ES2358331T3 - SPECIFIC PHOSPHODESTERASES OF CYCLIC NUCLEOTIDES FROM LEISHMANIA AND USES OF THE SAME. - Google Patents

Abstract

Substantially purified protein comprising a phosphodiesterase (PDE) catalytic domain of PDE-B1 from Leishmania comprising amino acids 647-880 as set forth in SEQ ID NO: 3 or variants and mutants of the catalytic domain that are at least 80% identical to amino acids 647-880 of SEQ ID NO: 3 and that hydrolyze cAMP.

Description

FIELD OF THE INVENTION

[0001] We request the priority of U.S. Provisional Applications No. 60 / 500,244 (filed on September 5, 2003), 60 / 504,070 (filed on September 19, 2003) and 60 / 582,584 (filed on June 25, 2004). 5

[0002] The present invention relates to novel amino acid and cyclic nucleotide specific phosphodiesterase nucleic acid sequences of a parasite of the Leishmania family, such as Leishmania major. The invention also relates to nucleic acid constructs, vectors and host cells comprising nucleic acid sequences as well as methods for producing and using amino acid and nucleic acid sequences. The invention further relates to the use of these sequences and antibodies directed against these sequences, in the diagnosis and treatment of disorders related to Leishmania infection, such as Leishmania major, including the identification of compounds that complex with the polypeptides and nucleic acids in the present invention.

BACKGROUND OF THE INVENTION

[0003] Leishmania major is one of several Leishmania parasites that, when introduced into a host organism, is the causative agent of leishmaniasis. Leishmania major is a species of the Leishmania tropica complex. Other complexes (comprising species and subspecies) include Leishmania donovani, Mexican Leishmania and Leishmania viannia. According to the World Health Organization, leishmaniasis is among the most infectious diseases globally and is endemic in 88 countries in Africa, Asia, Europe and in South and North America. It has been estimated that more than 12 million people suffer from Leishmanial infections globally, 20 with serious public health problems, particularly in countries such as Iran, Iraq, Afghanistan, Algeria, Brazil, India, Peru and Syria. Leishmaniasis is most commonly manifested as cutaneous leishmaniasis (i.e. skin) (CL) or visceral leishmaniasis (ie internal organs) (VL). CL is the most common form of leishmaniasis and is the result of the transmission of the Leishmania major parasite through the bite of an infected female sandfly. The symptoms of CL include large lesions or skin ulcers on exposed parts of the body, which cause severe disability and permanent scarring.

[0004] The methods of treating leishmaniasis have typically been limited to administering pentavalent antimony (Sbv) (Sundar et al. (2002) Curr. Opin. Infect. Dis. 15, 593-598). Due to the recent emergence of large-scale resistance to Sbv, however, the efficacy of this treatment is becoming increasingly limited. In addition, treatment with Sbv has several side effects that include nausea, vomiting, diarrhea and seizures. In addition, HIV coinfection with Leishmania species presents additional challenges since such coinfection can dramatically alter the epidemiology, diagnosis and response of leishmaniasis to therapy (Lee et al. (2003) Int. J. Infect. Dis. 7, 86 -93).

SUMMARY DESCRIPTION OF THE INVENTION

[0005] The invention provides new cyclic nucleotide specific phosphodiesterase polypeptides 35 (LmPDE) and nucleic acid molecules thereof that are useful in the diagnosis and treatment of leishmaniasis. The polypeptides of the present invention also include antibodies that recognize and bind to LmPDE polypeptides. The nucleic acid molecules of the invention also include peptide nucleic acids (PNAs) and antisense molecules that react with the nucleic acid molecules of the invention.

[0006] In one embodiment, the invention provides novel full-length phosphodiesterase (PDE) of the Leishmania major 40 parasite, designated LmPDE-A, LmPDE-B1 and LmPDE-B2, including polypeptide molecules, corresponding nucleic acid molecules and fragments of the same.

[0007] The present invention also encompasses various nucleotide and amino acid sequences that represent different forms and fragments of LmPDE genes, transcripts and proteins such as conservatively mutated proteins, different allelic forms, polymorphic forms, alternative precursor transcripts and mature transcripts. . Additionally, recombinant nucleic acid molecules are provided that are codon utilization variants of the new LmPDE sequences. The invention encompasses LmPDE of Leishmania major, as well as other Leishmania species and subspecies of the Leishmania tropica, Leishmania donovani, Mexican Leishmania and Leishmania viannia complexes.

[0008] The present invention further provides recombinant nucleic acid molecules encoding 50 wild-type sequences or mutants of LmPDE polypeptides that maintain the biological activity of PDE. These include naturally occurring and synthetic mutants, as well as semi-synthetic and recombinant polypeptides.

[0009] The present invention also includes polynucleotides encoding LmPDE in recombinant expression vectors and host-vector systems that include expression vectors. One embodiment provides various host cells transformed with recombinant vectors that include the nucleotide sequences of LmPDE of the invention. The invention also provides genetically modified organisms that comprise

a vector containing a recombinant LmPDE sequence in which at least one endogenous LmPDE gene has been disabled.

[0010] The present invention further provides methods for using substantially purified LmPDE polypeptides to identify compounds that modulate the expression or activity of LmPDE.

[0011] The present invention also provides methods for using LmPDE nucleotide sequences such as 5 nucleic acid probes and primers and for using LmPDE polypeptides as antigens for the production of new anti-LmPDE antibodies. LmPDE probes and primers and anti LmPDE antibodies are useful in diagnostic assays and kits for the detection of naturally occurring protein and nucleotide sequences of LmPDE present in biological samples.

[0012] Additional objects and advantages of the invention will be set forth in part in the description below and in part 10 will be obvious from the description or can be learned by practice of the invention. The objects and advantages of the invention will be realized and obtained by means of the elements and combinations particularly indicated in the appended claims. Furthermore, the advantages described in the body of the report, if not included in the claims, are not themselves limitations of the claimed invention.

[0013] The publications analyzed herein are provided only by their description prior to the date of submission of this application. Nothing herein should be construed as an admission that the present invention has no right to predate such publication by virtue of a prior invention. In addition, the publication dates provided may be different from the actual publication dates that may need to be independently confirmed.

BRIEF DESCRIPTION OF THE DRAWINGS 20

[0014] Figure 1 shows the deduced amino acid sequence of a full-length LmPDE-A polypeptide (SEQ ID NO: 1).

[0015] Figures 2A-2S show the DNA sequence (SEQ ID NO: 2) for the full length gene of LmPDE-A. The open reading phase begins with adenine at position 530 and ends with guanine at position 2425.

[0016] Figures 3A-3B show the deduced amino acid sequence of a full length LmPDE-B1 polypeptide (SEQ ID NO: 3).

[0017] Figures 4A-4L show the DNA sequence (SEQ ID NO: 4) for the full length gene of LmPDE-B1. The open reading phase begins with adenine at position 1267 and ends with adenine at position 4059.

[0018] Figures 5A-B show the deduced amino acid sequence of a full-length LmPDE-B2 polypeptide (SEQ ID NO: 5). 30

[0019] Figures 6A-6L show the DNA sequence (SEQ ID NO: 6) for the full length gene of LmPDE-B2. The open reading phase begins with adenine at position 2182 and ends with adenine at position 5004.

[0020] Figures 7A, 7B and 7C show the location of conserved domains found in the polypeptide sequences of LmPDE-A, LmPDE-B1 and LmPDE-B2, respectively.

[0021] Figure 8 shows the results of a Southern blot analysis for LmPDE-B1 using a conserved region of the sequence of LmPDE-B1 as a hybridization probe.

[0022] Figure 9 shows the results of a Southern blot analysis for LmPDE-B2 using a conserved region of the LmPDE-B2 sequence as a hybridization probe.

[0023] Figure 10 shows the results of a Southern blot analysis using a conserved region that was specific for both LmPDE-B1 and LmPDE-B2 as a hybridization probe. 40

[0024] Figure 11 shows a schematic drawing of the location of the LmPDE-B1 and LmPDE-B2 loci and their approximate relative locations on chromosome 15.

[0025] Figures 12A, 12B, 12C and 12D show the thermal shock test results of PDE-deficient Saccharomyces cerevisiae cells that have been transfected with LmPDE-B1 and LmPDE-A.

[0026] Figures 13A, 13B and 13C show the Michaelis-Menten kinetics of cAMP hydrolysis mediated by recombinant 45 LmPDE-B1. Figures 13A and 13B show the hydrolysis of cAMP in the absence and presence of a 100 fold excess of cGMP, respectively. Figure 13C shows the effect of a 50-fold excess of AMP. The insertions of figures represent the corresponding Eadie-Hofstee representations.

[0027] Figures 14A and 14B show the effect of various PDE inhibitors on LmPDE-B1 and LmPDE-B2, respectively, in the presence of 1 M cAMP and cilostamide (A), zaprinast (B), ethazolate (C), dipyridamole (D), Ro-20-1724 50

(E), rolipram (F), isobutylmethylxanthine (IBMX) (G), 8-methoxymethyl-IBMX (H), trequinsin (I), papaverine (K), milrinone (L), petoxyphillin (M) and erythro-9- (2-hydroxy-3-nonyl) adenine (EHNA) (N) 100 µM. Figures 14C and 14D show representative dose-response profiles of trequinsin against LmPDE-B1 and LmPDE-B2, respectively.

[0028] Figure 15 shows the inhibition of promastigote proliferation of L. major in the presence of increasing concentrations of dipyridamole (A), ethazolate (B) and trequinsin (C). 5

DEFINITIONS

[0029] The term "amino acid sequence" refers to amino acids encoding an oligopeptide, peptide, polypeptide or protein sequence and fragments thereof and includes molecules of natural or synthetic origin.

[0030] The term "antibody" refers to intact molecules as well as fragments thereof (eg, Fab), which can bind to an antigenic determinant in an antigen (eg, an antigenic determinant or determinants in an LmPDE) . The antibody can be "polyclonal," "monoclonal," "chimeric," "humanized," or human.

[0031] The term "antigenic determinant" refers to the fragment of a molecule (ie, an epitope) that induces an antibody and thereafter comes into contact with a particular antibody. When a protein or fragment of a protein is used to immunize a host animal, numerous regions of the protein can induce the production of antibodies that specifically bind to a given three-dimensional region or structure in the protein; These regions or structures are called antigenic determinants. An antigenic determinant can compete with the intact antigen (i.e., the immunogen used to induce the immune response) by binding with an antibody.

[0032] The term "antisense" refers to any composition that contains nucleotide sequences that are complementary to a specific DNA or RNA sequence. twenty

[0033] The term "antisense chain" is used in reference to a nucleic acid chain that is complementary to the "sense" chain. Antisense molecules include nucleic acids (which may include residues of modified nucleotide bases and modified sugars) and can be produced by any method including synthesis or transcription. Once introduced into a cell, complementary nucleotides combine with natural sequences produced by the cell to form double strands and block transcription or translation of the sequences.

[0034] The term "biological sample" is used in its broadest sense. A biological sample is suspected of containing LmPDE nucleic acid molecules, or fragments thereof, or an LmPDE polypeptide. The appropriate biological sample may be from an animal or a human being. The sample can be a cell sample or a tissue sample, including spleen, lymph node, thymus, bone marrow, liver, heart, testicle, brain, placenta, lung, skeletal muscle, kidney and pancreas samples. The sample may be a biological fluid, including urine, blood sera, blood plasma, phlegm or wash fluid. Alternatively, the sample may be a swab of the nose, ear or throat.

[0035] The term "biologically active" refers to a polypeptide that has structural, regulatory or biochemical functions of a naturally occurring molecule. Similarly, "immunologically active" refers to the ability of the natural, recombinant or synthetic LmPDEs of the invention, or any fragment thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies. For example, polypeptides of the invention can induce antibodies that specifically bind to an epitope associated with an LmPDE polypeptide of the invention. Accordingly, an LmPDE polypeptide is capable of inducing a specific immune response in appropriate animals or cells and / or binding with ligands such as specific antibodies.

[0036] The term "catalytic domain" refers to a subset of amino acids conserved within a PDE sequence that is responsible for catalyzing the hydrolysis reaction of the bound substrate.

[0037] The term "chimeric antibody" refers to an antibody in which the variable regions derived from one species are combined with the constant regions of an antibody derived from a different species. The chimeric antibodies are useful, since they are less likely to be antigenic to a human subject than antibodies with non-human constant regions and variable regions. The region that combines with antigen (variable region) of a chimeric antibody can be derived from a non-human source (for example murine) and the constant region of the chimeric antibody, which confers biological effector function to the immunoglobulin can be derived from a human source ( Morrison et al. (1985) Proc. Natl. Acad. Sci. USA 81, 6851; Takeda et al. (1985) Nature 314, 452; Cabilly et 50 al., U.S. Patent No. 4,816,567; Boss et al. ., U.S. Patent No. 4,816,397). The chimeric antibody may have the antigen binding specificity of the non-human antibody molecule and the effector function conferred by the human antibody molecule.

[0038] The term "complementary" refers to nucleic acid molecules that have purine and pyrimidine nucleotides that have the ability to associate through hydrogen bonds to form double-stranded nucleic acid molecules. The following base pairs are related by complementarity: guanine and cytosine; adenine

and thymine; and adenine and uracil. The term "complementary" applies to all base pairs comprising two double stranded nucleic acid molecules or to all base pairs comprising a single stranded nucleic acid molecule folded on itself. The complementarity between two single stranded molecules may be partial, in which only some of the nucleic acids bind or may be complete in which there is total complementarity between the single stranded molecules. The degree of complementarity between nucleic acid chains has significant effects on the efficiency and strength of hybridization between nucleic acid chains.

[0039] The expression "control cell" is a cell that is generally the same, for example, genotypically and phenotypically, to the cell with which it is compared (for example, the cells may be sister cells), but which is not exposed to a test compound.

