.7% decrease in the force of contraction compared to previous nicotine-induced contractions. Also strips of Guinea pig uterus, contracted with oxytocin, showed dose-dependent relaxation of 21.1% and 54.5% following treatment with 0.15 and 0.3 mg protein/ml medium, respectively. Preparations of all mammalian tissues regained their initial level of activity, or response to stimulation, shortly after removal of the tested cell lysate. The muscle-relaxing protein (MRP) was produced and purified from L. major lysates by a process comprising a first step of protein precipitation and four steps of sequential chromatography. Protein precipitation was carried out with ammonium sulfate and the sequential chromatography steps included hydrophobic interaction chromatography, first on Phenyl Sepharose™ nd then on Propyl Sepharose™ column, followed by size exclusion chromatography and RP-HPLC. In each step, the fractions were tested for their biological activity using the sand fly hindgut contractions bioassay described hereinafter, and the active fractions were pooled and subjected to the next purification step. By this process, MRP was purified > 14,000 fold over the crude cell-proteins preparation. Thus the concentration of purified protein that would cause the above mentioned effects on mammalian tissues may be proportionally smaller. According to analysis of highly purified preparations on SEC HPLC column, the estimated native molecular mass of MRP is approximately 12 kDa. Tandem MS/MS spectrometry revealed peptide sequences, listed in Table 2 hereinafter, that have no significant homology to any reported protein. In further experiments, MRP was found to be secreted in a relatively large quantity into the growth medium of the parasites. It was found tJhat MRP activity in the culture medium was about a 1000-fold higher than in the parasite cell extracts, with an increase of about a four orders of magnitude in its specific activity. Therefore, it is expected that the relaxing effect of the pure protein on the mammalian tissues would be higher compared to the L. major-lysate. The present invention also encompasses MRP in recombinant form, MRP fractions, and MRP homologues obtained by deletion of one o>r more amino acid residues, by replacement of one or more amino acids in the native molecule by a different amino acid, natural or non-natural, by addition of one or more amino acid residues at the amino and/or carbon terminal, and/or by substitution of one or more free hydroxy, thiol, amino or carboxyl radical by radicals not found in the native molecule, provided that the recombinant MRP, MRP fractions and MRP homologues exhibit substantially the same biological activity of the native MRP. The recombinant MRP is prepared by standard DNA recombinant techniques once a partial sequence, e.g. at the amino terminal, of MRP is known, and expression of said DNA after insertion in a suitable expression vector. In this aspect, the invention further encompasses a DNA molecule coding for MRP, and DNA molecules obtained by site mutagenesis, or deletion or addition of nucleotides that encode MRP homologues. In one aspect of the invention, the MRP secreted into the culture growth medium is purified to homogeneity and the amino acid sequence of the pure MRP is identified by mass spectroscopy analysis (MS/MS analysis). The data obtained is used for the construction of PCR primers for screening the L. major genome (complete genome sequence now available), for identification of the MRP gene. Strategies known in the art may be used for the cloning of the L. major MRP gene. The PCR protocols employed are well-known in the art (reviewed in Innis et al., 1990). A L. major genomic expression library made in λgtl 1 is available and can be used according to the invention. Cloning of the L. major MRP gene may be conducted by using oligonucleotides synthesized according to the nucleotide sequence deduced from the amino acid sequence of the purified MRP, as primers for the PCR cloning of the MRP gene from L. major genomic DNA. The PCR products are then analyzed and their sequences are used for screening the L. major genomic sequence. In another embodiment of the invention, the PCR products are used as probes in the screening of the genomic library, searching for overlapping inserts for the cloning of the complete MRP-encoding gene. The L. major expression library may also be exploited in accordance with published procedures (Sambrook et al., 1989) for the screening of clones, induced by and blot onto isopropyl-β-thiogalactopyranoside

(IPTG)-saturated nitrocellulose filters, by anti-MRP antibodies raised against the purified protein, using an Electrogenerated Chemiluminescence (ECL) technique. Expression and overproduction of MRP may also be carried out in a Leishmania expression system based on the species Leishmania tarentolae. For this purpose, the MRP gene is subcloned into the novel L. tarentolae expression system, which is commercially available (LEXSY, Jena Bioscience). This expression system is advantageous because it is homologous to the L.major system, in which the desired MRP gene is naturally expressed, and thus has the potential of similar protein expression, folding, and modification. Purification of the recombinant MRP may then be carried out using a 6 x histidine tag, that is made available in this vector. Other eukaryotic systems are also contemplated such as, for example, the use of an insect plasmid vector and its expression in insect cells, allowing the possible post-translational modification required and fast large scale production of the protein. Any of the several insect expression systems available van be used such as: (1) The Drosophila expression system (DES™, Invitrogen), where Drosophila Schneider S2 cells and simple, non-viral expression vectors allow stable or transient expression of recombinant proteins; (2) The Bacidlovirus expression system, that allows the high level of recombinant protein expression. The commercially available BTI-TN-5BI-4 (High Five™) cells and pBlueBacHis2™ vector (Invitrogen) system can be used, allowing convenient detection (by fusion to β- galactosidase) and affinity purification (through a fused polyhistidine) of the recombinant protein expressed. The invention further provides a pharmaceutical composition comprising

