Catalytically active recombinant memapsin and methods of use thereof

Abstract

Methods for the production of purified, catalytically active, recombinant memapsin 2 have been developed. The substrate and subsite specificity of the catalytically active enzyme have been determined. The substrate and subsite specificity information was used to design substrate analogs of the natural memapsin 2 substrate that can inhibit the function of memapsin 2. The substrate analogs are based on peptide sequences, shown to be related to the natural peptide substrates for memapsin 2. The substrate analogs contain at least one analog of an amide bond which is not capable of being cleaved by memapsin 2. Processes for the synthesis of two substrate analogues including isosteres at the sites of the critical amino acid residues were developed and the substrate analogues, OMR99-1 and OM99-2, were synthesized. OM99-2 is based on an octapeptide Glu-Val-Asn-Leu-Ala-Ala-Glu-Phe (SEQ ID NO:28) with the Leu-Ala peptide bond substituted by a transition-state isostere hydroxyethylene group (FIG. 1 ). The inhibition constant of OM99-2 is 1.6×10 −9 M against recombinant pro-memapsin 2. Crystallography of memapsin 2 bound to this inhibitor was used to determine the three dimensional structure of the protein, as well as the importance of the various residues in binding. This information can be used by those skilled in the art to design new inhibitors, using commercially available software programs and techniques familiar to those in organic chemistry and enzymology, to design new inhibitors to memapsin 2, useful in diagnostics and for the treatment and/or prevention of Alzheimer's disease.

We claim:  
     
         1 . Purified recombinant catalytically active memapsin 2.  
     
     
         2 . The memapsin 2 of  claim 1  having the amino acid sequence of SEQ ID NO. 2 or the sequence present in a homologous species.  
     
     
         3 . The memapsin 2  claim 2  of human origin and having the amino acid sequence of SEQ ID NO. 2.  
     
     
         4 . The memapsin 2 of  claim 1  not including the transmembrane domain.  
     
     
         5 . The memapsin 2 of  claim 1  expressed in a bacteria.  
     
     
         6 . The memapsin 2 of  claim 1  cleaving SEVKM/DAEFR (SEQ ID NO:4) and SEVNL/DAEFR (SEQ ID NO:5) at pH 4.0 with k cat /K m  of less than or equal to 39.9 s −1 M −1  and less than or equal to k cat , 2.45 s −1 , K m , 1 mM; k cat /K m , 2450 s −1 M −1 , respectively.  
     
     
         7 . A method for producing catalytically active recombinant memapsin 2 comprising refolding the recombinant memapsin 2 under conditions which dissociate and then slowly refold the enzyme into a catalytically active form.  
     
     
         8 . The method of  claim 7  wherein the memapsin 2 is first dissolved in 8 M urea solution including one or more reducing agents at a pH of greater than 8.0.  
     
     
         9 . The method of  claim 8  wherein the memapsin 2 is then diluted into an aqueous buffer like 20 mM-Tris, pH 9.0, the pH slowly adjusted to approximately 8 with 1 M HCl, and the solution maintained at low temperature for approximately 24 to 48 hours before proceeding with purification.  
     
     
         10 . The method of  claim 8  wherein the memapsin 2 is then rapidly mixed with an aqueous buffer like 20 mM-Tris, pH 9.0, containing oxidized and reduced glutathione, the process repeated, then the urea concentration decreased to approximately 0.4 M and the pH of the solution slowly adjusted to 8.0.  
     
     
         11 . The method of  claim 8  wherein the memapsin 2 is dissolved in 8 M urea, pH 10.0, then rapidly diluted into an aqueous buffer like 20 mM Tris base, pH 9.0, and maintained at low temperature several hours, maintained at room temperature for several hours, and then the process repeated at decreasing pH.  
     