[0040] The expression "expression control sequence" or "expression control element" refers to a regulatory polynucleotide sequence that can direct the transcription and translation of an open reading phase. Expression control elements are known in the art and include, without limitation, inducible promoters, constitutive promoters, donor and splice acceptor sites, secretion signals, enhancers, transcription terminators and other transcriptional regulatory elements. Other expression control elements that are involved in translation are known in the art, and include the Shine-Dalgarno sequence and 15 start and end codons.

[0041] The term "fragment" of an LmPDE polypeptide refers to a part of an LmPDE polypeptide. For example, a LmPDE fragment may be a polypeptide with fewer amino acids than a full-length LmPDE, but which has the biological activity of a full-length LmPDE-A, LmPDE-B1 or LmPDE-B2 polypeptide, for example, the ability to hydrolyze cAMP. A fragment may also be a part of an LmPDE polypeptide 20 that induces an immune response or that possesses any other biological or diagnostic property of the full-length LmPDE of the invention.

[0042] The term "fragment" of a LmPDE nucleic acid molecule refers to a part of a full-length LmPDE nucleotide sequence. For example, said nucleic acid fragment can encode a LmPDE polypeptide fragment that maintains the biological activity of a full-length LmPDE-A, LmPDE-B or 25 LmPDE-B2 polypeptide, for example, the ability to hydrolyze cAMP ( as determined by methods known in the art, for example, Schilling et al. (1994) Anal. Biochem. 216, 154-158).

[0043] The term "GAF domain" refers to a highly conserved domain that binds to low molecular weight ligands. It is known that the GAF domain in some PDEs binds to cGMP.

[0044] The term "humanized antibody" refers to an antibody molecule in which 30 amino acids have been replaced in the non-antigen-binding regions to closely resemble a human antibody, while still maintaining the ability to original union. As used herein, a humanized LmPDE antibody is an immunoglobulin molecule that is capable of binding to an LmPDE polypeptide and has variable regions substantially with the same amino acid sequence as a human immunoglobulin and has a hypervariable region substantially with the same amino acid sequence as a non-human immunoglobulin. 35

[0045] The term "hybridization" or "hybridization" refers to any process by which a nucleic acid sequence binds to a complementary chain through base pair formation.

[0046] The term "hydrolyze" refers to a chemical reaction in which a chemical bond is cleaved by a water molecule. The catalytic function of the LmPDEs of the invention, as in all PDEs, involves the hydrolysis of cAMP (as determined by methods known in the art, for example, Schilling et al. (1994) Anal. Biochem. 216, 40 154 -158).

[0047] As used herein, a first amino acid or nucleotide sequence is said to be "identical" to a second amino acid or reference nucleotide sequence, respectively, when a comparison of the first and second sequences are exactly same. Two sequences are said to be "X% identical" when a comparison of the first and second sequences has X% nucleotides or amino acids that are exactly the same. The percentage of identity can be determined electronically, for example, by using the MEGALIGN (DNASTAR) program that creates alignments between two or more sequences according to methods selected by the user, for example, the clustal method (for example, Higgins et al . (1988) Gene 73: 237-244). The percent identity between sequences can also be counted or calculated by other methods known in the art (eg, Hein (1990) Methods Enzymol. 183: 626-645). The identity between sequences can also be determined by other methods known in the art, for example, by varying the hybridization conditions.

[0048] The term "incubation" refers to a process of contacting a cell or a cell culture with the compound of interest or otherwise introducing the compound of interest into a cell.

[0049] The term "inhibitor" refers to an agent that, when bound to an LmPDE or some other polypeptide or nucleic acid, decreases the amount (expression) or biological activity of an LmPDE. Inhibitors can include proteins, nucleic acids, carbohydrates, antibodies or any other molecule that decreases the amount (expression)

or biological activity of the LmPDE present in a sample. The preferred inhibitor will selectively inhibit the biological activity of an LmPDE, while not affecting any other cellular protein.

[0050] The term "isolated" or "purified" refers to a specific nucleic acid, protein or polypeptide, or a fragment thereof, in which contaminants have been separated or substantially separated (ie substances that differ from the specific nucleic acid, protein or polypeptide molecule) of the specific nucleic acid, protein or polypeptide.

[0051] The term "LmPDE" means any of LmPDE-A, LmPDE-B1 or LmPDE-B2 and may refer to the polypeptide or nucleic acid sequences. LmPDE polypeptides can be natural, synthetic, semi-synthetic or recombinant. LmPDE includes Leishmania major polypeptides, as well as other Leishmania species and subspecies of the Leishmania tropica, Leishmania donovani, Mexican Leishmania and 10 Leishmania viannia complexes including Leishmania aethiopica, Leishmania brasiliensis, Leishmania d. Donovani, Leishmania d. infantum, Leishmania d. Chagasi, Leishmania Garnhami, Leishmania m. Mexican, Leishmania m. amazonensis, and Leishmania pifanoi.

[0052] The term "LmPDE-A nucleic acid molecule" refers to a nucleic acid molecule that encodes an LmPDE-A polypeptide. fifteen

[0053] The term "LmPDE-B1 nucleic acid molecule" refers to a nucleic acid molecule that encodes an LmPDE-B1 polypeptide.

[0054] The term "LmPDE-B2 nucleic acid molecule" refers to a nucleic acid molecule that encodes an LmPDE-B2 polypeptide.

[0055] The term "LmPDE expression" refers to the process whereby an RNA transcript or a translated polypeptide is produced from a nucleotide sequence of LmPDE.

[0056] The term "modulates" refers to a change in the activity of the LmPDE of the present invention. For example, modulation can cause an increase or decrease in the amount of protein (expression) or activity, binding characteristics or other biological, functional or immunological properties of the LmPDE of the invention.

[0057] The term "nucleic acid sequence" or "nucleic acid molecule" refers to an oligonucleotide, nucleotide or polynucleotide and fragments thereof; to DNA or RNA of genomic or synthetic origin, which can be double stranded or single stranded; and represents the sense or antisense chain.

[0058] The term "operably linked" refers to functionally related nucleic acid sequences. For example, a promoter is operably linked to a coding sequence if the promoter controls the translation of the encoded polypeptide. Although the operably linked nucleic acid sequences may be contiguous and in the same reading phase, certain genetic elements, for example, repressor genes, are not contiguously linked to the sequence encoding the polypeptide but still bind to the sequences. operators that control the expression of the polypeptide.

[0059] The term "PDEase domain" refers to the conserved catalytic domain of the PDE.

[0060] The term "stringent conditions" refers to conditions that allow hybridization between complementary polynucleotide sequences. Suitable stringent conditions can be defined by, for example, salt and / or formamide concentrations in the pre-hybridization and hybridization solutions or by the hybridization temperature, which is well known in the art. In particular, the rigor can be increased by reducing the salt concentration, increasing the concentration of formamide or increasing the hybridization temperature.

[0061] The term "substantially purified" refers to nucleic acid or polypeptide sequences that are removed from their natural environment and isolated or separated and are at least 60% without, 65% without, 70% without, 75% without , 80% without, 85% without, 90% without, 95% without, 96% without, 97% without, 98% without, or 99% without other components with which they are naturally associated.

[0062] The term "variants and mutants" refers to changes in a polypeptide or nucleic acid sequence such as substitutions, insertions, deletions of amino acids or nucleotides, changes of conservative amino acids, polymorphic changes, allelic changes, changes of alteration of phase, truncation or the like, in which the mutant variant protein maintains its native function (herein, at least AMPC hydrolysis) and in which the variant or mutant nucleic acid molecule encodes a protein that maintains its native function .

[0063] The term "vector" includes, without limitation, plasmids, cosmids and phagemids.

DETAILED DESCRIPTION OF THE INVENTION 50

[0064] Second holes such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) play important biological roles in modulating the effects of a wide variety of first messengers. For example cAMP and cGMP are involved in the propagation of a variety of signals

Extracellular originating from first messengers such as hormones, light and neurotransmitters. The intracellular steady-state levels of cAMP and cGMP are controlled by their rates of synthesis by cyclase and by their degradation rate by specific cyclic nucleotide phosphodiesterase (PDE).

[0065] PDEs are typically composed of a catalytic domain (approximately 270 amino acids), a N-terminal regulatory domain responsible for linking cofactors and, in some cases, a C-terminal domain of unknown function. A conserved motif, HDXXHXGXXN, has been discovered in the catalytic domain of all PDEs. Several families of PDEs have been identified (Beavo (1995) Physiol. Rev. 75, 725-748; Soderling et al. (1998) J. Biol. Chem. 273,15553-15558; and Fisher et al. (1998) J Biol. Chem. 273,15559-15564). PDE families have approximately 35% amino acid sequence identity with their catalytic domain. Isozymes within the same family typically have a sequence identity of 75-95% in this region. Within a family, 10 there is often a sequence identity greater than 60% outside the catalytic domain, while among different PDE families, there is little or no sequence similarity.

[0066] In eukaryotes, two distinct classes of PDEs have been identified. Class I enzymes all show significant amino acid sequence conservation within their catalytic domains. Class I PDEs include all currently known families of mammalian PDEs as well as several lower eukaryotic PDEs such as Saccharomyces cerevisiae PDE2 and the product of the RegA gene from Dictyostelium discoideum. Class II PDEs, however, share very little amino acid sequence identity with Class I PDEs and therefore probably have a different evolutionary origin. In addition, Class II PDEs are often distinguished by their generally higher KM values (Zoraghi et al. (2001) J. Biol. Chem. 276, 11559-11566). Class II PDEs have been identified in yeast (Nikawa et al. (1987) Mol. Cell Biol. 7, 3629-3636), Dictyostelium 20 discoideum mucilaginous mold (Lacombe et al. (1986) J. Biol. Chem. 261 , 16811-16817), Vibrio fisheri (Dunlap et al. (1993) J. Bacteriol. 175, 4615-4624), and Candida albicans (Hoyer et al. (1994) Microbiology 140, 1533-1542).

[0067] A variety of diseases are thought to result from decreased levels of cyclic nucleotides due to an increase in PDE activity. Consequently, PDEs have become highly attractive drug targets in recent years. A growing variety of family-specific and subtype-specific PDE inhibitors are being developed as therapeutic agents for a wide range of diseases such as autoimmune disease (Bielekova et al. (2000) J. Immunol. 164, 1117-1124), (Dal Piaz et al. (2000) Drug, 57 (2), 89-96), arthritis (Kiely et al. (1995) Eur. J. Immunol. 25, 2899-2906), asthma (Barnette (1999) Prog Drug Res. 53, 193-229), inflammatory diseases (Barnes (2001) Novartis Found. Symp. 234, 255-267), impotence (Langtry et al. (1999) Drugs 57, 967-989) and cancer (Marko et al. (2000) Chem. Res. Toxicol. 10, 944-948). So far, there is 30 limited information about PDE in kinetoplastea such as Leishmania major.

[0068] Rascón A et al. describes and characterizes a cyclic AMP phosphodiesterase in Mexican Leishmania and the purification of a soluble form thereof, but an amino acid sequence is not described herein (Mol Biochem Parasitol. 5 Mar 2000; 106 (2): 283-92 ).

[0069] Al-Chalabi KA et al. It indicates the isolation and partial purification of cAMP phosphodiesterase (EC 3.1.4.17) of the promastigote form of Leishmania tropica and a preliminary result of Leishmania donovani, but does not describe any amino acid sequence or nucleotide sequence encoding these proteins ( Comp Biochem Physiol B. 1989; 93 (4): 789-92).

Polypeptides of the Invention

[0070] One aspect of the present invention relates to new PDE polypeptides isolated from the Leishmania 40 major parasite, designated LmPDE-B1 (SEQ ID NO: 3) and LmPDE-B2 (SEQ ID NO: 5). Particular embodiments of the LmPDE polypeptides of the invention include LmPDE-B1 and LmPDE-B2 full length from Leishmania major. In another aspect, the invention relates to LmPDE fragments.

[0071] The present invention also relates to variants and mutants of LmPDE. LmPDE mutant alleles encode mutant forms of LmPDE polypeptides that have at least one amino acid substitution, insertion, deletion, truncation or phase change.

[0072] Another variant of LmPDE polypeptides may have amino acid sequences that differ in one or more amino acid substitutions. The variant may have conservative amino acid changes, when a substituted amino acid has similar structural or chemical properties. Amino acid residues that can be conservatively substituted with each other include, without limitation: glycine / alanine; valine / isoleucine / leucine; asparagine / glutamine; 50 aspartic acid / glutamic acid; serine / threonine; lysine / arginine and phenylalanine / tyrosine. Other substitutions can also be considered conservative, depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, methionine, which is relatively hydrophobic, can often be exchanged with leucine and isoleucine and sometimes with valine.

[0073] Alternatively, a variant may have non-conservative amino acid changes, such as, for example, replacing a glycine with a tryptophan or alanine with lysine. Similar minor variations may also include deletions and insertions of amino acids. Any amino acid substitution that does not significantly affect the biological and / or chemical properties of LmPDE polypeptides is encompassed by the present invention. May

guidance can be found in determining that and how many amino acid residues can be substituted, inserted and deleted using computer programs well known in the art such as the DNASTAR software package.

[0074] The present invention also encompasses various amino acid sequences that represent different forms and fragments of LmPDE polypeptides such as polypeptides with conservative mutations and fragments containing the PDEase domain. The LmPDE polypeptides can be from Leishmania major, or another species of 5 Leishmania and subspecies of the Leishmania tropica, Leishmania donovani, Mexican Leishmania, and Leishmania viannia complexes including Leishmania aethiopica, Leishmania brasiliensis, Leishmania d. Donovani, Leishmania d. infantum, Leishmania d. Chagasi, Leishmania Garnhami, Leishmania m. Mexican, Leishmania m. amazonensis, and Leishmania pifanoi.