MRP and a pharmaceutically acceptable earner. According to the biological activity demonstrated for MRP in the examples hereinafter, MRP can be used for relaxation of visceral muscles in various organs, including heart, uterus, and smooth muscles of arteries, veins and lungs. In one embodiment, MRP is useful for treatment of cardiac diseases and conditions including angina pectoris, cardiac arrythmia, hypertension, heart failure, or prevention of myocardial infarction, and can replace present drugs for these uses that are calcium-channel blockers, beta-adrenergic-receptor blockers or angiotensin- converting enzymes. In another embodiment, MRP is useful in the treatment of respiratory disorders, for example as bronchodilator for treatment of asthma, and to help expand the airways and improve the breathing capacity in emphysema, pneumonia or bronchitis patients. In a further embodiment, MRP is useful for the prevention of premature labor, which represents a major cause of infant morbidity and mortality. The invention will now be illustrated by the following non-limiting examples. EXAMPLES Materials and methods (i) Parasites and parasite cultures The experiments were carried out mainly with L. major MHOM/IL/86/Blum (Jordan Valley strain). L. braziliensis braziliensis MHOM/BR/75/M2903, L. infantum MCAN/IL/L760, L. tropica MHOM/IL/L590, and L. donovani (Khartoum and DD8 Indian strains). Herpetomonas muscarum and Leptomonas seymouri were used for comparative experiments or as a control. All the parasites were obtained from the W.H.O. Leishmania Reference Center, Department of Parasitology, The Hebrew University, Jerusalem, Israel. Parasites were grown in Dulbecco's modified Eagle's medium (DMEM) (Biological Industries, Beit Haemek, Israel) with high glucose content, 10% or 0.5% or no heat-inactivated fetal calf semm (FCS), 4 mM L-glutamine, 2 mM adenosine, and 2% (v/v) filter-sterilized human urine. Crithidia fasciciύata were grown in brain-heart infusion. Cultures were maintained at 28° C for rapid growth and passaged every 4 days. (ii) Experimental sand flies Sand flies were reared according to λ4odi and Tesh (1983). P. papatasi were from a colony originated with flies caught around Kfar Adumim, 10 km east of

Jerusalem. Constant insectary conditions were 26°C and 80% relative humidity on a 17:7 hi- light: dark cycle. Two to six day-old sugar-fed sand flies were used for all experiments.

(in) Preparation ofL. major cell lysate Late log-phase cultures with parasite density of 10 - 10 cells/ml were spun at 2000xg for 10 min at 8°C and washed twice with Aedes αβgvptz^'-buffered saline (ABS, final concentrations in mM: MgCl, 0.6, KCl 4.0, NaHC0₃ 1.8, NaCl 150, HEPES 25, CaCl₂Η₂0 1.7. Adjust to pH 7.4 with NaOH). A protease inhibitor cocktail (Sigma) was added 1 :1 to wet parasite volume, according to manufacturer's instructions. Pellets were frozen in liquid N₂ and thawed three times in a 30°C water-bath. Samples were checked under phase-contrast microscope to verify lysis of parasites. Crude cell lysates were frozen in liquid N₂ and stored at -70°C until use. Crude homogenates were thawed on ice and spun at 12,000xg to precipitate particulates. Both precipitate and supernatant were tested in the sand fly hindgut bioassay. The supernatant fraction is defined as L. major cell lysate, and was used as a starting material for further purification of MRP (designated as Fraction I [FI] in the purification scheme) and as the protein source in the bioassays described hereinafter (unless bioassays using purified MRP preparations are described). Protein was quantified using the Bradford assay (Bradford, 1976) using a bovine serum albumin (BS A) standard curve. Samples were read on an ELx 800 Universal Microplate Reader (Bio-Tek Instruments, Inc.) at 545 nm. Mass culturing of parasites for protein purification was repeated four times.

The final preparation yielded approximately 4.5xl0¹² L. major promastigotes grown in the total of 258 L of DMEM, containing 29 L of FCS, in 108 roller bottles (Corning, NY, USA). When protein samples from L. major growth medium were used, they were precipitated from the culture medium with 85% (of saturation at 0°C) (NH₄)₂S0 . and dialyzed (Spectra/Por Membrane 3.5 kDa) against ABS medium, and tested using the sand fly hindgut assay.

(iv) The standard bioassay with sand fly hindgut Hindguts from male and female sand flies were routinely dissected into 90-

100 μl oxygenated ABS warmed to 30°C, and their contractions were counted for 5 min increments. Each preparation was allowed to stabilize and regular contractions were then counted before adding and mixing the tested proteins. The activity in the pre-test five minutes was used as the control in all the experiments. A unit of MRP is defined as the amount of protein that causes one percent inhibition of the frequency of hindgut contractions in five minutes under the standard assay conditions. Specific activity is defined as units of activity per mg MRP. (v) Gut contractions measurement. Measurements of midgut and hindgut were taken under a phase-contrast microscope. Whole guts from unfed flies were dissected into 98 μl of oxygenated ABS warmed to 30°C. The width and length of the midgut and the hindgut were measured with an ocular graticule. 6.4 μg L. major lysate proteins was added (final concentration 64 μg/ml), and the guts were kept in a humid chamber until they were measured again at 5 and 30 min. Midgut and hindgut volumes were estimated considering their shape as cylindrical. Five male and five female flies were used in this experiment. An equal concentration of H. muscarum proteins was used as a control. Whole dissected guts before and after protein treatment were projected onto a light screen and traced using a Reichert 311 204 microscope.

(vi) Statistical analyses. Statistically significant differences in specific activity on insect hindgut and contractile activity in the mammalian muscle bioassays were tested using a two- sample t-test assuming equal variances. Gut volume calculations were tested for significant differences using the Mann- Whitney test. Measurements were independently and randomly drawn from their respective time points, and median values were different.

