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AssigneeOklahoma Medical Research Foundation
InventorsXinli Lin, Gerald Koelsch, Jordan J.N. Tang
Estimating the term…
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.
Purified recombinant catalytically active memapsin 2.
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.
A method of isolating inhibitors of cleavage by memapsin 2 comprising
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.
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.
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.
A crystallized memapsin 2 comprising the active site defined by the presence of two catalytic aspartic residues and substrate binding cleft.
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 kcat/Km of less than or equal to 39.9 s−1M−1 and less than or equal to kcat, 2.45 s−1, Km, 1 mM;
Dependencies are inferred from the claim text.