[0075] The LmPDE of the present invention can be performed in many ways, such as in isolated form or in purified form. One skilled in the art can readily employ conventional isolation and purification methods to obtain isolated LmPDE polypeptides (see, for example, Marchak et al. (1996) Strategies for Protein Purification and Characterization, Cold Spring Harbor Press, Plainview, NY). The nature and degree of isolation and purification will depend on the intended use. For example, purified LmPDE protein molecules will be substantially without other proteins or molecules that alter the binding of LmPDE proteins to antibodies or other ligands. Embodiments of LmPDE polypeptides include purified LmPDE polypeptides that have the biological activity of an LmPDE protein. In one form, said purified LmPDE polypeptides maintain the ability to bind to antibody or other ligand.

[0076] Various forms of a particular LmPDE polypeptide of the invention can be produced as a result of processes such as post-translational modifications. For example, the various forms of LmPDE 20 polypeptides isolated may include different precursor forms and mature forms of a LmPDE protein or polypeptide that result from post-translational events, such as glycosylation, phosphorylation and intramolecular cleavage.

[0077] The present invention provides isolated and purified polypeptides having an identical amino acid sequence to the predicted LmPDE polypeptide sequences described herein. Accordingly, the amino acid sequences of the present invention may be identical to LmPDE-B1 (SEQ ID NO: 3) or LmPDE-B2 25 (SEQ ID NO: 5). The LmPDE polypeptides of the invention may also comprise at least one sequence that is identical to a fragment of a full-length LmPDE polypeptide sequence. The present invention also provides isolated and purified polypeptides with at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with the sequences described herein.

Nucleic Acid Molecules of the Invention

[0078] The present invention provides LmPDE nucleic acid molecules corresponding to the full-length genes of LmPDE-B1 (SEQ ID NO: 4) and LmPDE-B2 (SEQ ID NO: 6). The present invention also provides nucleic acid molecules encoding the previously analyzed LmPDE polypeptides. The present invention also encompasses various nucleotide sequences representing different forms and fragments of LmPDE genes and transcripts, such as different allelic forms, polymorphic forms, alternative precursor transcripts 35 and mature transcripts. Additionally, recombinant nucleic acid molecules are provided that are codon usage variants of new LmPDE sequences.

[0079] A specific embodiment of a nucleic acid encoding an LmPDE-B1 polypeptide of the present invention comprises the nucleotide sequence of SEQ ID NO: 4, beginning with adenine at position 1267 and ending with adenine at position 4059 (which corresponds to amino acids 1-930 of SEQ ID NO: 3). Another embodiment 40 comprises a nucleotide sequence found in SEQ ID NO: 4, beginning with thymine at position 3205 and ending with cytosine at position 3906 corresponding to the PDEase domain of LmPDE-B1 (amino acids 647-880 of SEC ID No.: 3). Due to the degeneracy of the genetic code, the present invention also provides any nucleic acid molecule encoding a polypeptide comprising the LmPDE-B1 polypeptide sequence set forth in SEQ ID NO: 3, starting with methionine at amino acid position 1 and Terminating with valine at amino acid position 930. The invention also provides nucleic acids encoding fragments of a LmPDE-B1 polypeptide, for example a nucleic acid encoding the PDEase domain comprising amino acids 647-880 as set forth in SEQ ID NO: 3.

[0080] A specific embodiment of a nucleic acid encoding an LmPDE-B2 polypeptide of the present invention comprises the nucleotide sequence of SEQ ID NO: 6, beginning with adenine at position 2182 and ending 50 with adenine at position 5004 ( which corresponds to amino acids 1-940 of SEQ ID NO: 5). Another embodiment comprises a nucleotide sequence found in SEQ ID NO: 6, beginning with thymine at position 4150 and ending with cytosine at position 4851, which corresponds to the PDEase domain of LmPDE-B2 (amino acids 657-890 of SEQ ID NO: 5). Due to the degeneracy of the genetic code, the present invention also provides any nucleic acid molecule encoding a polypeptide comprising the LmPDE-B2 polypeptide sequence set forth in SEQ ID NO: 5, starting with methionine at amino acid position 1 and ending with valine at amino acid position 940. The invention also provides nucleic acids encoding fragments of a LmPDE-B2 polypeptide, for example a nucleic acid encoding the PDEase domain comprising amino acids 657-890 as set forth in SEC. ID Nº: 5.

[0081] One skilled in the art will appreciate that the nucleic acids of the present invention may encode domains or portions of LmPDE polypeptides other than those specifically mentioned above.

[0082] The present invention contemplates alternative allelic forms of LmPDE nucleotide sequences that are isolated from different subjects of the same species. Typically, isolated allelic forms of naturally occurring gene sequences include wild type and mutant alleles. A wild-type LmPDE gene sequence will encode an LmPDE protein that has normal PDE biological activity, such as, for example, a phosphodiesterase function. Alternatively, a mutant of an LmPDE gene sequence can encode an LmPDE polypeptide that has normal activity. Accordingly, the present invention provides wild-type allelic forms and mutants of LmPDE nucleotide sequences.

[0083] The present invention further contemplates polymorphic forms of nucleotide sequences of LmPDE. 10 Typically, isolated polymorphic forms of naturally occurring gene sequences are isolated from different subjects of the same species. Polymorphic forms include sequences that have one or more nucleotide substitutions that may or may not result in changes in the amino acid codon sequence. These substitutions may result in a wild-type LmPDE gene that encodes a protein that has the biological activity of wild-type LmPDE proteins. fifteen

[0084] The present invention further provides isolated codon usage variants (see Table 1) that differ from the described LmPDE nucleotide sequences, but do not alter the predicted LmPDE protein sequence or its biological activity. The codon usage variants can be generated by recombinant DNA technology. Codons can be selected to optimize the production level of the LmPDE transcript or LmPDE protein in a particular prokaryotic or eukaryotic expression host, according to the frequency of 20 codons used by the host cell. Alternative reasons for altering the nucleotide sequence encoding an LmPDE protein include the production of RNA transcripts that have more desirable properties, such as a prolonged half-life or increased stability.

Table 1

 Amino acid

 Symbol One Letter Symbol Codons

 To the girl

 Wing A GCU, GCC, GCA, GCG

 Cysteine

 Cys C UGU, UGC

 Aspartic acid

 Asp D GAU, GAC

 Glutamic acid

 Glu E GAA, GAG

 Phenylalanine

 Phe F UUU, UUC

 Glycine

 Gly G GGU, GGC, GGA, GGG

 Histidine

 His H CAU, CAC

 Isoleucine

 I have AUU, AUC, AUA

 Lysine

 Lys K AAA, AAG

 Leucine

 Leu L UUA, UUG, CUU, CUC, CUA, CUG

 Methionine

 Met M AUG

 Asparagine

 Asn N AAU, AAC

 Proline

 Pro P CCU, CCC, CCA, CCG

 Glutamine

 Gin Q CAA, CAG

 Arginine

 Arg R CGU, CGC, CGA, CGG, AGA, AGG

 Serina

 Ser S UCU, UCC, UCA, UCG, AGU, AGC

 Threonine

 Thr T ACU, ACC, ACA, ACG

 Valine

 Val V GUU, GUC, GUA, GUG

 Tryptophan

 Trp W UGG

 Tyrosine

 Tyr and UAU, UAC

 25

[0085] The present invention additionally provides new purified and isolated polynucleotides (ie DNA sequences and fragments thereof), which may be in isolated form, including DNA and RNA transcripts (complementary sense and antisense strands) encoding LmPDE , DNA / RNA hybrids and related molecules. The nucleic acid molecules of the present invention include nucleotide sequences substantially identical or complementary to the LmPDE nucleotide sequences described herein.

[0086] The invention encompasses genomic, cDNA, ribozyme and antisense molecules as well as nucleic acids based on an alternative main chain and including alternative bases and modified sugar moieties, derived from natural sources or completely or partially synthesized. As used herein, synthesized DNA

"Completely" means that DNA is produced completely by chemical means and synthesized "partially" means that only parts of the resulting DNA are produced by chemical synthesis. The antisense molecules can be RNA or other molecules, including nucleic peptide acids (PNA) or non-nucleic acid molecules such as phosphorothioate derivatives (described in greater detail below) that specifically bind to DNA or RNA in a pair dependent manner. bases. One skilled in the art can easily obtain these classes of 5 nucleic acid molecules using the sequences described herein.

[0087] The present invention additionally provides nucleotide sequences that selectively hybridize with LmPDE nucleotide sequences under highly stringent hybridization conditions. Typically, hybridization under highly stringent conventional conditions will occur between two complementary nucleic acid molecules that differ in sequence complementarity from about 70% to about 10 100%. It is immediately apparent to one skilled in the art that highly rigorous hybridization between nucleic acid molecules depends on, for example, the degree of identity, the rigor of the hybridization and the length of the chains that hybridize. Methods and formulas for performing highly stringent hybridizations are well known in the art and can be found, for example, in Sambrook, et al. (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). fifteen

[0088] In general, stringent hybridization conditions are those that: (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl, 0.0015 M sodium citrate and 0.1% SDS at 50 ° C ; or (2) employ a denaturing agent during hybridization such as formamide, for example, 50% formamide (vol / vol) with 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinyl pyrrolidone %, 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl and 75 mM sodium citrate at 42 ° C. twenty

[0089] Another example of stringent conditions is the use of 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate , 5x Denhardt solution, sonic salmon sperm DNA (50 mg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C, with 42 ° C washes in 0.2x SSC and 0.1 SDS %. A person skilled in the art can easily determine and vary the conditions of rigor in an appropriate manner to obtain a clear and detectable hybridization signal. Typical ranges of stringent conditions include: (1) under 25 rigor (SSC 2x / SDS 0.1% (w / v), room temperature), (2) moderate rigor (SSC 0.2x / SDS 0.1% ( p / v), 42 ° C) and (3) high rigor (SSC 0.1x / SDS 0.1% (p / v), 68 ° C).

[0090] The present invention provides RNA molecules encoding LmPDE polypeptides. In particular, the RNA molecules of the invention can be isolated in full length or as partial mRNA molecules or RNA oligomers encoding an LmPDE polypeptide. 30

[0091] The nucleic acid molecules of the invention also include derived nucleic acid molecules that differ from DNA or RNA molecules and antisense molecules. Derived molecules include nucleic peptide acids (PNA) and non-nucleic acid molecules that include phosphorothioate, phosphotriester, phosphoramidate and methyl phosphonate molecules, which bind to single-stranded DNA or RNA in a base pair-dependent manner (Zamecnik et al. ( 1978) Proc. Natl. Acad. Sci. USA 75, 280-284; Goodchild et al. (1986) Proc. Natl. Acad. Sci. USA 83, 4143-4146). 35

[0092] Nucleic acid peptide molecules comprise a nucleic acid oligomer to which an amino acid residue, such as lysine and an amino group, has been added. These small molecules (also known as antigenic agents) stop elongation of the transcript by binding to its complementary nucleic acid chain (template) (Nielsen et al., (1993) Anticancer Drug Des. 8, 53-63). Reviews of methods for synthesis of DNA, RNA and their analogs can be found in Oligonucleotides and Analogues (ed. F. Eckstein (1991) IRL Press, New York) and 40 Oligonucleotide Synthesis (ed. MJ Gait (1984) IRL Press, Oxford, England). Additionally, methods for antisense RNA technology are described in U.S. Patent Nos. 5,194,428 and 5,110,802. One skilled in the art can readily obtain these classes of nucleic acid molecules using the LmPDE nucleotide sequences described herein (see, for example, Innovative and Perspectives in Solid Phase Synthesis (1992) Egholm, et al. P. 325-328, or U.S. Patent No. 5,539,082). Four. Five

[0093] Embodiments of LmPDE nucleic acid molecules of the invention include DNA and RNA primers that allow specific amplification of LmPDE sequences or any fragment thereof and probes that selectively or specifically hybridize with LmPDE sequences. or with any fragment thereof. As used herein, amplification refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technology that is well known in the art. (Dieffenbach et al. (1995) PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, NY). Nucleic acid probes can be labeled with a detectable label. Examples of a detectable label include, but are not limited to, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator or an enzyme. The technology for generating labeled DNA and RNA probes is well known in the art (see, for example, Sambrook et al. (1989) in Molecular Cloning). 55

Recombinant Nucleic Acid Molecules Encoding the LmPDE

[0094] The invention also includes recombinant nucleic acid molecules encoding LmPDE polypeptides. Such molecules may have regulatory sequences operably linked to the nucleotide sequences of

LmPDE of the invention.

[0095] The present invention also encompasses recombinant nucleic acid molecules such as DNA molecules (rDNAs) that comprise nucleotide sequences encoding LmPDE polypeptides. As used herein, a rDNA molecule is a DNA molecule that has undergone molecular manipulation in vitro. Methods for generating rDNA molecules are well known in the art (see, for example, Sambrook et al. (1989) Molecular Cloning, mentioned above). In one embodiment of the present invention, the rDNA sequences encoding an LmPDE polypeptide, or fragments thereof, are operably linked to one or more expression control sequences and / or vector sequences.

Vectors that comprise the new LmPDE

[0096] The nucleic acid molecules of the present invention may be recombinant molecules, each comprising the sequence, or part thereof, of an LmPDE nucleotide sequence linked to a non-LmPDE nucleotide sequence. For example, the LmPDE sequence can be operatively linked to a vector to generate a recombinant molecule.

[0097] A possible vector for expression is a vector that replicates autonomously that comprises a replicon that directs the replication of the rDNA within the appropriate host cell. Alternatively, the vector directs the integration of the recombinant vector into a host cell. Various viral vectors, such as several well-known adeno-associated retroviral, adenoviral and viral (AAV) vectors (Berkner (1988) Biotechniques 6, 616-629), can also be used.

[0098] The vectors of the present invention may allow the expression of a transcript or polypeptide sequence of LmPDE in prokaryotic or eukaryotic host cells. Such vectors include expression vectors that comprise an expression control element, described above. Vectors used for the expression of LmPDE nucleotide sequences in eukaryotic host cells may include expression control elements such as the baculovirus polyhedrin promoter for expression in insect cells. Other possible expression control elements include promoters or enhancers derived from plant cell genomes (e.g., heat shock, RUBISCO, storage protein genes), viral promoters or leader sequences of plant viruses and promoters or enhancers of mammalian or mammalian virus genes.