(vii) Testing the effect of MRP on mammalian tissues. L. major cell lysate was tested on cultured rat ventricular myocytes that had been prepared according to Pinson et al. (1985). Newborn rats were decapitated, ventricles were dissected out, washed in PBS, sliced finely, and digested with 1% trypsin. The first 10-20 supernatants were centrifuged at 1000 g and resuspended in Ham F-10 (nutrient solution for cell line maintenance with L-glutamine, supplemented with horse serum; Ham, 1963). Diluted suspensions, in 1 ml aliquots, were used to seed each well of sterile 24-well plates as described (Pinson et al., 1985). Monolayers of ventricular myocytes were maintained at 35°C and 3.1% C0₂. Samples of BSA, L. major (0.034 mg protein/ml) or C. fasciculata cell lysate proteins (0.042 mg protein/ml) were overlaid on cultured myocytes. Samples were evenly distributed by gently swirling after application, and allowed to incubate 30 min. Contractions were counted for 10 min on a warm plate. All wells were then washed with sterile saline, re-incubated for 30 min, and observed again. Data for 4- 6 observations are presented. The effect of lysate proteins was tested also on visceral muscle preparations of Guinea pigs. Animals were starved 2-3 days before the experiment. To avoid extraneous effects of drugs, they were then euthanized by concussion, in accordance with the Report of the Federation of European Laboratory Animal Science Associations. Segments of ileum and uterus were dissected and cleared of adhering fat and mesentery. Segments of 2-3 cm of each organ were tied with a silk thread to the base of a 20 ml isolated organ bath containing Tyrode's solution. Tyrode's solution was prepared according to Webster and Prado, 1970: 11.90 mM NaHC0_3> 1.05 mM MgCl₂, 5.55 mM glucose, 2.68 mM KCl, 167.0 mM NaCl, 0.42 mM NaH₂P0₄, 1.80 mM CaCl₂. All items, except for CaCl₂, were prepared, bubbled in 95% 0₂: 5% C0₂ for 10 min until precipitate formed, and adjusted to pH 7.2 with NaOH. CaCl₂ was added, and the mixture was sterilized through 0.22 μ filter at 35°C, and bubbled with 95% 0₂: 5% C0₂. Bubbling in the organ bath allowed for effective mixing of drugs and proteins. The other end of the tissue was hooked to a force-displacement transducer (Coulbourn Instruments) attached to a computerized polygraph to record contractions. Both ileum and uterus preparations were allowed 30 min equilibration before addition of drugs or L. major lysate proteins. Segments of ileum were pre-contracted with 30 μl nicotine or acetylcholine per 20 ml Tyrode's solution. After repeated contractions reached a plateau, 2.0 ml (8.8 mg protein/ml) of L. major lysate (final concentration 0.88 mg protein/ml) were added and contractile force measured after 2 min (to allow bath temperature to equilibrate to 35°C). The tissue samples were washed with fresh Tyrode's solution and nicotine or acetylcholine was added again to check if muscle relaxation was reversible. Tests were repeated six times. Additionally, the ileal strips were stimulated to contract with regular cycles of 40 V. Samples of 0.22, 0.43 and 0.82 mg protein/ml L. major cell lysate were added, and 1.0 ml of the above proteins (8.8 mg/ml) were added to test for dosage response. Tests were repeated three times. Segments of uterus were pre-contracted with 10 μl oxytocin (10 units/ml) per 20 ml Tyrode's solution. One ml samples (3.3 mg protein/ml) of L. major-lysate were added 3 and 6 min after initial equilibration and allowed to act on the tissue for 3 min. Final protein concentrations in the organ bath after each application were 0.17 mg/ml and 0.31 mg/ml. The bath was washed afterwards with fresh Tyrode's solution and the same volume of oxytocin was added again to check if muscle relaxation was reversible. Data are presented for two repetitions.

Example 1. Initial characterization of the muscle-relaxing protein (MRP). L. major cell lysate proteins or, alternatively, proteins precipitated from the growth medium after removal of parasites, were treated with trypsin, chymotrypsin, or proteinase-K to test the sensitivity of MRP to proteases. Aliquots of parasite proteins (3 mg/ml) or culture medium proteins (1-2 mg/ml) were treated with 0.5 mg/ml trypsin and chymotrypsin, or 0.05 mg/ml proteinase-K, incubated for 30 min at 37°C, and reactions halted with proteinase inhibitors (Sigma). Treated proteins were frozen in liquid N₂ and stored at -70° C until tested in the sand fly hindgut assay. To determine the culture stage in which MRP is expressed, parasite cells and culture growth medium were harvested from day 1 to 7 of growth (4 samples per day) by centrifugation at 2000xg for 10 min at 8°C and the parasite pellets were washed twice with ABS. Daily parasite density was calculated. Protein samples from culture medium were precipitated with 85% (of saturation at 0°C) (NH )₂S0 and preparations were made from parasites pellets as described above. Both precipitate and supernatant (lysate) were dialyzed (Spectra/Por Membrane 3.5 kDa) against ABS medium, and tested using the sand fly hindgut bioassay.

Example 2. L. major lysate proteins inhibit P. papatasi hindgut contractions Dissected sand fly hindguts in warmed ABS spontaneously contracted for

1-6 hours. L. majorAysaXe proteins (0.05-0.07 mg/ml) were tested in the standard bioassay with sand fly hindgut as described in sections (iv) and (v) above. As shown in Fig. 1, the lysate inhibited 60% of spontaneous P. pαpαtαsi hindgut contractions within 5 min of application. Hindguts exposed to proteins for 20 min, rinsed in ABS, and returned to ABS, resumed contracting (Return in Fig. 1). Different dosages of L. mαjor- ysatc proteins were then tested in the same bioassay. As shown in Fig, 2, application of 1.2, 2.4, 4.8 and 9.6 μg/ml L. major cell lysate proteins caused dose-dependent inhibition of hindgut contractions by 20, 34, 60 and 80%, respectively. Hindguts completely distended within seconds of a concentration of 48 μg protein/ml or more. Based on these observations, midgut and hindgut distension was quantified 5 and 30 min after addition of 64 μg protein (64 μg/ml) of L. major and H. muscarum. The increase of P. papatasi midgut volume (average 3.2 μl) was insignificant after 5 min incubation with 64 μg/ml L. major-\y sate proteins, saline, or 64 μg/ml H. muscarum proteins. However, the difference was significant (p < 0.01) after 30 min incubation with L. major-lysate proteins (48.7%), saline (19.2%), and H. muscarum proteins (7.8%). Average hindgut volume increased 5 min after application of 64 μg/ml L. m /or-lysate proteins, saline, and 64 μg/ml H. muscarum proteins by 20.7, 7.3, and 13.1%, respectively. After 30 min incubation, hindgut volume increased by 57.1, 11.0, and 13.1%, respectively. The increase in hindgut volume (average 0.1 μl) incubated with L. major-lysate proteins was significantly greater than the other two applications (p < 0.01) at both 5 and 30 min. Example 3. Inhibition of P. papatasi hindgut contractions by different parasite lysates. Nine trypanosomatid parasites were tested: L. major, L braziliensis braziliensis, L. donovan (Sudan), L. donovan (India), L. infantum, L. tropica, L. seymouri, H. muscarum and C. fasciculata. As shown in Fig. 3, among the nine parasites tested, only proteins of L. b. braziliensis and L. major consistently inhibited sand fly hindgut contractions. The Sudanese L. donovani strain inhibited hindgut contractions, while the Indian strain had no muscle relaxing effect on P. papatasi hindgut.

