Cyclic nucleotide-specific phosphodiesterases from leishmania and uses thereof

Abstract

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

1 - 6 . (canceled) 
     
     
         7 . A substantially purified protein 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. 
     
     
         8 - 16 . (canceled) 
     
     
         17 . A substantially purified protein comprising a PDE catalytic domain of LmPDE-B2 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. 
     
     
         18 - 30 . (canceled) 
     
     
         31 . A method of identifying a compound that modulates LmPDE activity, the method comprising:
 a) contacting a sample that has LmPDE activity with a compound under conditions and for a time sufficient for the sample to express LmPDE activity absent the compound;   b) incubating a control sample under the same conditions and for the same time absent the compound;   c) measuring LmPDE activity in the cell in the presence of the compound;   d) measuring LmPDE activity in the control sample; and   e) comparing the amount of LmPDE activity in the presence and absence of the compound, wherein a difference in the level of activity indicates that the compound modulates LmPDE activity.   
     
     
         32 . The method of  claim 31 , wherein the compound decreases LmPDE activity. 
     
     
         33 - 39 . (canceled) 
     
     
         40 . The protein of  claim 7 , wherein the protein has a K M  value of about 1 to 2 μM for cAMP. 
     
     
         41 . The protein of  claim 7 , wherein the protein does not hydrolyze cGMP. 
     
     
         42 . The protein of  claim 7 , wherein the protein substantially hydrolyzes cAMP in the presence of up to about 100 μM of a phosphodiesterase inhibitor chosen from cilostamide, zaprinast, etazolate, Ro-20-1724, rolipram, isobutylmethylxanthine (IBMX), 8-methoxymethyl-IBMX, papaverine, milrinone, petoxifylline, and erythro-9-(2-hydroxy-3-nonyl)adenine. 
     
     
         43 - 45 . (canceled)