[0099] Specific initiation signals may also be required for efficient translation of the LmPDE nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an LmPDE initiation codon and upstream sequences are inserted into the appropriate expression vector, additional translation control signals may not be necessary. However, in cases where only the coding sequence (or a part thereof) is inserted, exogenous translational control signals that include the ATG initiation codon must be provided. In addition, the initiation codon must be in the correct reading phase to ensure translation of the complete insert. The translational elements and exogenous initiation codons can be of various origins, both natural and synthetic. The effectiveness of the expression can be enhanced by the inclusion of enhancers suitable for the cellular system in use (Scharf et al (1994) Results 35 Probl. Cell. Differ. 20,125-162; Bittner et al. (1987) Methods in Enzymol. 153 , 516-544).

[0100] A possible vector includes at least one selectable indicator gel encoding a gene product that confers drug resistance such as ampicillin or tetracycline resistance. The vector can also comprise multiple endonuclease restriction sites that allow convenient insertion of exogenous DNA sequences. Methods for generating a recombinant expression vector encoding an LmPDE protein of the invention are well known in the art (see, for example, Sambrook et al. (2001) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York NY).

[0101] The vectors of the present invention for generating transcripts of LmPDE and / or encoded LmPDE polypeptides may be expression vectors that are compatible with prokaryotic host cells. Prokaryotic cell expression vectors 45 are well known in the art and are available from various commercial sources. For example, pET vectors (eg, pET-21, Novagen Corp.), pQE vectors (Qiagen, Chatsworth, CA), phagemid BLUESCRIPT (Stratagene, LaJolla, CA), pSPORT (Gibco BRL) or ptrp- hybrids can be used. lac to express LmPDE polypeptides in bacterial host cells.

[0102] Alternatively, the expression vectors of the present invention for generating transcripts of LmPDE and / or the 50 encoded LmPDE polypeptides may be expression vectors that are compatible with eukaryotic host cells, such as vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Such vectors may contain convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-1 / pML2d (International Biotechnologies, Inc.), pTDT1 (ATCC, No. 31255) and similar eukaryotic expression vectors.

Host-Vector Systems that comprise LmPDE

[0103] The invention further provides a host-vector system comprising a vector, plasmid, phagemid or cosmid comprising a nucleotide sequence of LmPDE, or a fragment thereof, introduced into a suitable host cell. A variety of vector / host expression systems can be used to carry and express LmPDE nucleotide sequences. The host-vector system can be used to express (for example, produce) LmPDE polypeptides encoded by LmPDE nucleotide sequences. The host cell can be prokaryotic or eukaryotic. Examples of suitable prokaryotic host cells include bacterial strains of genera such as Escherichia, Bacillus, Pseudomonas, Streptococcus, and Streptomyces. Examples of suitable eukaryotic host cells include yeast cells, plant cells or animal cells such as mammalian cells and insect cells. Several possible embodiments provide a host-vector system 10 comprising the pcDNA3 vector (Invitrogen, Carlsbad, CA) in COS-7 mammalian cells, the pGEX vector (Promega, Madison, Wl) in bacterial cells, the vector pLT1 (S Kunz, unpublished) in Saccharomyces cerevisiae cells or the pFastBac HT (Gibco / BRL) baculovirus vector in Sf9 insect cells (ATCC, Manassas, VA).

[0104] The introduction of the recombinant DNA molecules of the present invention into an appropriate host cell can be achieved by well known methods that depend on the type of vector and host system employed. For example, prokaryotic host cells (for example, transformed) with nucleic acid molecules are introduced by electroporation or salt treatment methods (see, for example, Cohen et al. (1972) Proc Natl Acad Sci USA 69, 2110; Sambrook et al. (1989) Molecular Cloning, mentioned above). Vertebrate cells can be transformed with vectors containing recombinant DNA by various methods, including electroporation or treatment with cationic lipids or salt (Graham et al. (1973) Virol. 52,456; Wigler et al. (1979) Proc. Natl. Acad. Sci. USA 76,1373-1376).

[0105] Successfully transformed cells, that is, cells containing a rDNA molecule of the present invention, can be identified by techniques well known in the art. For example, cells resulting from the introduction of recombinant DNA of the present invention are selected and cloned to produce unique colonies. The 25 cells of these colonies are collected, lysed and their DNA content is examined for the presence of the rDNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503, or Berent et al. (1985) Biotech. 3, 208. The proteins produced from the cell can also be assayed by a biochemical assay or an immunological method.

[0106] In bacterial systems, several expression vectors may be selected depending on the intended use for LmPDE polypeptides. For example, when large amounts of LmPDE are needed for antibody induction, vectors that direct a high level of fusion protein expression that are soluble and easily purifiable may be desirable. Such vectors include, but are not limited to, cloning and expression vectors of multifunctional E. coli such as BLUESCRIPT (Stratagene, San Diego, CA), in which a nucleotide sequence of LmPDE may be phase linked with sequences for the Met amino terminal and the 7 subsequent residues of 35-35 galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J. Biol. Chem. 264, 5503-5509) and the like. The pGEX vectors (Promega, Madison, Wl) can also be used to express LmPDE polypeptides as fusion proteins with glutathione S-transferase (GST). In general, said fusion proteins are soluble and can be easily purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins prepared in such systems are designed to include cleavage sites of heparin, thrombin or factor XA protease so that the cloned protein of interest can be released from the rest of GST at will.

[0107] In yeast (Saccharomyces cerevisiae) several vectors containing constitutive or inducible promoters such as beta factor, alcohol oxidase and PGH can be used. For reviews, see Ausubel et al. (mentioned above) and Grant et al. (1987) Methods in Enzymology 153, 516-544. Four. Five

[0108] In cases where expression vectors are used in plants, the expression of a sequence encoding an LmPDE polypeptide can be directed by several different promoters. For example, viral promoters such as CaMV 35S and 19S promoters (Brisson et al. (1984) Nature 310, 511-514) can be used alone or in combination with the TMV omega leader sequence (Takamatsu et al. (1987) EMBO J 6, 307-311). Alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al (1984) EMBO J 3, 1671-1680; 50 Broglie et al. (1984) Science 224, 838-843), or heat shock promoters can be used (Winter et al. (1991) Results Probl. Cell Differ. 17, 85-105). These constructs can be introduced into plant cells by transformation with direct DNA or pathogen-mediated transfection. For reviews of these techniques, see Hobbs (1992) McGraw Yearbook of Science and Technology, McGraw Hill New York N. Y., p. 191-196; or Weissbach and Weissbach (1988) Methods for Plant Molecular Biology, Academic Press, New York N. Y., p. 421-463. 55

[0109] An alternative expression system that can be used to express LmPDE polypeptides is an insect system. In such a system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia larvae (Smith et al. (1983) J. Virol. 46, 584; Engelhard et al. (1994) Proc. Nat. Acad. Sci. USA 91, 3224-3227). The sequence encoding an LmPDE polypeptide can be cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the virus.

polyhedrin promoter. The successful insertion of a nucleotide sequence of LmPDE will make the polyhedrin gene inactive and will produce recombinant viruses that lack coat protein. Recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which the LmPDE polypeptide is expressed.

[0110] In mammalian host cells, several virus-based expression systems are used. In cases where an adenovirus is used as an expression vector, a nucleotide sequence of LmPDE is linked to an adenovirus translation / translation vector consisting of the late promoter and the tripartite leader sequence. Insertion into a non-essential E1 or E3 region of the viral genome results in a viable virus (Logan et al. (1984) Proc Natl. Acad. Sci. USA 81, 3655-3659) capable of expressing an LmPDE protein in cells Infected hosts In addition, transcription enhancers, such as the rous sarcoma virus (RSV) enhancer, can be used to increase expression in mammalian host cells. 10

[0111] A strain of host cells can also be selected for their ability to modulate the expression of inserted LmPDE nucleotide sequences or to process the expressed LmPDE polypeptide in a particular way. Such modifications of LmPDE polypeptides include, without limitation, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation. Post-translational processing that cleaves a precursor form of the protein may also be important for proper insertion, folding and / or function. Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc. They have specific cellular machinery and characteristic mechanisms for such post-translational activities and can be selected to ensure the correct modification and processing of the introduced foreign protein.

[0112] For long-term high yield production of recombinant proteins, stable expression is preferred. For example, cell lines that stably express LmPDE polypeptides are transformed using 20 expression vectors containing viral replication sources or endogenous expression elements and a selectable reporter gene. After the introduction of the vector, the cells are cultured in an enriched medium before switching to selective medium. The purpose of the selectable marker is to confer resistance to selection and its presence allows the growth and recovery of cells that successfully express the introduced sequences. Resistant groups of stably transformed cells can be made to proliferate using tissue culture techniques appropriate to the type of cells used.

[0113] Any number of selection systems can be used to recover transformed cell lines. These include, without limitation, the herpes simplex virus (tk) thymidine kinase genes (Wigler et al. (1977) Cell 11, 223-232) or adenine phosphoribosyltransferase (aprt) (Lowy et al. (1980 ) Cell 22, 817-823) which can be used in tk-less or aprt-minus cells, respectively. Resistance to antimetabolites, antibiotics or herbicides can also be used as the basis for selection. Examples include: dhfr that confers resistance to methotrexate (Wigler et al. (1980) Proc Natl Acad Sci USA 77, 3567-3570); npt, which confers resistance to aminoglycoside neomycin and G-418 (Colbere-Garapin et al. (1981) J. Mol. Biol. 150,1-14) and also pat, which confers resistance to chlorosulfaron and phosphinothricin acetyltransferase, respectively (Murry, mentioned above).

[0114] Additional selectable genes have been described, for example UpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol instead of histidine (Hartman et al. (1988) Proc Natl. Acad. Sci. USA 85, 8047-8051). Recently, the use of visible markers has gained popularity. Such markers include anthocyanins, -glucuronidase and its substrate, GUS and luciferase and its substrate, luciferin. These markers are widely used not only to identify transformants but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes et al. (1995) Methods 40 Mol. Biol. 55,121-131).

Reactive Antibodies Against LmPDE Polypeptides

[0115] The present invention also provides antibodies that bind to the LmPDE of the invention. These antibodies can be used for diagnostic and therapeutic purposes.

[0116] The invention provides antibodies, such as polyclonal, monoclonal, chimeric, humanized and human antibodies, as well as fragments thereof (eg, Fab), which bind to LmPDE polypeptides. Such antibodies can selectively bind to an LmPDE polypeptide but will not bind (or weakly bind) a protein other than LmPDE. These antibodies can be from any source, for example, rabbit, sheep, rat, dog, cat, pig, horse, mouse and human.

[0117] As those skilled in the art will understand, epitopes of an LmPDE polypeptide to which an antibody is directed may vary with the intended application. LmPDE polypeptides can be targets for therapeutic methods such as targeted antibody therapy and immunotherapy to treat conditions associated with the presence or absence of an LmPDE of the invention. Additionally, some of the antibodies of the invention may be internalization antibodies, which are internalized (eg, enter) in the cell before or after binding. Internalization antibodies are useful for inhibiting cell growth and / or inducing cell death and for detecting or directing LmPDEs within damaged or dying cells.

[0118] The invention also encompasses antibody fragments that specifically recognize an LmPDE polypeptide. As used herein, an antibody fragment is defined as at least a portion of the

variable region of the immunoglobulin molecule that binds to its target, that is, the antigen binding region. Part of the constant region of immunoglobulin may be included. Monoclonal antibody fragments or polyclonal antisera include Fab, F (ab ') 2, Fv fragments, single chain antibodies and fusion proteins that include the immunologically significant part (i.e., a part that recognizes and binds to an LmPDE).

[0119] The chimeric antibodies of the invention may be immunoglobulin molecules comprising at least 5 two antibody parts of different species, for example a human and non-human part. The invention also provides chimeric antibodies that have different effector functions (Neuberger et al. (1984) Nature 312, 604), immunoglobulin constant regions of other species and constant regions of another immunoglobulin chain (Sharon et al. (1984) Nature 309 , 364; Tan et al. (1985) J. Immunol. 135, 3565-3567). Additional procedures for modifying antibody molecules and for producing chimeric antibody molecules using homologous recombination for target gene modification have been described (Fell et al. (1989) Proc. Natl. Acad. Sci. USA 86, 8507-8511).

[0120] Humanized antibodies directed against LmPDE polypeptides are also useful. Humanized antibodies can be prepared according to several methods known in the art (Teng et al. (1983) Proc. Natl. Acad. Sci. USA 80, 7308-7312; Kozbor et al. (1983) Immunology Today 4, 7279; Olsson et al. (1982) Meth. Enzymol. 92, 3-16).

[0121] Various methods for the preparation of antibodies are well known in the art. For example, antibodies can be prepared by immunization of a suitable mammalian host with an immunogen such as an isolated LmPDE polypeptide (Harlow (1989) Antibodies, Cold Spring Harbor Press, NY). In addition, LmPDE proteins can also be used as immunogens, such as an LmPDE fused with GST, human Ig or His-labeled fusion proteins. Cells that express or overexpress LmPDE polypeptides can also be used for immunizations. This strategy can result in the production of monoclonal antibodies with 20 enhanced capacities for the recognition of endogenous LmPDE polypeptides (Harlow et al. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press).

