Inhibitors of MEMAPSIN2 and use thereof

An inhibitor, the technology of MMI-071, is applied in the field of treatment and/or prevention of Alzheimer's disease, and the treatment of Alzheimer's disease patients, and can solve the problems such as inability to prepare active enzymes

Inactive Publication Date: 2006-05-17
OKLAHOMA MEDICAL RES FOUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although many enzymes have been identif...

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  • Inhibitors of MEMAPSIN2 and use thereof
  • Inhibitors of MEMAPSIN2 and use thereof
  • Inhibitors of MEMAPSIN2 and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0133] Example 1 Proteolytic activity and the priority of the fragmentation site of recombinant memapsin2

[0134] To confirm the specificity of memapsin2, the amino acid sequence around the APP proteolytic cleavage site was determined. Recombinant pro-memapsin2-T1 was incubated in pH 4.0, 0.1M sodium acetate solution for 16 hours at room temperature to generate autocatalytic cleavage. Products were analyzed using SDS-polyacrylamide gel electrophoresis. Many bands with molecular weights smaller than pro-memapsin2 were found. The bands were blotted onto PVDF membranes. Four swimming bands were selected, and their N-terminal sequences were determined in a protein sequencer. The N-terminal sequence of these bands identifies the location of the pro-memapsin2 proteolytic cleavage site.

[0135] In addition, the oxidized beta chain of bovine insulin and two different synthetic peptides were used as substrates for memapsin2 to determine the extent of other hydrolysis sites. Pu...

Embodiment 2

[0140] Example 2 Activation and enzyme kinetics of pro-memapsin2

[0141] Pro-M2 at 22°C pd Incubate in 0.1M sodium acetate solution at pH 4.0 for 16 hours to transform into M2 by autocatalysis pd . To determine the initial rate of hydrolysis, two synthetic peptides were mixed with pro-M2 pd Cultured in 0.1M sodium acetate solution at pH 4.0, and maintained for 2-18 hours. Cultured samples were analyzed using liquid chromatography (LC) and mass spectrometry (MC) to identify hydrolysates. For kinetic studies, identified HPLC (Beckman System Gold) product peaks were integrated for quantitative analysis. Determination of K of Sensenin 1 and Swedish APP Peptides (Table 1) Using Static Kinetics m and K cat value. Since the K of the APP peptide cannot be accurately determined by standard methods m and K cat single value, so its K was determined by competitive hydrolysis of mixed substrates with senescentin 1 peptide cat / K m Value (Fersht A "Emzyme Structure and Mechani...

Embodiment 3

[0143] Example 3 Design and Synthesis of Memapsin2 Inhibitors OM99-1 and OM99-2

[0144] Based on the results of memapsin2 specificity, it was predicted that the residues suitable for P1 and P1' positions should be leucine and alanine. Next, as determined by the specificity data, P1' is preferably a small residue such as alanine and serine. However, the crystal structure (determined below) indicates that this site is also capable of accommodating a variety of large residues. It has been confirmed that P1' of memapsin2 is the most stringent position for specificity, and it is desirable to have small side chain residues, such as alanine, serine, aspartic acid. Alanine was selected for P1' mainly because its hydrophobicity is better than that of serine and aspartic acid, which is conducive to penetrating the blood-brain barrier and meets the design requirements of a memapsin2 inhibitor for the treatment of AD. Therefore, the inhibitor was designed to place a transition state ...

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Abstract

The substrate and subsite specificity of the catalytically active enzyme was determined by measuring the initial hydrolysis rate of the substrate using MALDI-TOF/MS. In addition, memapsin subsite specificity was determined by probing the inhibitor library with memapsin2 and detecting bound memapsin2 with a memapsin2 antibody and alkaline phosphatase-conjugated secondary antibody. Substrate and subsite specificity information can be used to design analogs of natural memapsin2 substrates capable of inhibiting memapsin2 function. Substrate analogs are based on peptide sequences related to the native peptide substrate of memapsin2. Substrate analogs contain at least one analog whose amide bond cannot be broken by memapsin2. A synthetic method for analogues containing isosteric substrates at key amino acid residue positions has been developed, and more than 70 substrate analogues have been synthesized, including MM1-005, MM1-012, MM1-017, MM1- 018, MM1-025, MM1-026, MM1-037, MM1-039, MM1-040, MM1-066, MM1-070 and MM1-071 correspond to recombinant pro-memapsin2 with inhibition constants in the range of 1.4-61.4×109M . These inhibitors are useful in the diagnosis, treatment and/or prevention of Alzheimer's disease.

Description

[0001] related application [0002] This application claims the benefit of U.S. Patent Application Nos. 60 / 275,756 and 60 / 258,705, filed March 14, 2001, and December 28, 2000, the entire contents of which are hereby incorporated by reference in this article. Background of the invention [0003] The present invention relates to the design and synthesis of a specific inhibitor of aspartic acid protease Memapsin2 (beta-secretase or β-secretase), which is used for treating and / or preventing Alzheimer's disease. [0004] Alzheimer's disease (AD) is a degenerative brain disorder that was first documented in 1907 by Alios Alzheimer after examining a patient who suffered a dramatic decline in cognitive abilities and was diagnosed as dementia (The early story of Alzheimer's Disease, edited by Bick et al (Raven Press, New York 1987)). AD is the main cause of senile dementia. Its patients continue to lose memory, intelligence declines, and eventually lose the functions of normal peopl...

Claims

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Application Information

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IPC IPC(8): C07K14/81A61K38/55A61P25/28C07K5/03C12Q1/37
Inventor 乔丹·J·N·唐杰拉尔德·科埃尔施阿鲁安·K·格霍什
Owner OKLAHOMA MEDICAL RES FOUND
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