Example 4. L. major lysate proteins inhibit mammalian cardiomyocyte contractions. L. major-lysate proteins were tested on cultured neonatal rat ventricular myocytes as described in section (vii) above. Fig. 4 shows that contractions of rat myocytes incubated 30 min with 34 μg/ml L. mαjor-lysate proteins stopped completely (triangles). The plates were gently agitated after application of the proteins so that they were spread on the entire cell monolayer surface and myoinhibition they caused was general. The cessation of contraction was accompanied by clumping and swelling of cells. Cells resumed contracting normally after a saline wash and a further 30 min incubation. Neither ABS nor C. fasciculate lysate proteins (42 μg/ml) had such an effect on cardiomyocyte contractions.

Example 5. L. major lysate proteins inhibit mammalian ileum contractions. L. major lysate proteins were further tested on segments of Guinea pig ileum, as described in section (vii) above. Fig. 5A shows that strips of Guinea pig ileum contract spontaneously for 3 min without significant change in frequency or amplitude, when incubated in oxygenated Tyrode's solution at 35°C. The ileum strips were then precontracted with nicotine or acetylcholine (30 μl per 20 ml Tyrode's solution) and then treated with L. major lysate proteins (final concentration of 0.88 mg/ml) and contractile force was measured after 2 min. As shown in Fig. 5B, ileum strips precontracted with nicotine exerted 2.8 g tension. Strips treated with 0.88 mg/ml L. major lysate proteins and retreated with nicotine exerted 1.8 g tension, a rapid 35.7% decrease in force of contraction. After washing in fresh Tyrode's solution, and reapplication of nicotine, the tension of the strips increased to 3.0 g more. As shown in Fig. 5C, ileum strips precontracted with acetylcholine exerted 4.3 g tension. The tension decreased insignificantly to 3.8 g in strips treated with

0.88 mg/ml L. major lysate proteins and acetylcholine. After washing in fresh

Tyrode's solution, and reapplication of acetylcholine, the strips resumed contracting at 4.2 g or greater. Fig. 5D shows that the mean peak value (3.7 g) during regular 40 V electric stimulation was immediately reduced to 2.6 g after addition of 0.22 mg/ml L. major lysate proteins (p< 0.01), a 29.7% decrease in contractile strength without changes in the frequency. Increasing concentration to 0.44 mg/ml proteins resulted in a mean peak value of 0.9 g, a total decrease of 75.6% from the original and a decrease in frequency. A further increase in concentration to 0.88 mg/ml proteins practically obliterated contractions despite regular electric stimulation of 40 V. The mean peak value of 0.5 g was close to the baseline value of 0.2 g.

Example 6. L. wfα ør-lysate proteins inhibit mammalian uterus contractions. The experiments were conducted as described in section (vii) above. Strips of uterus precontracted with oxytocin (an hormone that induces uterine contractions in mammals) repeatedly exerted 3.1 g tension (result not shown). As shown in Fig. 6, treatment with 0.17 mg/ml L. major lysate proteins immediately decreased the tension of contraction by 21.2%, to 2.6 g. An increase to 0.31 mg/ml proteins resulted in a further significant (p < 0.01) reduction of 54.5% to a tension of 1.5 g. After washing in fresh Tyrode's solution, and reapplication of 10 μl oxytocin, the strips continued contracting at 4.0 g tension or greater.

Example 7. Purification and identification of MRP from L. major crude lysate. A series of five steps, including one ammonium sulfate precipitation step and four column steps, described below, was used to purify MRP from L. major crude lysate. The purification scheme was performed four times; results for the final trial are presented herinbelow and summarized in Table 1. Briefly, cell lysate proteins (Fraction 1) were precipitated with 85% (of saturation at 0°C) of (NH )₂S0₄ (Fraction 2) and subjected to two steps of hydrophobic chromatography (Fractions 3 and 4), size exclusion chromatography (Fraction 5), and finally reversed phase high performance liquid chromatography (RP-HPLC) (Fraction 6), with purification of > 14,000 fold over the crude cell lysate. Chromatography, centrifugation, and dialysis were performed at 4°C .

7(a). Ammonium sulfate precipitation Fraction 1, containing cell lysates proteins from freeze/thawed L. major cells (as described above, Materials and Methods, section iii), was precipitated with 85%) (of saturation, at 0°C) (NH₄)₂S0₄ (AS), and the pH adjusted to 7.2 with NH₄OH. Samples were centrifuged 5 x 10 xg for 30 min and the supernatant was discarded. Pellets were pooled, immediately frozen in liquid nitrogen, and stored at -70°C (Fraction 2). Aliquots of Fraction 1, Fraction 2, and supernatant were bioassayed. Fraction 2 and supernatant were dialyzed overnight against ABS medium.

7(b). Hydrophobic interaction chromatography: Phenyl Sepharose ™. Fraction 2 samples thawed on ice, were diluted with ABS medium to 1.5 M

(NH₄)₂S0₄, and centrifuged 5 x 10³xg for 30 min, to remove insoluble particulates.