[0122] The invention also provides human antibodies. There are several well-known strategies that one skilled in the art could use to produce human recombinant antibodies. One is the generation of antibodies using phage display technologies (Low et al. (1996). J Mol Biol 260 (3): 359-368; Winter et al. (1994). Annu 25 Rev Immunol 12: 433-455 ). Specifically, human RNA is used to produce a cDNA library of heavy and light chain fragments of antibodies expressed on the surface of a bacteriophage. These libraries can be used to explore the antigen of interest. The binding phage, due to an antibody expressed on the surface, can then be isolated. The DNA encoding the variable regions is sequenced and cloned for antibody expression. 30

[0123] Another method of producing human antibodies employs "humanized" mice. The genes of these transgenic mice themselves have been replaced with a part of the human antibody gene complex so that after inoculation with an antigen they produce human antibodies (Green et al. (1994) Nat. Genet. 7: 13- 21; Low et al. (1996). J Mol Biol 260 (3): 359-368; Wagner et al. (1994) Eur. J. Immunol. 24 (11): 2672-2681; Wagner et al. (1994 ) Nuc. Acids Res. 22 (8): 1389-1393; Winter et al. (1994) Annu Rev. Immunol. 12: 433-455). The resulting antibody producing cells can then be incorporated into conventional hybridoma technology for the establishment of specific monoclonal antibody producing cell lines.

[0124] Recombinant human antibodies are also produced by isolation of B lymphocytes producing antibodies from human volunteers that have a robust response against antigen of interest. Using fluorescence activated cell selection (FACS) and fluorescence labeled antigen, cells that produce antibodies directed against the antigen from the other cells can be separated. The RNA can then be extracted and the sequence of the variable regions of the reactive antibody determined (Kantor et al. (1995) Ann. NY Acad. Sci. 764: 224-227, Wang et al. (2000) J. Immunol. Methods 244: 217-225). The DNA sequence of the functional variable regions can be synthesized or cloned into mammalian expression vectors to produce large-scale human recombinant antibodies. Four. Five

[0125] The amino acid sequence of LmPDE polypeptides can be used to select specific regions of an LmPDE protein to generate antibodies. For example, hydrophobicity and hydrophilicity analysis of an amino acid sequence of LmPDE can be used to identify hydrophilic regions in a protein structure of LmPDE. The regions of an LmPDE polypeptide that show immunogenic structure, as well as other regions and domains, can be easily identified using various different methods known in the art such as Chou-50 Fasman, Garnier-Robson, Kyte-Doolittle, Eisenberg, Karplus- analyzes. Schultz or Jameson-Wolf (Rost et al. (1994) Protein 19, 55-72).

[0126] Methods for preparing a protein for use as an immunogen and for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH or other carrier proteins are well known in the art. Techniques for conjugating or binding therapeutic agents to antibodies are well known (Amon et al. (1985) 55 "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (Eds.) P. 243-56, Alan R. Liss, Inc; Hellstrom et al. (1987) "Antibodies For Drug Delivery", Controlled Drug Delivery (2nd Ed.), Robinson et al. (Eds.) Pp. 623-53, Marcel Dekker , Inc .; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", Monoclonal Antibodies '84 - Biological And Clinical

Applications, Pinchera et al. (eds.) p. 475-506; Thorpe et al. (1982) "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62,119-158; and Sodee et al. (1997), Clin. Nuc. Med. 21, 759-766). In some circumstances, for example, direct conjugation can be used using carbodiimide reagents; in other cases binding reagents such as those provided by Pierce Chemical Co., Rockford, IL may be effective.

[0127] Administration of an LmPDE immunogen is generally performed by injection for a suitable period of time and with the use of a suitable adjuvant, as generally understood in the art. (Harlow et al. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press). During the immunization program, antibody titres can be taken to determine if antibody formation is sufficient.

[0128] Although polyclonal antisera produced in this way may be satisfactory for some applications, monoclonal antibody preparations are preferred for pharmaceutical compositions. Immortalized cell lines 10 that secrete a desired monoclonal antibody can be prepared using the conventional method of Kohler and Milstein (Nature 256, 495-497) or modifications that effect immortalization of lymphocytes or spleen cells, as is generally known in the art. Immortalized cell lines that secrete the desired antibodies are screened by immunoassay in which the antigen is a LmPDE polypeptide. When the appropriate immortalized cell culture secreting the desired antibody is identified, the cells can be cultured in vitro or by ascites production. The desired monoclonal antibodies are recovered after the culture supernatant or ascites fluid supernatant.

[0129] Antibodies or fragments can also be produced by recombinant means. Antibody regions that specifically bind to the desired regions of an LmPDE polypeptide can also be produced in the context of chimeric antibodies of multiple species origin. twenty

[0130] The antibodies of the invention can specifically bind to LmPDE polypeptides. In one embodiment, the LmPDE antibodies can specifically bind to the GAF domain of an LmPDE protein. In another embodiment, the antibodies of the invention can specifically bind to the C-terminal domain of an LmPDE protein. In a further embodiment, the antibodies can specifically bind to the PDE catalytic domain of an LmPDE polypeptide. In other embodiments, the antibodies of the present invention can bind to other domains of an LmPDE polypeptide, for example the antibodies can bind to the N-terminal domain of an LmPDE polypeptide.

Use of Antibodies Against LmPDE

[0131] LmPDE polypeptides can be used to induce the generation of antibodies, including fragments, that specifically bind to an epitope associated with an LmPDE polypeptide, using methods described herein (Kohler et al. Mentioned above). Antibodies that are immunoreactive with selected domains or regions of an LmPDE polypeptide are particularly useful. In one embodiment, LmPDE antibodies are used to explore expression libraries to obtain polypeptides related to LmPDE polypeptides (eg, homologs).

[0132] In another embodiment, LmPDE antibodies are used to enrich or purify LmPDE polypeptides from a sample that has a heterologous population of polypeptides. Enrichment and purification methods include conventional techniques, such as immunoaffinity methods. In general, immunoaffinity methods include the following steps: preparing an affinity matrix by ligating a solid support matrix with an LmPDE antibody, the affinity matrix specifically binding with an LmPDE polypeptide; contacting the linked affinity matrix with the sample under conditions that allow an LmPDE polypeptide in the sample to bind to the linked affinity matrix; remove non-LmPDE polypeptides that did not bind the bound affinity matrix, thereby enriching or purifying an LmPDE polypeptide. An additional step may include eluting an LmPDE polypeptide from the affinity matrix. The general stages and conditions for affinity enrichment for a desired protein or protein complex can be found in Antibodies: A Laboratory Manual (Harlow et al. (1988) CSHL, Cold Spring, NY).

[0133] In addition, there are multiple diagnostic uses of the antibodies of the invention. The invention provides methods for diagnosing in a subject, for example, an animal or a human subject, a disease associated with the presence or deficiency of at least one LmPDE polypeptide. In one embodiment, the method comprises quantitatively determining the amount of at least one LmPDE polypeptide in the sample (eg, cell or biological fluid sample) using any one or a combination of the antibodies of the invention. The amount determined in this way can then be compared with the amount in a sample of a normal subject. The presence of an appreciably different amount in the sample (ie, the amount of an LmPDE polypeptide in the test sample exceeds or is reduced from the amount of an LmPDE polypeptide in a normal sample) indicates the presence of the disease. .

[0134] The anti-LmPDE antibodies of the invention may also be useful in diagnostic imaging methodologies, in which the antibodies have a detectable label. The invention provides various immunological assays useful for the detection of LmPDE polypeptides in a suitable biological sample. Suitable detectable markers include, without limitation, a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a chromophore, a metal chelator, biotin or an enzyme. Sayings

Assays generally comprise one or more labeled LmPDE antibodies that recognize and bind to an LmPDE polypeptide and include various immunological assay formats well known in the art, including without limitation various types of precipitation, agglutination, complement fixation, radioimmunoassays (RIA ), enzyme-linked immunoabsorption assays (ELISA), enzyme-linked immunofluorescent assays (ELIFA) (H. Liu et al. (1998) Cancer Research 58, 4055-4060), immunohistochemical analyzes and the like. 5

Methods to Generate LmPDE Polypeptides

[0135] The LmPDE polypeptides of the present invention can be generated by chemical synthesis or by recombinant methods. If high performance is desired, recombinant methods can be used, as discussed above. The LmPDE polypeptides of the invention can also be generated by chemical synthetic methods. The principles of chemical solid phase synthesis of polypeptides are well known in the art and can be found in 10 general texts related to this area (see, for example, Dugas et al. (1981) Bioorganic Chemistry, pp. 54-92, Springer-Verlag, New York).

[0136] The present invention also provides derived polypeptide molecules, such as chemically modified LmPDE polypeptides. Illustrative of such modifications is the replacement of hydrogen with an alkyl, acyl or amino group. LmPDE polypeptide derivatives retain the biological activities of LmPDE of natural origin.

Exploration of Compounds that Modulate the Activity / Expression of LmPDE

[0137] The LmPDE polypeptides of the present invention are Leishmania phosphodiesterases, such as Leishmania major, which function as key components in the regulation of intracellular cAMP levels by catalysis of their hydrolysis. Together with adenylyl cyclase, these phosphodiesterases ultimately control the 20 biological responses mediated by messenger cAMP. The regulation of intracellular cAMP levels is crucial in the processes of transformation and cell proliferation. Therefore, LmPDE polypeptides are important targets for compounds that modulate their biological activity or that modulate their expression. Compounds that effectively modulate the biological functions of LmPDE can serve as important therapeutic agents for the treatment of parasitic diseases such as leishmaniasis. The invention also provides a method for obtaining compounds that modulate the activity or expression of LmPDE.

[0138] The present invention relates to scanning methods for identifying compounds that bind to LmPDE polypeptides (eg ligands) and modulate the biological activity of LmPDE polypeptides. These scanning methods can also identify compounds that do not necessarily bind directly to LmPDE, but nevertheless modulate the activity of LmPDE. Such scanning methods can also be used to identify compounds that modulate the expression of LmPDE polypeptides.

[0139] Typically, the objective of the scanning methods is to identify compounds that bind to the target LmPDEs and cause changes in the biological activity of the target nucleic acid polypeptide or molecule. The compounds of interest are identified from a formulation of candidate compounds. For example, a compound that effectively binds to the target nucleic acid molecule can reduce the expression of the LmPDE polypeptide and thereby reduce the proliferation of cells expressing LmPDE polypeptides. Reduction of proliferation of cells expressing LmPDE polypeptides may be an effective method to treat diseases associated with infection of parasites such as Leishmania major.

[0140] Several assays and scans can be used to identify compounds that modulate the activity and / or expression of LmPDE. The compounds identified in the tests and explorations can modulate the activity of 40 LmPDE in various ways. For example, the compounds may bind directly to an LmPDE polypeptide or may bind to intracellular proteins that bind to an LmPDE. The compounds can also modulate the activity of an LmPDE gene or modulate the expression of an LmPDE gene or an LmPDE polypeptide. For example, such compounds can bind to an LmPDE regulatory sequence and thereby modulate gene expression (see, for example, Piatt (1994) J. Biol. Chem. 269, 28558-28562). Four. Five

[0141] Compounds that can be screened by the methods described in the present invention include, without limitation, peptides and derivatives thereof (eg peptidomimetics) that bind to an LmPDE polypeptide or otherwise modulate their activity in any way. . Such compounds may include peptides, such as soluble peptides, including members of random peptide libraries (Lam et al. (1991) Nature 354, 82-84; Houghten et al. (1991) Nature 354, 84-86), and libraries molecules derived from combinatorial chemistry composed of 50 amino acids D and / or L, phosphopeptides (including, without limitation, members of phosphopeptide libraries directed partially degenerated or randomized; see, for example Songyang et al. (1993) Cell 72, 767-778 ), organic and inorganic small carbohydrates and molecules. Compounds that can be explored include, without limitation, natural and synthetic products. One skilled in the art can readily recognize that there is no limit with respect to the structural nature of the compounds tested with respect to their binding to LmPDE polypeptides. 55

[0142] The candidate compounds that are tested for their binding to LmPDE polypeptides and / or modulation of LmPDE polypeptide activity can be randomly selected or rationally selected. As used herein, it is said that a compound is randomly selected when the compound is

randomly selects without considering the specific sequences of the LmPDE polypeptide or nucleic acid. Examples of randomly selected agents are members of a chemical library, a combinatorial peptide library, a culture broth of an organism or plant extract.

[0143] As used herein, it is said that a compound is rationally selected when the compound is selected non-randomly that is based on the sequence of the target site and / or its conformation in relation to the action of the compound . The compounds can be rationally selected using the peptide sequences that make up the LmPDE polypeptide or by analyzing the nucleotide sequence encoding an LmPDE polypeptide.

[0144] Methods for rationally selecting a compound that modulates the activity and / or expression of an LmPDE polypeptide include computer modeling or search techniques. For example, 10 compounds that probably interact with the active site of an LmPDE polypeptide are identified. The active site of an LmPDE polypeptide can be identified using methods known in the art including, for example, analysis of the amino acid sequence of a molecule and from a study of complexes formed by an LmPDE polypeptide and a native substrate (for example CAMP). Methods such as X-ray crystallography and NMR can be used to resolve the three-dimensional structure of a protein to identify possible binding sites, including the active site of the natural substrate.

[0145] Computer-based model formation can be used to complete an incomplete or insufficiently accurate structure. Modeling methods that can be used are, for example, models with specific set parameters for particular biopolymers such as proteins or nucleic acids, molecular dynamics modeling based on molecular motion calculations, statistical mechanical models based on thermal assemblies or combined models. For most types of models, conventional molecular force fields, representing the forces between atoms and constituent groups, are necessary and can be selected from force fields known in the art. Information on incomplete or less precise structures determined as indicated above can be incorporated as constraints on the structures calculated by these modeling methods. 25

[0146] Once the structure of the active site of an LmPDE polypeptide has been determined, candidate modulation compounds can be identified by searching databases containing compounds together with information on their molecular structure. The compounds identified in such a search are those that have structures that match the active site structure, fit the active site or interact with groups that define the active site. The compounds identified by the search are potential LmPDE modulating compounds. 30

[0147] These methods can be used to identify improved modulating compounds based on compounds known to modulate other PDEs. The structure of the known compound is modified and modulatory effects are determined using experimental and computer methods as described herein. The altered structure is compared with the active site structure of an LmPDE polypeptide to determine or predict how a particular modification of the compound will affect its interaction with that protein. Systematic variations in the composition, such as varying side groups, can be evaluated to obtain modified modulating compounds of specificity or preferred activity.

