Catalytically active recombinant memapsin and methods of use thereof

• US 20080112946A1
• Filed: 08/03/2007
• Published: 05/15/2008
• Est. Priority Date: 06/28/1999
• Status: Active Grant

• Current Assignee: Board of Trustees of The University of Illinois, Oklahoma Medical Research Foundation
• Original Assignee: Oklahoma Medical Research Foundation
• Inventors: Tang, Jordan J. N., Ghosh, Arun K., Lin, Xinli, Hong, Lin, Koelsch, Gerald
• Granted Patent: US 7,829,669 B2
• Time in Patent Office: Days
• US Class Current: 424/94.66
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 1/1136 by reversible modification of the secondary, tertiary or quarternary structure, e.g. using denaturating or stabilising agents
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes
• CPC Class Codes: C07K 5/06026 the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
• CPC Class Codes: C07K 5/06043 Leu-amino acid
• CPC Class Codes: C07K 5/0806 the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
• CPC Class Codes: C07K 5/1021 with the first amino acid being acidic
• CPC Class Codes: C12N 9/6421 from mammals
• CPC Class Codes: C12N 9/6478 Aspartic endopeptidases (3.4.23)
• CPC Class Codes: Y02A 90/10 Information and communication technologies [ICT] supporting adaptation to climate change, e.g. for weather forecasting or climate simulation

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 bond 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.

Claims

1. :

A memapsin 2 protein consisting of amino acid residues 1-456 of SEQ ID NO;

3.

2. :

The memapsin 2 protein of claim 1, wherein the memapsin 2 protein is expressed in a bacterial cell.

3. :

The memapsin 2 protein of claim 1, wherein the memapsin 2 protein is refolded after expression in a bacterial cell.

4. :

The memapsin 2 protein of claim 3, wherein the memapsin 2 protein is refolded by dissolution in a urea solution having a urea concentration of about 8M and at least one reducing agent at a pH of at least about pH 8.0.

5. :

The memapsin 2 protein of claim 4, wherein the memapsin 2 protein is refolded by dilution in an aqueous buffer having a pH of at least about pH 9, and wherein the pH subsequently is adjusted to about pH 8, and then maintained at a temperature of about 4°

C. for a period of time in a range of between about 24 hours and about 48 hours.

6. :

The memapsin 2 of claim 4, wherein the memapsin 2 protein is refolded by dilution in an aqueous buffer having a pH of at least about pH 9 and including an oxidized and reduced glutathione, and wherein the urea concentration of the diluted solution of memapsin 2 protein decreases to about 0.4M, and wherein the pH is adjusted to about pH 8.

7. :

The memapsin 2 protein of claim 4, wherein the memapsin 2 protein is refolded by dissolution in urea having a urea concentration of about 8M and a pH of about pH 10, wherein a resulting solution subsequently is diluted in an aqueous buffer having a pH of about pH 9.0, and maintaining the solution at a temperature of about 4°

C. for a period of time in a range of between about 24 hours and about 48 hours.

8. :

A memapsin 2 protein consisting of amino acid residues 43-456 of SEQ ID NO;

3 or residues 45-456 of SEQ ID NO;

3.

9. :

A composition comprising a memapsin 2 protein consisting of amino acid residues 43-456 of SEQ ID NO;

3, or a memapsin 2 protein consisting of amino acid residues 45-456 of SEQ ID NO;

3 or a mixture of memapsin 2 protein consisting of amino acid residues 43-456 of SEQ ID NO;

3 and a memapsin 2 protein consisting of amino acid residues 45-456 of SEQ ID NO;

3.

10. :

The composition of claim 9, wherein the mixture is produced by autocatalytic cleavage of memapsin 2 protein consisting of amino acids residues 1-456 of SEQ ID NO;

3.

11. :

A composition comprising memapsin 2 protein crystallized with a substrate, wherein said crystallized memapsin 2 protein and substrate diffract to about 3.5 Å

or less, and

wherein the memapsin 2 protein is a mixture of memapsin 2 protein consisting of amino acid residues 43-456 of SEQ ID NO;

3 and memapsin 2 protein consisting of residues 45-456 of SEQ ID NO;

3.

12. :

The composition of claim 11, wherein the crystallized memapsin 2 protein and substrate have the parameters defined in Table 2.

13. :

The composition of claim 11, wherein the crystallized memapsin 2 protein and substrate diffract to at least 2 Å

or less.

14. :

The composition of claim 11, wherein said memapsin 2 protein is bound to the substrate.

15. :

The composition of claim 14, wherein the substrate includes a peptide.

16. :

The composition of claim 14, wherein the substrate includes a transition state isostere.

17. :

The composition of claim 16, wherein the transition state isostere incorporates hydroxyethylene.

18. :

The composition of claim 14, wherein the substrate is selected from the group consisting of OM99-1 and OM99-2.