A total of 2.9 g of Fraction 2 were loaded onto a Phenyl Sepharose™ 6 Fast Flow column (6.5 x 23 cm), equilibrated with 1.5 M (NH₄)₂S0₄ in ABS (Kennedy, 1990). The column was eluted stepwise with the same buffer containing decreasing concentrations of (NH₄)₂S0₄, as follows: 1.0, 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.25, and without (NH₄)₂S0₄. Aliquots of 100 μl were dialyzed against 4.0 1 of ABS medium overnight, tested in sand fly hindgut bioassays and pooled active fractions were termed Fraction 3.

7(c). Hydrophobic interaction chromatography: Propyl Sepharose ™ Fraction 3 (40 mg) was loaded onto a Fractogel EMD Propyl Sepharose™ column (1 .5 x 9 cm), equilibrated with 1.5 M (NH )₂S0₄ in ABS. The sample was eluted stepwise with the same buffer containing decreasing concentrations of (NH₄)₂S0₄, as follows: 1.25, 1.00, 0.75, 0.50, 0.25, and no (NH₄)₂S0₄. Aliquots of 50 μl were dialyzed against 4.0 1 of ABS overnight and tested by the sand fly hindgut bioassay. Active fractions from Propyl Sepharose™ chromatography were defined as Fraction 4. Before proceeding to size exclusion chromatography, Fraction 4 was concentrated on a 1 ml bed-volume Phenyl Sepharose™ 6 Fast Flow column, equilibrated with 1.5 M (NH₄)₂S0₄ in ABS. The column was washed with 3 bed-volume of the equilibration buffer and eluted with ABS buffer containing no (NH₄)₂S0 , dialyzed, and bioassayed for activity. This concentrated preparation was defined as Fraction 4a.

7(d). Size exclusion chromatography. Fraction 4a was filtered (0.22 μm) and loaded onto a Merck Fractogel EMD BioSEC (S) column for size exclusion chromatography (Darmstadt, Germany). Chromatography was performed using a Varian 5000 Liquid Chromatograph equipped with Rheodyne solvent delivery module (Cotati, CA, USA). The following proteins were used as molecular weight (MW) markers: β-amylase (200 kDa), BSA (66 kDa), chymotrypsinogen (25 kDa) and aprotinin (6.5 kDa). The column was equilibrated and washed with ABS at 0.5 ml/min. Ninety fractions were collected on ice and aliquots were bioassayed. Fractions with muscle relaxing activity were pooled and defined as Fraction 5. Fig. 7 shows the elution pattern of the MW markers and of the MRP protein. The arrow denotes the retention time of MRP (134.9 min). The preliminary estimation of the protein apparent native mass, deduced only from the preparative BioSEC column (Fig. 7), is approximately 12 kDa.

7(e). Reversed-phase HPLC. Fraction 5 was vacuum concentrated, re-suspended in TDW (triple-distilled water), and filtered (0.22 μm) before loading onto a Vydac C4 (214TP) RP- HPLC column (0.46 x 25 cm). The column was eluted using a gradient of 5-95% acetonitrile containing 0.1% TFA, at a flow rate of 0.5 ml/min for 60 min. Ninety fractions were collected and immediately frozen in liquid nitrogen; aliquots were concentrated as above, re-dissolved in triple distilled water and tested in the sand fly hindgut bioassay. Active fractions, eluted at 48-50 min (black arrow) with about 33% acetonitrile were pooled to yield Fraction 6 (Fig. 8). The purification steps described in this Example are summarized below and in Table 1 (Phenyl Sepharose™ 6 Fast Flow: 0.65M (NH₄)₂S0₄; EMD Propyl Sepharose™: 1.0M (NH₄)₂S0_4; Phenyl Sepharose™ 6 Fast flow (to concentrate sample): ABS with no (NH₄)₂S0₄. Fractogel EMD BioSEC: 134.9 min retention time; Vydac C4 RP-HPLC: -33.5 % acetonitrile):

Table 1. L. major MRP purification

Example 8. Purification of MRP secreted into L. major culture medium MRP was purified from the parasites' culture medium, in which the MRP is secreted in a relatively large quantity. L. major parasites were grown in culture medium 199 (powder, M199, cat. No. M0393; Sigma, St. Louis, MO) containing FCS (containing per liter: 350 mg NaHC0₃, 5.96 g HEPES (adjusted by HC1 to pH 7.1), 0.1 mM adenosine, 10 mg folate, 10 ml BME vitamins solution 100-fold concentrate (Beit Haemek, Israel, Cat. # 01-316-1), 5.25 mg xanthin, 5 mg hemin, 2 mM L-glutamine, 10 ml penicillin-streptomycin mixture (Beit-Ha-Emek), 1% FCS) (Lira et al. 1998), and transferred into FCS-free medium, which was used as a source for the isolation of MRP. Surprisingly, it was found that after transfer into FCS-free medium, at least one generation of parasites was able to grow and secrete approximately the same amount of MRP, as in medium containing 10% FCS. Removal of the serum increased the specific activity in the growth medium about a 100-fold, with no measurable effect on the total MRP activity. It was also found that, unlike most other proteins, MRP does not precipitate from the culture medium in the presence of high concentrations of ammonium sulfate. We used this unusual property of MRP to remove the majority of the contaminating proteins in the growth medium, simply by their precipitation in the presence of high (80% of saturation at 0°C) ammonium sulfate, followed by precipitation of MRP by a saturated ammonium sulfate solution (100%, 0°C). The rest of the purification protocol was kept as established in Example 7.