[0148] Examples of molecular modeling systems are the QUANTA programs, for example, CHARMm, MCSS / HOOK and X-LIGAND (Molecular Simulations, Inc., San Diego, Ca.). QUANTA provides a modeling environment for the simulation and analysis of macromolecules and small organic molecules. 40

[0149] The process of using experimental or predicted structural information in a computer simulation to predict the interactions of potential modulating compounds is well known in the art. For example, see Rotivinen et al. (1988) Acta Pharm. Fenn 97, 159-166; and McKinaly et al. (1989) Ann. Rev. Pharmacol. Toxicol 29, 111-122. Computer programs designed to explore and represent chemical compounds are available from companies such as MSI, Allelix, Inc., and Hypercube, Inc. These applications are designed largely for specific drugs of particular proteins; however, they can be adapted to the design of specific drugs for identified regions of DNA or RNA. Commercial sources of chemical libraries can be used as sources of candidate compounds. Such chemical libraries can be obtained from, for example, ArQule, Inc.

[0150] Compounds that modulate the activity and / or expression of an LmPDE polypeptide may also be based on antisense constructs. Therapeutic techniques based on an antisense approach involve the design of oligonucleotides that are complementary to LmPDE mRNAs. These oligonucleotides bind to complementary transcripts and prevent translation. Absolute (or total) complementarity is not required. An oligonucleotide can function as an effective antisense construct as long as its sequence is sufficiently complementary to be able to hybridize with RNA and form a stable double chain. In the case of a double stranded antisense nucleic acid molecule, a single strand of the double strand can be tested or a triple strand formation can be tested. The ability to hybridize will depend on the degree of complementarity and the length of the antisense nucleic acid. In general, the longer the nucleic acid that hybridizes, the more mismatches of bases with an RNA can contain and still form a stable double chain (or triple chain, as the case may be). A person skilled in the art can determine a tolerable degree of mismatch through the use of procedures

conventional to determine the melting point of the hybridized complex.

[0151] In addition, several in vitro assays can be used to identify compounds that modulate the expression and / or activity of an LmPDE polypeptide. Such assays typically involve the preparation of a reaction mixture comprising an LmPDE polypeptide and a test compound under conditions sufficient to allow the two components to interact and bind, thereby forming a complex that can be detected and / or isolated. Binding of a compound with an LmPDE polypeptide can be assayed using a change in molecular weight or a change in the biological activity of the unbound LmPDE polypeptide or expression of a reporter gene in a two yeast hybrid system (Fields et al. (1989) Nature 340, 245-246). The method used to identify whether a compound binds to an LmPDE polypeptide will be based primarily on the nature of the LmPDE polypeptide used. For example, a gel delay assay can be used to determine if a compound binds to an LmPDE polypeptide. Alternatively, immunodetection and biochip technologies (eg, US Patent No. 4,777,019) can be adopted for use with an LmPDE polypeptide. An alternative method to identify compounds that bind to an LmPDE polypeptide employs TLC overlay assays using glycolipid extracts from immune cells (Abdullah et al. (1992) Infect. Immunol. 60, 56-62). In addition, a reduction in the cAMP hydrolytic activity of LmPDE can be measured to determine whether or not a particular compound inhibits an LmPDE. One skilled in the art can readily employ numerous techniques known in the art to determine if a particular compound binds to an LmPDE polypeptide of the invention. Such assays will typically use a control cell.

[0152] It is also possible to use cell-based assays to identify compounds that interact with LmPDE polypeptides. Cell lines that naturally express LmPDE or that have been engineered to express LmPDE can be used. For example, test compounds can be added to 20 cell cultures after which cAMP hydrolysis can be measured using conventional techniques known in the art. A reduction in the amount of hydrolysis in the presence of a test compound compared to control cells that do not contain the test compound indicates that the test compound is an inhibitor of LmPDE activity.

[0153] LmPDE expression inhibitors can be identified by the use of a chimeric gene in which a nucleotide sequence of LmPDE is fused to an indicator, such as firefly luciferase. Cultured cells that have been transformed with the chimeric gene can be screened for the expression of luciferase activity in the presence of test compounds. Compounds that inhibit luciferase activity in this high-performance assay can also be confirmed by direct measurement with respect to the presence of the endogenous LmPDE polypeptide (for example, by Western blotting) and LmPDE mRNA (e.g., by transferring Northern) using methods that are well known in the art (see, for example, Ausubel et al. (1994) Current Protocols in Molecular Biology, John Wiley & Sons). Candidate compounds can be further tested in cell cultures or titres as well as in animal models. For example, cells expressing an LmPDE polypeptide can be incubated with a test compound, after which cell lysates are prepared and screened for the presence of the LmPDE polypeptide (for example, using Western blot transfer techniques. ). A reduction in the amount of LmPDE expression in cultures treated with the test compound compared to control cultures without the test compound indicates that the test compound is an inhibitor of LmPDE expression.

[0154] In vivo assays can also be used to test these compounds in animal models of Leishmania infection. Test compounds that are predicted to inhibit the activity and / or expression of LmPDE are administered to the animals. The treated animals can then be tested for the inhibition of LmPDE activity. Such tests may be indirect or inferential. Improved health, for example, may indicate the efficacy of a test compound. Direct assays can also be performed when a reduction in LmPDE expression can be measured by Northern blot analysis. A reduction in the amount of LmPDE mRNA present in the sample of treated animals compared to untreated control animals indicates that the test compound inhibits the expression of LmPDE. A direct test that measures the hydrolytic activity of an LmPDE in cAMP can also be performed. A reduction in cAMP hydrolysis in the sample of treated animals compared to untreated control animals indicates that the test compound inhibits the activity of LmPDE.

[0155] The LmPDE polypeptides that are used in the screening assays described herein include, without limitation, an isolated LmPDE polypeptide, a host cell that expresses a 50 LmPDE polypeptide or a fraction of a host cell that expresses a polypeptide of LmPDE.

[0156] The cell extracts to be tested for binding with LmPDE polypeptides and / or modulation of LmPDE polypeptide activity may be, for example, aqueous cell or tissue extracts, organic cell or tissue extracts or partially cellular fractions. purified. One skilled in the art can readily recognize that there is no limit with respect to the source of cell extracts used in the scanning methods of the present invention.

[0157] Compounds that are identified as candidates for inhibiting the activity and / or expression of an LmPDE polypeptide, when administered in a therapeutically effective amount, may be useful for treating diseases and reducing symptoms associated with Leishmania infection, such as leishmaniasis The toxicity and therapeutic efficacy of identified compounds that modulate the activity and / or expression of an LmPDE polypeptide can be determined.

by conventional pharmaceutical procedures. For example, using cells in culture or experimental animals, the lethal dose can be determined for 50% of the population (LD50). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compounds with a high therapeutic index are preferred. Although compounds that have toxic side effects may be used, care should be taken to design a delivery system that directs such compounds to the site of affected tissue to minimize potential damage to uninfected cells and thereby reduce side effects.

[0158] Data obtained from cell culture assays and animal studies can be used in the formulation of a series of dosages for use in humans. The dosage of said compounds is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration 10 used. For any compound used in the method of the present invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a concentration range in circulating plasma that includes IC50 as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography. fifteen

Generation of transgenic organisms

[0159] Another aspect of the invention provides transgenic organisms comprising LmPDE nucleic acids. As used herein, a genetically modified organism refers to an organism that has been altered from its natural state by manipulation of the native nucleic acid sequences. For example, in one application, PDE-deficient organisms can be generated using conventional knock-out methods 20 to inactivate an endogenous PDE. Alternatively, inducible PDE antisense molecules can be used to regulate the activity and / or expression of endogenous PDE. An organism can also be produced to contain a nucleic acid molecule encoding the LmPDE or an antisense LmPDE expression unit that directs the expression of an LmPDE polypeptide or the antisense molecule. In such cases, an organism is generated in which the expression of the endogenous PDE gene is altered by inactivation and / or replaced by an LmPDE gene. This can be achieved using a variety of methods known in the art such as directed recombination. Once generated, the endogenous PDE-deficient organism that expresses an LmPDE polypeptide can be used to (1) identify biological and pathological processes mediated by LmPDE polypeptides, (2) identify proteins and other genes that interact with LmPDE polypeptides, (3 ) identify compounds that can be provided exogenously to inhibit an LmPDE polypeptide and (4) serve as an appropriate screening to identify mutations within the LmPDE genes that increase or reduce activity.

[0160] For example, in one embodiment the endogenous PDE genes in S. cerevisiae can be suppressed, which results in an intracellular accumulation of cAMP. Organisms that accumulate high levels of intracellular cAMP stop growing when exposed to thermal shock. LmPDE nucleic acid molecules can then be cloned into a yeast expression vector and transfected into the PDE-deficient strain in S. Cerevisiae. The heat-insensitive growth restoration is thus an indicator of LmPDE activity. The observation of whether heat-insensitive growth is restored or not in the strain transfected under various conditions may indicate the effects that these conditions have on the activity of the LmPDE.

Uses of Polypeptides in LmPDE

[0161] As discussed previously, the present invention provides specific PDEs of Leishmania cAMP, such as Leishmania major, including LmPDE-A, LmPDE-B1 and LmPDE-B2 and fragments, variants and mutants thereof. It is known that cAMP plays a key role in cell growth and differentiation in this parasite and that PDEs are responsible for the hydrolysis of this messenger. Therefore, as discussed above, LmPDEs are targets for drug screening assays and are useful for achieving a selective drug design.

[0162] Additionally, the invention provides methods for controlling the course of the disease or disorders associated with the presence of LmPDE in a test subject by measuring the amount of a LmPDE polypeptide in a sample of the test subject in various points in time. This is done in order to determine a change in the amount of an LmPDE over time. The control of the course of a disease or disorder over time can optimize the timing, dosage and type of treatment. In one embodiment, the method comprises quantitatively determining in a first sample of the subject the presence of a LmPDE polypeptide and comparing the amount determined in this way with the amount present in a second sample of the same subject taken at a different point in time. , being a difference in the determined amounts indicative of the course of the disease. The measurement of the amount of LmPDE polypeptide present in a sample can be performed using a variety of techniques well known in the art, for example, by the use of immunoassays as discussed below. 55

[0163] The present invention further provides methods for using isolated and substantially purified LmPDE polypeptides as antigens for the production of new anti-LmPDE antibodies and for using LmPDE polypeptides to obtain and detect new LmPDE ligands. Anti-LmPDE antibodies are useful in assays and diagnostic kits for the detection of naturally occurring LmPDE protein sequences present in

biological samples.

Uses of Nucleic Acid Molecules Encoding LmPDE

[0164] Nucleic acid molecules encoding LmPDE polypeptides of the invention are useful for a variety of purposes, including their use in diagnosis and / or prognostic methods. The nucleic acid molecules and polypeptides of the invention can be used to test for the presence and / or amount of 5 nucleotide sequences of LmPDE and LmPDE polypeptides in a suitable biological sample.

[0165] The nucleic acid molecules of the present invention can be used in various hybridization methods to identify and / or isolate nucleotide sequences related to LmPDE nucleotide sequences, such as different polymorphic forms and genomic sequences. Sequences related to a nucleotide sequence of LmPDE are useful for the development of additional ligands and antibodies. Hybridization methods are used to identify / isolate DNA and RNA sequences that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97% , 98% or 99% identical to LmPDE nucleotide sequences, such as new homologs, allelic variants and mutant forms of LmPDE.

[0166] Nucleotide sequences encoding LmPDE polypeptides described herein can be used as nucleic acid probes to recover nucleic acid molecules having sequences related to LmPDE.

[0167] In one embodiment, an LmPDE nucleic acid probe is used to explore genomic libraries, such as lambda phage-constructed libraries or BAC (artificial bacterial chromosomes) or YAC (artificial yeast chromosomes), to isolate a genomic clone of an LmPDE gene. LmPDE nucleotide sequences from genomic libraries are useful for isolating non-coding sequences upstream or downstream, such as promoter, enhancer and transcriptional determination sequences. The upstream sequences of an LmPDE gene can bind to non-LmPDE sequences to construct a recombinant DNA molecule that expresses the non-LmPDE sequence upon introduction into an appropriate host cell. In another embodiment, an LmPDE probe is used to explore cDNA libraries to isolate cDNA clones expressed in certain types of tissues or cells. 25

[0168] Additionally, pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively amplify or clone nucleic acid molecules encoding LmPDE proteins or fragments thereof. PCR methods (US Patent No. 4,965,188) that include numerous stages of denaturation / hybridization / polymerization steps are well known in the art and can be readily adopted for use in the isolation of nucleic acid molecules encoding LmPDE. . 30

[0169] In addition, the nucleic acid molecules of the invention can also be employed in diagnostic embodiments, using LmPDE nucleic acid probes to determine the presence and / or the amount of LmPDE sequences present in a biological sample. One embodiment encompasses determining the amount of LmPDE nucleotide sequences present within the appropriate biological sample such as in specific cell types, tissues or body fluids, using an LmPDE probe in a hybridization procedure. The amount of LmPDE nucleic acid molecules in the test sample can be compared with the amount of LmPDE nucleic acid molecules in a reference sample of a normal subject. The presence of an appreciably different amount of LmPDE nucleic acid molecules between the test and reference samples can be correlated with the presence or severity of a disease associated with abnormal (high or low) levels of LmPDE nucleic acid molecules in Comparison with normal levels. 40

[0170] In another embodiment, control of the amount of LmPDE RNA transcripts over time is effected by quantitative determination of the amount of LmPDE RNA transcripts in test samples taken at different times in time. A difference in the amounts of LmPDE RNA transcripts in the various samples is indicative of the course of the disease associated with the expression of an LmPDE transcript.

