Inhibitors of memapsin 2 and 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 tliree 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 . An inhibitor of catalytically active 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.  
     
     
         2 . The inhibitor of  claim 1  comprising an isostere of the active site of memapsin 2.  
     
     
         3 . The inhibitor of  claim 2  comprising a molecule having the general form X- L 4 -P 4 -L 3 -P 3 -L 2 -P 2 -L 1 -P 1 -L 0 -P 1 ′-L 1 ′-P 2 ′-L 2 ′-P 3 ′-L 3 ′-P 4 ′L 4 ′-Y, 
 wherein Px represent the substrate specificity position relative to the cleavage site which is represented by an -LO-, and Lx represent the linking regions between each substrate specificity position, Px, and  
 wherein L 0  is a non-hydrolyzable bond and P1′is -R 1 CR 3 -, wherein R 1  is a group smaller than CH 2 OH (side chain of serine), and at least two other P positions are a hydrophobic group.  
 
     
     
         4 . The inhibitor of  claim 3  which is OM99-1.  
     
     
         5 . The inhibitor of  claim 3  which is OM99-2.  
     
     
         6 . The inhibitor of  claim 3  having the structure of FIG. 11.  
     
     
         7 . The inhibitor of  claim 3  having the structure of FIG. 12.  
     
     
         8 . The inhibitor of  claim 3  having the structure of FIG. 13.  
     
     
         9 . The inhibitor of  claim 3  having the structure of FIG. 14.  
     
     
         10 . The inhibitor of  claim 1  having an K i  of less than or equal to 10 −7  M.  
     
     
         11 . The inhibitor of claim I which binds to crystallized enzyme characterized by the parameters in Table 2 when bound to OM-99-2.  
     
     
         12 . The inhibitor of  claim 13  having a K i  of less than or equal to 10 −6  M.  
     
     
         13 . The inhibitor of  claim 11  having a K i  of less than or equal to 2 nM.  
     
     
         14 . The inhibitor of  claim 13  having a K i  of less than or equal to 2 nM.  
     
     
         15 . The inhibitor of  claim 11  having a root mean square difference of less than or equal to 0.5 Å for the side chain and backbone atoms for amino acids 18-379 of memapsin 2.  
     
     
         16 . The inhibitor of  claim 1  which is permeable to the blood brain barrier.  
     
     
         17 . The inhibitor of  claim 1  which blocks cleavage by memapsin 2 under physiological conditions.  
     
     
         18 . The inhibitor of  claim 1  which is a non-amino acid small molecule.  
     
     
         19 . The inhibitor of  claim 18  having a molecular weight of less than 800 Daltons.  
     
     
         20 . A method of synthesis of a Leu*Ala dipeptide isostere.  
     
     
         21 . A method for treating a patient to decrease the likelihood of developing or the progression of Alzheimer's disease comprising administering to the individual an effective amount of an inhibitor of memapsin 2 having an K; of less than or equal to 10 −7  M or which binds to crystallized enzyme characterized by the parameters in Table 2 when bound to OM-99-2.  
     
     
         22 . The method of  claim 21  wherein the inhibitor is administered orally.  
     
     
         23 . The method of  claim 21  wherein the inhibitor blocks cleavage of APP.