Example 9. Proteolysis-derived MRP peptides and their identification. Fraction 6 of Example 7(e) was brought to a final concentration of 25 mM NH₄HC0₃ and submitted to proteolytic digestion, as follows: A first sample, enzymatically digested with trypsin overnight at 37°C, was prepared using a ZipTip C₁₈ pipette tip (Millipore Corp.), eluted with 75% CH₃CN, 1% formic acid, and digested with the endoproteinase Asp-N (see below). A second sample was enzymatically digested overnight at 37°C with Asp-N protease in 25 mM NH HC0₃ at pH 8.0. After enzymatic digestion, the sample was eluted using a ZipTip Cis pipette tip and 75% CH₃CN, 1% formic acid. A third sample underwent reduction and alkylation. It was treated with DTT (1,4-dithiothreitol) for 30 min at 60°C and IAA (iodoacetamide) for 30 min in the dark at room temperature. It was then eluted with a ZipTip Cι₈ tip and 75% CH₃CN, 1% formic acid. After alkylation reduction, the sample was divided into two fractions, which were enzymatically digested. The first was treated with Asp-N in 25 mM NH₄HCO₃ at pH 8.0 overnight at 37°C and eluted using a ZipTip Cι₈ tip and 75% CH₃CN, 1% formic acid. The second fraction was treated with trypsin and incubated overnight at 37°C. It was then eluted using a ZipTip Cι₈ tip (Millipore Corp.) and 75% CH₃CN, 1% formic acid. These samples were submitted to a tandem MS/MS spectrometry (Micromass, Mass Spectrometry, London UK), at the Bletterman Laboratory of the Interdepartmental Division, the Faculty of Medicine, The Hebrew University of Jerusalem. Active fractions were submitted to electrospray ionization and tandem mass spectrometry analysis to determine amino acid sequence. These sequences, identified from peptide fragmentation data after mass spectrometry, were matched to protein and genome sequence databases. The MS-MS spectra were matched against non-redundant database sequences using Mascot (www.matrixscience.com). A sequence tag search (Mascot) and a full sequence search (BLAST and FASTA) were done using small identified peptide fragments. Individual and combinations of sequences were initially checked for homology with known proteins from L. major and the trypanosomatid parasites Tiypanosoma b. briicei and T. cruzi, and then they were checked against all recorded proteins in the database. The tandem mass spectrometry of 10 major proteoly sis-derived MRP- peptides yielded the respective m/z values, molecular weights, and putative sequences of SEQ ID NOs: l-15 listed in Table 2. The sequences were used to search for sequence homologies in known proteins as described above. By searching the entire database, three organisms yielded sequences with >45% homology to L. major myoinhibiting peptide MRP and low probability that the search sequence is a random string (an expected value, or E, less than 1.0). A putative gene product of Pseudomonas aeruginosa had a 53% homology with identified fragments and an E-value of 0.13 (Stover et al., 2000). One hypothetical protein of a Halobacterium sp. had 73% homology with identified fragments and an E-value of 0.35. Three putative gene products in Drosophila melanogaster had 45 % homology with identified fragments and E-values of 0.84 (Adams et al., 2000). The functions for these proteins in P. aeruginosa, Halobacterium, and D. melanogaster are unknown.

Table 2. Peptide sequences obtained by tandem MS/MS spectrometry

X denotes an unidentified amino acid; - denotes a run of unidentified amino acids; * Sequences most likely from the same peak. Example 10. Cytotoxicity of pure L. major MRP

10(a). Cell cultures To examine the cytotoxicity of pure L. major MRP, its effect is tested on several cell lines and primary cultures from human origin, including: keratinocytes, fibroblasts, skeletal muscle cells, smooth muscle cells and lymphoid cells. Cytotoxicity assays are designed according to methods known in the art as previously disscribed by Arbel et al., 2003; Ben-Bassat et al., 2002; Shushan et al., 2004.

10(b). General experimental design Cells are seeded at subconfluent densities in 96-well microplates and grown for 3 days. Thereafter, the growth medium is replaced with medium that contains pure MRP, at the range of concentrations that is found to be effective in the relaxation of mammalian smooth muscles. Cells are treated for 4 days and then the

MRP is washed. Cell growth (survival/rescue) is monitored for an additional 3 days.

10(c). Automated microculture methylene blue assay Cell growth is determined by the automated microculture methylene blue assay. The MRP-treated cultures and controls are fixed in 0.05% glutaraldehyde for 10 minutes at room temperature. The microplates are washed, stained with 0.01% methylene blue in 0.1 M borate buffer for 60 minutes, at room temperature. The microplates are washed again extensively and rigorously to remove excess dye and then dried. The dye that is taken up by the cells is then eluted in 0.1N HC1 for 60 minutes at 37°C and is read at 620 nm. Each point of the growth curve experiments is calculated from eight wells. Each experiment is repeated 2-3 times. For each MRP concentration used, medium that contained only the dilution solution (PBS) is used as a control for a 100% growth. 10(d). Automated microculture XTT-PMS Assay The XTT-tetrazolium salt with the addition of intermediate electron acceptor, phenazine methosulphate (PMS) (XTT-PMS assay) is used to monitor cell growth and drug sensitivity when cell culture in suspension is studied. Briefly, XTT and PMS are added to the MRP-treated and control cultures and incubated for 4 h. After mixing on a mechanical plate-mixer, absorbance at 450 nm is measured by using a microplate reader.

10(e). Fluorescence-activated cell sorter analysis of DNA content and determination of apoptotic cells Selected samples of the cell cultures are submitted to fluorescence-activated cell sorter (FACS) analysis for their cell cycle and DNA content. Samples of cells that are treated with MRP for predetermined periods are dispersed with a trypsin/EDTA solution and stained with propidium iodide in suspension. To visualize apoptotic cells, selected samples are DAPI-stained. Briefly, the cell cultures are washed twice with PBS, fixed with 4% formaldehyde for 20 minutes at room temperature, washed extensively with PBS, stained with 0.05 mg/ml DAPI for 30 minutes in the dark, and then washed again. The apoptotic cells are visualized using fluorescent microscope.