[0171] To perform such diagnostic methods, a suitable biological sample of a test subject is contacted with a labeled LmPDE probe, under conditions effective to allow hybridization between the nucleic acid molecules of the sample and the probe. . Similarly, a biological sample from a normal subject is contacted with an LmPDE probe and hybridized under similar conditions. The presence of nucleic acid molecules hybridized with the probe is detected. The relative and / or quantified amount of the hybridized molecules can be compared between the test and reference samples. LmPDE probes can be labeled with any of 50 several known detectable markers, including radioactive, enzymatic, fluorescent or chemiluminescent markers.

[0172] Many suitable variations of hybridization technology are available for use in the detection of nucleic acids encoding LmPDE polypeptides. These include, for example, Southern and Northern procedures. Other hybridization techniques and systems are known that can be used in relation to the detection aspects of the invention, including diagnostic assays such as those described in Falkow et al. (U.S. Patent No. 4,358,535). Another hybridization procedure includes in situ hybridization, in which the target nucleic acids are located within one or more cells and come into contact with the LmPDE probes. How I know

Knows well in the art, cells are prepared for fixation hybridization, for example chemical hybridization, and are placed under conditions that allow hybridization of an LmPDE probe with nucleic acids located within the fixed cell.

[0173] The nucleic acid molecules of the present invention additionally provide antisense molecules that recognize and hybridize with an LmPDE nucleic acid. Antisense polynucleotides are particularly useful in regulating the expression of an LmPDE protein in cells expressing an LmPDE mRNA. A useful embodiment for this approach is an antisense molecule that corresponds to the N-terminal sequence of the gene. The present invention includes said full length antisense polynucleotides and fragments.

[0174] The polynucleotides of the present invention additionally provide reagents for developing animal models using knock-out strategies through homologous recombination. Methods for generating knockout animals that do not express a functional protein molecule are well known in the art (Capecchi (1989) Science 244, 1288-1292), and can be used in the study of LmPDE in vivo functions.

[0175] It should be understood that both the above general description and the examples below are exemplary and explanatory only and are not restrictive of the invention, as claimed. Furthermore, it should be understood that the invention is not limited to the particular embodiments described. In addition, the terminology used to describe particular embodiments is not intended to be limiting, since the scope of the present invention will be limited only by its claims.

[0176] The following examples are presented to illustrate the present invention and to assist a habitual expert in the preparation and use thereof. The methodology and results may vary depending on the intended objective of treatment and the procedures used. The examples are not intended to limit the scope of the invention in any other way.

EXAMPLES

Example 1

[0177] The following example provides the methods used to identify three specific cyclic nucleotide phosphodiesterase from Leishmania major. 25

[0178] Two families of cAMP-specific PDEs of the protozoan parasite Trypanosoma brucei have been previously characterized and identified (Zoraghi et al. (2001) J. Biol. Chem. 276, 11559-11566; Zoraghi et al. (2002) Proc. Natl. Acad. Sci. USA 99, 4343-4348; Gong et al. (2001) Mol. Biochem. Parasitol. 116, 229-232); and Rascon et al. (2002) Proc. Natl Acad. Sci. U.S.A. 99, 4714-4719. Sequences representing each family were then used to explore the Leishmania genomic database (http://www.genedb.org/genedb/leish). A BLAST 30 search was performed using the nucleotide sequence corresponding to the trypanosomal enzyme TbPDE1, which identified a nucleotide sequence in Leishmania major that shares an amino acid sequence identity of 45.1% (determined using the BESTFIT utility of the set of GCG programs with default parameters). This new sequence was labeled LmPDE-A. The gene for LmPDE-A is located within a group of sequences consisting of chromosomes 18, 20 and 22. A BLAST search was also performed using the nucleotide sequence that corresponds to the trypanosomal enzyme TbPDE2C, which identified two closely related sequences that were subsequently labeled LmPDE-B1 and LmPDE-B2. The LmPDE-B1 compares an amino acid sequence identity of 70.2% with the search sequence TbPDE2C, while the LmPDE-B2 shares a global sequence identity of 69.8% with TbPDE2C. The overall amino acid sequence identity between LmPDE-B1 and LmPDE-B2 is 84.5%. According to the Leishmania genome database, the LmPDE-B1 and LmPDE-B2 40 loci identified are located on chromosome 15.

Example 2

[0179] The following example provides the methods used to clone and sequence the new sequences identified in the Leishmania genome database.

[0180] Based on the sequences identified in the Leishmania genome database (as discussed in Example 1), PCR primers were designed to amplify the three full-length genes of the Leishmania major genomic DNA. PCR primers were also designed to amplify only the portion of the open reading phase of the three full-length genes. Primers were designed to contain a SaII site for phase cloning in the pLT1 vector (the ATG initiation codon being provided by the vector) and to encode a C-terminal hemagglutinin marker. For LmPDE-A, the direct and reverse primers were designed as 50 follows: direct - 5 ’-gtggtcgactcgactttcttgagcag-3 '(nucleotides 4-21 of the open reading phase, corresponding to nucleotides 533-550 of Figure 2); reverse - 5’-ctgggaatcctaagcataatctggaacatcatatggatacgagtcgtcgtggttgg-3 '(nucleotides 1896-1877 of the open reading phase, corresponding to nucleotides 2425-2406 in Figure 2 - and HA marker). For LmPDE-B1, the direct and reverse primers were designed as follows: direct - 5'-gatgtcgactggcatatttcacggcca-3 '(nucleotides 2-19 of the open reading phase, which correspond to nucleotides 1268-55 1286 in Figure 4) ; reverse - 5 '-ctgggaatcctaagcataatctggaacatcatatggataaacaatcgagggtcggatg-3' (nucleotides 2792-2772 of the open reading phase, corresponding to nucleotides 4058-4038 in Figure 4, and HA marker). For

LmPDE-B2 direct and reverse primers were designed as follows: direct - 5'-gatgtcgacattcagcggtcttttcc-3 '(nucleotides 3-21 of the open reading phase, which corresponds to nucleotides 2184-2202 in Figure 6); reverse - 5'-ctgggaatcctaagcataatctggaacatcatatggataaacaatcgaggatcggatg-3 '(nucleotides 2822-2803 of the open reading phase, which corresponds to nucleotides 5003-4984 in Figure 6, and HA marker). Leishmania major genes, as with other kinetoplastea, do not contain introns. Consequently, amplification of open reading phases 5 directly from genomic DNA is performed routinely (Beverley (2003) Nat. Rev. Genet. 4, 11-19). Both chains of the three PCR fragments (LmPDE-A, LmPDE-B1 and LmPDE-B2) were then cloned by the TA protrusion into the vector pCR2.1-TOPO following the instructions of the kit provided by Invitrogen. The clones were then sequenced using the reaction kit prepared for ABI PRISM Big Dye Terminator v3.0 cycle sequencing (Applied Biosystems). Both strands of each clone were sequenced at least twice to verify. 10

[0181] The full-length LmPDE-A nucleotide sequence, with a length of 10,966 nucleotides, is shown in Figure 2 in which the open reading phase begins with adenine at position 530 and ends with guanine at position 2425. The corresponding amino acid sequence is set forth in Figure 1. The full length nucleotide sequence of LmPDE-B1, which is 7095 nucleotides in length is shown in Figure 4, in which the open reading phase begins with adenine at position 1267 and ends with adenine at position 4059 and the corresponding amino acid sequence is set forth in Figure 3. The full length nucleotide sequence of LmPDE-B2 which is 6,945 nucleotides in length, is shown in Figure 6, in which the open reading phase begins with adenine at position 2182 and ends with adenine at position 5004. The amino acid sequence corresponding to this sequence is set forth in Figure 5.

[0182] The sequence of the cloned LmPDE-A coincided 100% with the sequence present in the Leishmania database. The predicted amino acid sequence of LmPDE-A (Figure 1) shares a global sequence identity of 45.1% with the amino acid sequence of the trypanosomal TbPDE1. The predicted amino acid sequence of LmPDE-B1 (Figure 3) shares a global sequence identity of 70.2% with the amino acid sequence of trypanosomal TbPDE2C, while the predicted amino acid sequence of LmPDE-B2 (Figure 5) shares a 69.8% global sequence identity with TbPDE2C. Using the conserved domain search service 25 provided by NCBI (see http://www.ncbi.nlm.nih.gov/Structure/ccd/cdd.shtml) several domains conserved in the amino acid sequences of LmPDE were identified. As indicated in Figure 7, LmPDE-A contains a highly conserved catalytic domain (PDEase) that begins with tyrosine at amino acid position 384 and ends with proline at amino acid position 609. LmPDE-B1 and LmPDE-B2 also contain each a highly conserved PDEase domain, which begins with phenylalanine at amino acid position 647 and ends with phenylalanine at amino acid position 880 (LmPDE-B1) and begins with phenylalanine at amino acid position 657 and ends with phenylalanine at amino acid position 890 (LmPDE-B2). In addition to the PDEase domain, LmPDE-B1 and LmPDE-B2 both have two GAF domains as shown in Figures 7B and 7C.

Example 3

[0183] The following example describes the methods used to evaluate the genomic organization of LmPDE-B1 and LmPDE-B2.

[0184] When the nucleotide sequences of the cloned LmPDE-B1 and LmPDE-B2 genes were compared with the sequences present in the Leishmania genome database, it became apparent that the sequences of the database had been assembled from wrong way. The 3 ’untranslated regions of the two genes had been involuntarily exchanged. The correct organization of these two genes was then established by Southern blot analysis of a series of restriction digestions of genomic DNA of L. major (as shown in Figures 8 and 9). Lane 8 of each hybridization (double digestion NotI / ScaI) will be used as an example of analytical reasoning. If the sequence assembly in the database is correct, a double digestion of NotI / ScaI should produce a fragment of at least 8 kb when hybridized with a specific LmPDE-B2 probe (since a restriction site is not present of ScaI in the immediate 3 'region of the LmPDE-B2 gene). Unlike this prediction, the data shows that a 4 kb fragment is generated, demonstrating the presence of a ScaI restriction site near the 3 ’end of the LmPDE-B2 gene. All additional digestions shown in Figures 8 and 9 support or are compatible with this conclusion. In addition to establishing the genomic organization of the LmPDE-B1 and LmPDE-B2 genes, these experiments demonstrated that each is a single copy gene. The hybridization probes used in Southern blot analysis were the following: specific 50 LmPDE-A: nucleotides 462-910 of the open reading phase (corresponding to nucleotides 991-1439 of Figure 2); LmPDE-B1 specific: nucleotides 96-489 of the open reading phase (corresponding to nucleotides 1362-1755 in Figure 4); specific for LmPDE-B2: 106-417 of the open reading phase (corresponding to 2287-3936 in Figure 6). This analysis also confirmed that LmPDE-B1 and LmPDE-B2 are arranged in tandem and are separated by approximately 5 kb on chromosome 15 as shown in Figure 11. 55

Example 4

[0185] The following example provides the methods used to produce a transgenic yeast strain, which contains an LmPDE-A and an LmPDE-B1, in which the endogenous PDE activity was suppressed.

[0186] The verified open reading phases of LmPDE-A and LmPDE-B1 were cloned into an expression vector of

yeast (pLT1, S. Kunz, unpublished) and were transfected into a strain of Saccharomyces cerevisiae in which both endogenous PDE genes had been previously suppressed (strain PP5: MATa leu2-3 Ieu2-112 ura3-52 his3-532 his4 cam pde1 :: URA3 pde2 :: HIS3; Colicelli et al. (1993) Proc. Natl. Acad. Sci. USA 90,11970-11974). The pLT1 expression vector is a 2-based yeast vector that carries a LEU2 selector gene. The cloning site is flanked by a TEF2 promoter followed by an optimized Kozak box and by a Cyc1 terminator. S. cerevisiae 5 deficient in PDE accumulates intracellular cAMP, which results in a heat-sensitive phenotype. In particular, no growth was observed after a 15-minute thermal shock at 55 ° C. As shown in Figure 12, both LmPDE-A and LmPDE-B1 completely complemented this phenotype and restored growth insensitive to thermal shock. For a thermal shock test, stretch marks were seeded on SC-met-ura plates and allowed to grow for 2 days at 30 ° C. The patches were replicated on a preheated plate at 55 ° C. The plate was incubated for an additional 15 minutes at 55 ° C, allowed to cool to room temperature and then incubated for 1-2 days at 30 ° C. These findings demonstrate that the LmPDE-A and LmPDE-B1 constructs produce active enzymes and that both enzymes are capable of hydrolyzing cAMP. Subsequent experiments demonstrated that LmPDE-B2 also completely complemented the heat-sensitive phenotype and restored thermal shock resistance (Johner et al. (Presented) J. Biol. Chem.). fifteen

[0187] The open reading phases of LmPDE-A, LmPDE-B1 and LmPDE-B2 were independently cloned into a yeast expression vector (pLT1, S. Kunz, unpublished) and transfected into a strain of Saccharomyces cerevisiae in which the endogenous PDE genes had been previously suppressed (strain PP5: MATa leu2-3 Ieu2-112 ura3-52 his3-532 his4 cam pde1 :: URA3 pde2 :: HIS3; Colicelli et al. (1993) Proc. Natl. Acad. Sci. USA 90, 11970-11974). These strains were deposited in the American Type Culture Collection (ATCC) on August 25, 2004 and have been assigned the numbers PTA-6167, and, respectively.

Example 5

[0188] The following example provides the methods used to characterize the catalytic activities of recombinant LmPDE-A, LmPDE-B1 and LmPDE-B2. The example also provides the methods used to evaluate the sensitivity of recombinant LmPDE-B1 and LmPDE-B2 to commercially available PDE inhibitors. 25

Example 5.1: Preparation of Yeast Lysates

[0189] PP5 yeast cells expressing LmPDE-A, LmPDE-B1 and LmPDE-B2 were lysed as previously described (Zoraghi et al. (2001) J. Biol. Chem. 276, 11559-11566). Cells were cultured to final logarithmic medium to logarithmic growth stages in SC-leu medium, collected, resuspended in the original volume of preheated YPD medium and incubated for an additional 3.5 hours at 30 ° C to maximize expression. of protein The cells were then collected, washed twice in H2O, pelleted by centrifugation and stored overnight at -70 ° C. The cell pellet was thawed on ice and resuspended in ice-cold extraction buffer (50 mM Hepes pH 7.5, 100 mM NaCI, Complete® 1X protease inhibitor cocktail without EDTA (Roche)). Cells were lysed by grinding with glass beads (0.45-0.50 mm) in 2 ml Sarstedt tubes using a FastPrep FP120 cell disruptor (3 x 45 seconds in setting 4). The lysed cells were centrifuged and glycerol was added to the resulting supernatant to a final concentration of 15% (v / v). The aliquots were rapidly frozen in liquid nitrogen and stored at -70 ° C.