     
         12 . A method of isolating inhibitors of cleavage by memapsin 2 comprising 
 adding to one or more potential inhibitors catalytically active recombinant memapsin 2 cleaving SEVKM/DAEFR (SEQ ID NO:4) and SEVNL/DAEFR (SEQ ID NO:5) at pH 4.0 with k cat /K m  of less than or equal to 39.9 s −1 M −1  and less than or equal to k cat , 2.45 s −1 , K m , 1 mM; k cat /K m , 2450 s −1 M −1 , respectively, and a substrate for memapsin 2, and    screening for decreased cleavage of the substrate by the inhibitors.    
     
     
         13 . The method of  claim 12  wherein the inhibitors are in a library of small synthetic molecules.  
     
     
         14 . The method of  claim 12  wherein the inhibitors are compounds selected from the group consisting of proteins and peptides.  
     
     
         15 . The method of  claim 12  wherein the recombinant memapsin 2 is expressed in genetically engineered cells and the inhibitors and substrate are added to the cells.  
     
     
         16 . The method of  claim 15  wherein the inhibitors are oligonucleotides preventing or decreasing expression of catalytically active memapsin 2.  
     
     
         17 . The method of  claim 14  wherein the compounds are isosteres of the memapsin 2 active site defined by the presence of two catalytic aspartic residues and substrate binding cleft.  
     
     
         18 . The method of  claim 12  further comprising identifying the inhibitors maximally decreasing cleavage of substrate by the memapsin 2.  
     
     
         19 . A method for designing or obtaining inhibitors of catalytically active memapsin 2 comprising modeling an inhibitor based on the crystallization coordinates of memapsin 2 or parameters of Table 2.  
     
     
         20 . The method of  claim 19  comprising using a computer program to model a compound to determine its binding to the memapsin 2 active site defined by the presence of two catalytic aspartic residues and substrate binding cleft.  
     
     
         21 . The method of  claim 19  comprising using a computer program to design a compound which binds to the memapsin 2 active site defined by the presence of two catalytic aspartic residues and substrate binding cleft.  
     
     
         22 . The method of  claim 19  further comprising screening compounds which bind to the active site defined by the presence of two catalytic aspartic residues and substrate binding cleft for inhibition of memapsin 2 catalytic activity.  
     
     
         23 . A data base comprising binding properties and chemical structures of compounds designed or screened by modeling an inhibitor based on the crystallization coordinates of memapsin 2 or parameters of Table 2.  
     
     
         24 . A method of treating or preventing Alzheimer's disease comprising administering administering to a patient in need thereof an inhibitor of memapsin 2 which binds to the active site of the memapsin 2 defined by the presence of two catalytic aspartic residues and substrate binding cleft.  
     
     
         25 . The method of  claim 24  wherein the inhibitor has an K i  of less than or equal to 10 −7  M.  
     
     
         26 . The method of  claim 24  wherein the inhibitor is selected from the group consisting of proteins, peptides, oligonucleotides, and small synthetic molecules.  
     
     
         27 . The method of  claim 24  wherein the inhibitor is modeled based on the crystallization coordinates of memapsin 2 or parameters of Table 2.  
     
     
         28 . A crystallized memapsin 2 comprising the active site defined by the presence of two catalytic aspartic residues and substrate binding cleft.  
     
     
         29 . The memapsin 2 of  claim 28  not including the transmembrane and/or intracellular domains.  
     
     
         30 . The memapsin 2 of  claim 28  having the parameters defined in Table 2.  
     
     
         31 . The memapsin 2 of  claim 28  having a diffraction to at least 3.5 Å or less.  
     
     
         32 . The memapsin 2 of  claim 31  having a diffraction to at least 2 Å or less.  
     
     
         33 . A method for treatment or preventing Alzheimer's disease comprising immunizing an individual in need thereof with catalytically active memapsin 2 cleaving SEVKM/DAEFR (SEQ ID NO:4) and SEVNL/DAEFR (SEQ ID NO:5) at pH 4.0 with k cat /K m  of less than or equal to 39.9 s −1 M −1  and less than or equal to k cat , 2.45 s −1 , K m , 1 mM; k cat /K m , 2450 s −1 M −1 , respectively, to elicit an effective amount of antibodies to reduce cleavage by endogenous memapsin 2.