Example 11. Mammalian muscle bioassays 11(a). Neonatal rat cardiomyocytes. According to the present invention, isolated cardiomyocytes rapidly stopped contracting when incubated with crude L. major MRP, and resumed contracting normally when rinsed with saline (as shown in Fig. 4). Cardiomyocytes contract essentially like striated muscle, except for the 94- extraordinarily large influx of Ca" during the action potential plateau. Both voltage-gated Ca²⁺ channels and the Ca²⁺-Na⁺ exchange pump control intracellular

Ca" concentration. Voltage-gated Ca channels and the Ca" -Na exchange pump are members of a related gene family and are functionally analogous. Both channels are inhibited when antagonists enter the cell and bind to the intracellular side. By testing a series of the ATPase blockers ouabain, bafilomycin Ai, and N,N'-dicyclohexylcarbodiimide (DCCD), only DCCD paralyzed and distended the sand fly hindgut (results not shown). DCCD specifically blocks FjFo ATPases, which use a proton-Na⁺ gradient to synthesize ATP. ATPases facilitate both ion and water transport across epithelia. Damaged or inhibited ATPases result in increased intracellular ionic concentrations. This, in turn, leads to membrane hyperpolar- ization, the first critical step in epithelial swelling. A Leishmania protein might interfere with the Ca²⁺-Na⁺ pump, which controls the efflux of Ca²⁺. This increase in intracellular Ca"⁺ would cause an influx of water across the plasma membrane by osmosis, resulting in the swollen cells seen after 30 min incubation. The same distension was seen in the sand fly midgut and hindgut which depend on voltage- gated Ca channels to maintain osmolarity and neuroendocrine-mediated peristalsis. In accordance with the present invention, the effect of pure MRP on cardiomyocytes culture contraction is also determined. Ventricular cardiomyocytes in culture contract rhythmically for three weeks if critical ions (Na⁺, K⁺, and Ca²⁺) are replenished frequently. The cardiomyocytes culture is prepared according to Pinson et al, 1985, and treated with pure L. major MRP (recombinant or endogenous) and incubated, and the changes in its contractility are recorded. Then the cells are washed twice in serum-free medium, incubated again as previously described and the contractility is monitored.

11(b). Guinea pig ileum. Crude L. major MRP significantly inhibited nicotine-induced contractions in guinea pig ileum. No significant myoinhibition was detected on acetylcholine- induced ileum contractions. This may show that MRP acts on ganglionic receptors in the muscle and not on cholinergic synapses. Acetylcholine acts through muscarinic and nicotinic receptors. Muscarinic stimulation is largely o 5 parasympathetic, and plays a role in gastrointestinal motility and smooth muscle contraction. If muscarinic receptors are blocked, nicotinic receptors stimulate autonomic ganglia and voluntary muscle. Nicotinic receptors in the guinea pig ileum are localized in the somatodendritic region of excitatory longitudinal muscle motoneurons. They are inhibited by a number of antagonists, such as dihydro-β- erythroidine and tetrazepam, which is believed to reduce ileal contractions through 94- 94- a reduction of Ca" influx through Ca channels. L. major lysate MRP was as effective (35.7%) in reversibly inhibiting nicotine-induced contractions as the quinoline alkaloid pteleprenine, a specific antagonist of nicotinic receptors. The precise mechanism by which nicotinic receptor antagonists functions is unclear even in the thoroughly studied guinea pig ileum contractile system. A number of myoinhibiting peptides might function against several different receptors. The effect of pure L. major MRP on Guinea pig ileum following nicotine stimulation is determined. A segment of guinea pig ileum is stimulated with nicotine and the increase in peak contractility is then recorded with each application. The ileum segment is washed and treated with L. major pure MRP. The system is allowed to equilibrate for several minutes, and then treated again with nicotine, and the ileum contractility is measured. The segment is then electrically stimulated and contractility is recorded. Then, increasing doses of pure L. major MRP are added and changes in the gut contractility are measured The dose-dependent effect seen on guinea pig ileum electrically stimulated 94- with 40 V also might affect Ca" uptake. Electric stimulation of 30 V or greater on guinea pig myenteric plexus longitudinal muscle activates nerve impulses, 9 - potentiating muscarinic transmission and Ca" uptake.

11(c). Guinea pig uterus. Crude L. major MRP showed a significant, reversible, dose-dependent inhibition of oxytocin-induced uterine contractions in vitro (as shown in Fig. 6). The hormone oxytocin, produced by the hypothalamus and stored in the posterior pituitary, is the sole physiological inducer of uterine contractions. Oxytocin is active only in the presence of adequate oxytocin receptors and acts by inhibiting Ca²⁺-Mg²⁺ ATPase, the pump system that extrudes Ca²⁺ from uterine 94- smooth muscle. By inhibiting the efflux of Ca , oxytocin allows a temporary, but 94- sustained rise in intracellular Ca" concentration, thereby prolonging a contractile state. Uterine muscle contractions originate in the muscle itself and are not abolished by interference with the nerve supply. Drugs that inhibit oxytocin- induced contractions are called tocolytic agents. They act as selective β₂- adrenoreceptor agonists, promoting cAMP-mediated inhibition of contractility. 94-

Tocolytic agents inhibit Ca -calmodulin function, or prostaglandin biosynthesis, or

Ca"⁺ influx by Ca"⁺-channel blockers. Finally, tocolytic peptides directly interfere with myometrial contractile function. Recently a number of different agents - including erythromycin, human chorionic gonadotrophin, and atosiban - have been tested as tocolytic drugs.