Example 5.2: PDE activity assay in yeast lysates

[0190] PDE activity was determined in 50 mM HEPES, pH 7.5, 0.5 mM EDTA, 10 mM MgCI2 and 50 mg / ml BSA in a final assay volume of 100 µl. Each assay contained 50,000 cpm of 3 H labeled cAMP, with no labeled cAMP added to adjust the desired total substrate concentration. The reactions were performed at 30 ° C and were linear for at least 60 minutes. The conventional reaction time was adjusted to 15 minutes and the amount of enzyme was always selected so that no more than 15% substrate was hydrolyzed. Inhibitor studies were performed at a cAMP concentration of 1 µM. The inhibitors were dissolved in DMSO, but the final concentration of DMSO in the assays never exceeded 1%. Control reactions with DMSO were always included only. Reactions 45 were stopped by the addition of 25 µl of 0.5 N HCI. For subsequent dephosphorylation of the AMP, the stopped reactions were neutralized with 20 µl of 1 M Tris base, followed by the addition of 10 µl of alkaline phosphatase (Roche Diagnostics; 1 unit / 10 l). Dephosphorylation reactions were incubated for 15 minutes at 37 ° C and then applied to 1 ml columns of QAE-Sephadex A25 in 30 mM ammonium formate, pH 6.0. The 3H-adenosine formed as a reaction product was eluted with 1.6 ml of 30 mM ammonium formate, pH 6.0 and collected in 3.5 ml of water-soluble scintillation fluid 50 (Packard Ultima Flo). Triplicate assays were performed and the data analyzed using the Graph Pad Prism software package.

Example 5.3: Activity and specificity of LmPDE

[0191] Lysates prepared from yeast expressing LmPDE-A consistently showed no appreciable PDE activity. This observation is consistent with the finding that LmPDE-A is less effective than LmPDE-B1 and LmPDE-B2 in complementing the PDE deficiency of the host strain PP5. In addition, these findings are very similar to the observations made with the trypanosomal homologue TbPDE1. For example, TbPDE1 complemented the PDE deficiency of PP5 cells but its effect was less than that of other PDEs.

trypanosomal or human and no enzymatic activity could be detected in the corresponding yeast cell lysates (Kunz et al. (2004) Eur. J. Biochem. 271, 637-647).

[0192] Unlike the lysates of cells expressing LmPDE-A, the lysates of yeast strains expressing LmPDE-B1 and LmPDE-B2 showed strong PDE activities. Both enzymes showed very similar KM values for cAMP that were within the range of other Class I PDEs (Zoraghi et al. (2001) J. Biol. Chem. 276, 11559-11566; Rascon et al. (2002) Proc Natl. Acad. Sci. USA 99, 4714-4719; Zoraghi et al. (2002) Proc. Natl. Acad. Sci. USA 99, 4343-4348; Francis et al. (2001) Prog. Nucleic Acid Res. Mol Biol. 65, 1-52; Mou and Cote (2001) J. Biol. Chem. 276, 27527-27534). In addition, the presence of a 100 fold excess of cGMP did not affect the rate of cAMP hydrolysis by LmPDE-B1 (compare Figure 13B with Figure 13A). A 50-fold excess of the 5’-AMP reaction product also had no effect on KM (or Kcat) (Figure 13C). Similar results were obtained with LmPDE-B2 (data not shown). Therefore, LmPDE-B1 and LmPDE-B2 are cAMP specific PDEs.

[0193] These data agree well with the finding that no cGMP hydrolysis activity can be detected in whole cell strata of Leishmania major (data not shown). Because PDE-catalyzed hydrolysis is the only mechanism by which a cell can dispose of its cyclic nucleotides (except for possible export mechanisms; Guo et al. (2003) J. Biol. Chem. 278, 29509-29514) , the absence of a PDE 15 activity that hydrolyzes cGMP from Leishmania cells suggests that cGMP signaling may not exist in Leishmania major.

Example 5.4: LmPDE activity in the presence of PDE inhibitors

[0194] Figure 14 shows the effects of several commercially available PDE inhibitors (100 M) on the activity of LmPDE-B1 and LmPDE-B2 in the presence of 1 M cAMP. Both enzymes were insensitive to the IBMX broad spectrum PDE inhibitor but were partially sensitive to trequinsin, dipyridamole and ethazolate. Trequinsin and dipyridamole inhibited the activity of LmPDE-B1 with IC50 values of 96.6 and 22.6 µM respectively.

[0195] The inhibitory profile observed, including the fact that dipyridamole and trequinsin were the most potent compounds, closely corresponds to that previously indicated for trypanosomal PDE (Zoraghi et al. (2001) J. Biol. Chem. 276, 11559- 11566; Zoraghi et al. (2002) Proc. Natl. Acad. Sci. USA 99, 4343-4348; Rascon et al. (2002) 25 Proc. Natl. Acad. Sci. USA 99, 4714-4719). The fact that several potent and specific inhibitors of different human PDEs had no effect on Leishmania PDEs strongly suggests that the development of Leishmania-specific PDE inhibitors is feasible.

Example 6

[0196] The following example describes the methods used to determine the effect of PDE 30 dipyridamole, ethazolate and trequinsin PDE inhibitors on the proliferation of L. major promastigotes in vitro.

[0197] Promastigote forms of L. major MHRO / IR / 75 / ER or LV39 were grown at 27 ° C in SDM medium containing 5% heat-inactivated fetal bovine serum (R. Brun and M. Schonenberger (1979 ) Minutes Trop. 36, 289-292). Cell proliferation was assayed in 5 ml cultures containing various concentrations of dipyridamole, ethazolate or trequinsin dissolved in DMSO (final concentration of 1% v / v) or DMSO 1% v / v as control. At various times, 150 µL aliquots were removed and absorbance at 600 nm was measured in a microtiter plate reader. The correlation between DO600 and the number of cells was strictly linear for at least the range of 3 x 105 to 4 x 107 cells / ml.

[0198] As shown in Figure 15, the three PDE inhibitors strongly inhibited the proliferation of promastigotes with IC50 values of approximately 50 µM. The extent of inhibition was independent of cell density and the effect of the inhibitors was not reduced by prolonged incubation of the cultures.

[0199] These results are consistent with the ability of dipyridamole, ethazolate and trequinsin to inhibit the activity of recombinant LmPDE-B1 (see, for example, Figure 14). In addition, the data strongly suggests that LmPDE-B1 and LmPDE-B2 are involved in the growth of L. major and further support the development of Leishmania-specific PDE inhibitors for leishmaniasis therapy. Four. Five

[0200] With respect to value ranges, the invention encompasses each intermediate value between the upper and lower limits of the range up to at least one tenth of the unit of the lower limit, unless the context clearly indicates otherwise. In addition, the invention encompasses any other indicated intermediate value. In addition, the invention also encompasses intervals that exclude the upper or lower limits or both of the range, unless specifically included in the indicated range. fifty

[0201] Unless otherwise defined, the meanings of all technical and scientific terms used herein are those commonly understood by one skilled in the art to which the present invention belongs. A person skilled in the art will also appreciate that any method and material similar or equivalent to those described herein can be used to practice or test the invention.

[0202] It should be noted that, as used herein and in the appended claims, forms 55

Singular "a", "or" and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, the reference to "an object polypeptide" includes a plurality of such polypeptides and the reference to "the agent" includes references to one or more agents and equivalents thereof known to those skilled in the art, and so on.

[0203] Although the above invention has been described in some detail by means of illustrations and examples in order that there is a clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Therefore, the description and examples of the description should not be construed as limiting the scope of the invention, which is defined by the scope of the appended claims.

SEQUENCE LIST

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REFERENCES CITED IN THE DESCRIPTION

This list of references cited by the applicant is solely for the convenience of the reader. This list is not part of the European patent document. Although great care has been taken in the collection of references, errors or omissions cannot be excluded and the EPO disclaims all responsibility in this regard.

Patent documents cited in description 5

image 1

 10

Non-patent bibliography cited in the description

Claims (31)

1. Substantially purified protein comprising a Leishmania PDE-B1 phosphodiesterase (PDE) catalytic domain comprising amino acids 647-880 as set forth in SEQ ID NO: 3 or variants and mutants of the catalytic domain that are at least 80 % identical to amino acids 647-880 of SEQ ID NO: 3 and that hydrolyze cAMP. 5 2. Substantially purified protein comprising a PDE-B2 PDE catalytic domain of Leishmania comprising amino acids 657-890 as set forth in SEQ ID NO: 5 or variants and mutants of the catalytic domain that are at least 80% identical to amino acids 657-890 of SEQ ID NO: 5 and that hydrolyze cAMP. 3. Substantially purified protein of claim 1 comprising the amino acid sequence set forth in SEQ ID NO: 3 or variants and mutants thereof that are at least 80% identical to SEQ ID NO: 3 and hydrolyze 10 cAMP. 4. Substantially purified protein of claim 2 comprising the amino acid sequence set forth in SEQ ID NO: 5 or variants and mutants thereof that are at least 80% identical to SEQ ID NO: 5 and that hydrolyze cAMP. 5. A substantially purified nucleic acid molecule comprising a nucleic acid sequence encoding a protein of claim 1 or 2. 6. Nucleic acid molecule according to claim 5 comprising a nucleic acid sequence encoding a protein of claim 3. 7. Nucleic acid molecule according to claim 6, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 4 which begins with adenine at position 1267 and ends with adenine at position 4059. 8. Nucleic acid molecule according to claim 5 comprising a nucleic acid sequence encoding a protein of claim 4. 9. Nucleic acid molecule according to claim 8, wherein the nucleic acid molecule comprises the nucleotide sequence set forth in SEQ ID NO: 6 which begins with adenine at position 2182 and ends with adenine at position 5004. 10. Nucleic acid molecule of claim 6 or 8, the nucleic acid molecule being a DNA molecule. 11. Protein of claim 3 or claim 4, or the nucleic acid of claim 6 or 8, wherein the protein has a KM value of 1 to 2 µM for cAMP. 12. Protein of claim 3 or claim 4, or the nucleic acid of claim 6 or 8, wherein protein 30 does not hydrolyse cGMP. 13. Protein of claim 3 or 4, or the nucleic acid of claim 6 or 8, wherein the protein substantially hydrolyzes cAMP in the presence of up to about 100 µM of a phosphodiesterase inhibitor selected from cilostamide, zaprinast, ethazolate, Ro- 20-1724, rolipram, isobutylmethylxanthine (IBMX), 8-methoxymethyl-IBMX, papaverine, milrinone, petoxifylline and erythro-9- (2-hydroxy-3-nonyl) adenine. 35 14. Vector comprising the nucleic acid molecule of claim 6 or 8. 15. Vector of claim 14 wherein the nucleic acid molecule is operably linked to at least one expression control sequence. 16. Host vector system comprising the vector of claim 14 in a suitable host cell. 40 17. Host vector system of claim 16 wherein the suitable host cell is a bacterial cell. 18. Host vector system of claim 16 wherein the suitable host cell is a eukaryotic cell. 19. Host vector system of claim 18 wherein the eukaryotic cell is Saccharomyces cerevisiae. Four. Five 20. Antibody or antibody fragment comprising an antigen binding site that selectively recognizes and binds to a protein of claim 1, 2, 3 or 4. 21. Antibody of claim 20, wherein the antibody is a monoclonal antibody or a polyclonal antibody. 22. Antibody of claim 21, wherein the antibody is chimeric, humanized or human. 23. Method for producing a PDE protein comprising culturing the host vector system of claim 16 under conditions suitable for producing the PDE protein in the host and recovering the PDE protein produced in this manner. 24. Method for identifying a compound that modulates the expression of a protein according to claim 3 or 5, the method comprising: (a) incubating a cell that can express said protein with a compound under conditions and for a time sufficient for the cell to express said protein without the compound; (b) incubating a control cell under the same conditions and during the same time without the compound; (c) measure the expression of said protein in the cell in the presence of the compound; 10 (d) measure the expression of said protein in the control cell; Y (e) compare the amount of expression of said protein in the presence and absence of the compound, where a difference in the level of expression indicates that the compound modulates the expression of said protein. 25. The method of claim 24 wherein the compound reduces the expression of said protein. 26. Method for identifying a compound that modulates Leishmania PDE activity of the protein according to claim 3 or 4, the method comprising: (a) contacting a genetically engineered cell line to express said protein, with a compound under conditions and for a time sufficient for the sample to express said protein without the compound; (b) incubating a control sample under the same conditions and during the same time without the compound; twenty (c) measuring said PDE activity of said protein in the sample in the presence of the compound; (d) measuring said PDE activity of said protein in the control sample; Y (e) comparing the amount of said PDE activity of said protein in the presence and absence of the compound, wherein a difference in the level of activity indicates that the compound modulates the PDE activity of said protein. 27. The method of claim 24 or 26, wherein the compound reduces PDE activity. 25 28. Genetically modified non-human organism comprising the vector of claim 14 and wherein at least one endogenous PDE gene has been deactivated. 29. Method for detecting infection by the Leishmania parasite comprising determining the amount present in a sample of a subject of at least one protein of any of claims 1 to 4. 30. Method for detecting infection by the Leishmania parasite comprising determining the amount present in a sample of a subject of at least one nucleic acid molecule of any one of claims 5 to 9. 31. Method of claim 29 or 30, wherein the Leishmania parasite is Leishmania major.