According to the invention, L. major lysate proteins acted as effectively as atosiban and erythromycin by reversibly reducing oxytocin-induced uterine contractions by

21.2% and 54.5% in a dose-dependent fashion. Given the specific mechanisms by which tocolytic agents function, it appears reasonable that Leishmania MRP either decreases Ca²⁺ influx or increases Ca²⁺ efflux to inhibit uterine muscle contraction. In accordance with the present invention, the effect of pure L. major MRP on

Guinea pig uterus is tested. A segment of guinea pig uterine muscle, stimulated with oxytocin, has constant, regular contractions. Increasing doses of pure L. major

(endogenous or recombinant) MRP are added and the changes in contractility are monitored with time. The role of oxytocin in stimulating uterine contraction and the specificity of β₂-adrenoreceptors in uterine muscle relaxation provide a simple and accurate tool for evaluating other myoinhibiting agents in Leishmania. REFERENCES

Adams, M. D., et al. and J. C. Venter. (2000). The genome sequence of Drosophila melanogaster. Science. 287: 2185-2195. Arbel, R., Rojansky, N., Klein, B. Y., Levitzki, R., Hartzstark, Z., Laufer, N., and Ben-Bassat, H. (2003). Inhibitors that target protein kinases for the treatment of ovarian carcinoma. Am J Obstet Gynecol 188, 1283-1290 Ben-Bassat, H., Hartzstark, Z., Levitzki, R., Klein, B. Y., Shlomai, Z., Gazit, A., and Levitzki, A. (2002). Tyrosine kinase inhibitors suppress the growth of non- Hodgkin B lymphomas. J Pharmacol Exp Ther 303, 163-171. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein dye-binding. Analyt. Biochem. 72: 248-254. Ham, R. G. (1963). An improved nutrient solution for diploid Chinese hamsters and human cell lines. Exptl. Cell Res. 29: 515-519. Innis, M. A., Gelfand, D. H., Snisky, J. J., and White, T. J. (eds.) (1990).

PCR protocols: A guide to methods and applications. Academic Press (San Diego, California). Lira R. Memdez S. Carrera L. Jaffe C. Neva F. and Sacks D. (1998). Leishmania tropica: The identification and purification of metacyclic promastigotes and use in establishing mouse and hamster models of cutaneous and visceral disease. Exp. Parasitol. 89:331-342. Modi, G. and R. Tesh. (1983). A simple technique for mass rearing Lutzomyia longipalpis and Phlebotomus papatasi (Diptera: Psychodidae) in the laboratory. J. Med. Entomol. 20; 568-569. Pinson, A., P. Padieu, and I. Harary. (1985). Techniques for culturing heart cells. In The heart cell in culture. A. Pinson and I. Harary, eds. Academic Press. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2^nd ed. (Cold Spring Harbor Press, Cold Spring Harbor, NY). Stover, C. K., et al., and I. T. Paulsen. (2000). Complete genome sequence of Pseudomonas aeruginosa PAOl, an opportunistic pathogen. Nature. 40 : 947-948. Shushan, A., Rojansky, N., Laufer, N., Klein, B. Y., Shlomai, Z., Levitzki, R., Hartzstark, Z., and Ben-Bassat, H. (2004). The AG1478 tyrosine kinase inliibitor is an effective suppressor of leiomyoma cell growth. Hum Reprod 19, 1957-1967. Webster, M. E. and E. S. Prado. (1970). Glandular kallikreins from horse and human urine and from hog pancreas. In Methods in enzymology vol. XIX. Proteolytic enzymes. G. E. Perlman and L. Lorand, eds. Academic Press, New York. 681-699.

Claims

1. A protein, herein designated muscle-relaxing protein (MRP), obtainable from the lysate or from the growth medium of Leishmania major organisms and capable of causing reversible muscle relaxation.

2. The protein according to claim. 1, capable of causing reversible muscle relaxation and distention of P. papatasi l indgut.

3. The protein according to claim 1 , capable of causing reversible relaxation of mammalian heart cells and smooth muscles of different organs, e.g. intestine and uterus.

4. The protein according to any one of claims 1 to 3, having an estimated native molecular mass of about 12 kDa as determined by size exclusion chromatography.

5. A protein obtainable from lysed Leishmania major organisms having the following characteristics: (i) it has an estimated native molecular mass of about 12 kDa as determined by size exclusion chromatography; (ii) it inhibits P. papatasi hindgut contractions in a reversible manner; (iii) it inhibits mammalian cardiomyocyte contractions in a reversible manner; (iv) it inhibits mammalian ileum strips contractions in a reversible manner when the strips have been precontracted with nicotine, but not when the strips have been precontracted with acetylcholine; (v) when eluted from a RP-ITPLC column using a linear acetonitrile gradient (5% to 95%), the muscle-relaxing activity, as verified by bioassays with P. papatasi hindgut, is eluted at by acetonitrile concentration of around 33%.

6. The protein according to claim 5 which, after tandem mass spectrometry and proteolysis, provides the peptides of the putative sequences of SEQ ID NOs:l-15.

7. A muscle-relaxing protein according to any one of claims 1 to 6 in recombinant fonn.

8. A pharmaceutical composition comprising a muscle-relaxing protein according to any one of claims 1 to 7, and a pharmaceutically acceptable earner.

9. A pharmaceutical composition according to claim 8 for treatment of angina pectoris, cardiac arrythmia, hypertension, heart failure, or prevention of myocardial infarction.

10. A pharmaceutical composition according to claim 8 for treatment of asthma, emphysema, pneumonia or bronchitis.

11. A pharmaceutical composition according to claim 8 for the prevention of premature labor.

12. A method for treatment or prevention of a cardiac disease or condition which comprises administering to a subject in need an effective amount of a muscle- relaxing protein of claim 1.

13. The method according to claim 12, wherein the cardiac disease or condition is angina pectoris, cardiac arrythmia, heart failure, or myocardial infarction.

14. A method for treatment of hypertension which comprises administering to a subject in need an effective amount of a muscle-relaxing protein of claim 1.

15. A method for treatment or prevention of a respiratory disease or condition which comprises administering to a subject in. need an effective amount of a muscle-relaxing protein of claim 1.

16. The method according to claim 15, wherein the respiratory disease or condition is asthma, emphysema, pneumonia or bronchitis.

17. A method for the prevention of premature labor which comprises administering to a female subject in need an effective amount of a muscle-relaxing protein of claim 1.