WO2013004556A1 - A molecule regulating activity of insulin degrading enzyme (ide) - Google Patents

A molecule regulating activity of insulin degrading enzyme (ide) Download PDF

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Publication number
WO2013004556A1
WO2013004556A1 PCT/EP2012/062404 EP2012062404W WO2013004556A1 WO 2013004556 A1 WO2013004556 A1 WO 2013004556A1 EP 2012062404 W EP2012062404 W EP 2012062404W WO 2013004556 A1 WO2013004556 A1 WO 2013004556A1
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Prior art keywords
compound
ide
amyloid
degrading enzyme
degradation
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PCT/EP2012/062404
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French (fr)
Inventor
Metin TURKAY
Seda Kizilel
Halil KAVAKLI
Bilal CAKIR
Onur DAGLIYAN
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Koc Universitesi
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to the treatment of Alzheimer's disease and type 2 diabetes mellitus by a small molecule changing activity of IDE.
  • the molecule of the invention binds to the exosite of insulin degrading enzyme. Therefore, the molecule accelerates amyloid- ⁇ cleavage.
  • IDE Prior Art Insulin degrading enzyme
  • IDE Prior Art Insulin degrading enzyme
  • N- terminal has an exosite.
  • Said exosite plays a role as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules which bind to the exosite of IDE. Thus, its proteolytic activity towards different substrates can be enhanced.
  • Amyloid- ⁇ protein and insulin are substrates of the insulin degrading enzyme.
  • IDE consists of two 56 kDa N- and C-terminal domains. These domains have four structurally homologous ⁇ roll domains. These two N- and C-terminal domains are connected by 28 amino acid residues loop and constitute a large catalytic chamber where peptides smaller than 70 residues can fit.
  • Ensuring the regulation of IDE by using molecules such as medicament is very important approach for the treatment of Alzheimer's disease and type 2 diabetes mellitus.
  • Amyloid- ⁇ protein and insulin which are two substrates of insulin degrading enzyme is known to be important in the pathogenesis of Alzheimer's disease and type 2 diabetes mellitus.
  • Type 2 diabetes mellitus has a strong relationship with insulin resistance. Recent studies in the state of the art attract attention to the relationship of insulin resistance and the disorder glucose tolerance with Alzheimer's disease. It is believed that elimination of insulin resistance decreases the risk of Alzheimer's disease.
  • Cabrol et al. introduced two novel compounds that stimulated the proteolysis of short peptides of IDE using high-throughput screening. These two molecules are the precursor molecules that enhance the catalytic activity of IDE in the presence of ATP; however said molecules accelerate cleavage of small substrates and amyloid- ⁇ .
  • the molecules found to be efficient are shown in formula 1 and formula 2:
  • the present invention discloses a compound which accelerates cleavage of amyloid- ⁇ . Brief Description of the Invention
  • the present invention relates to high binding affinity of the compound shown in formula 3 to exosite of the insulin degrading enzyme.
  • the present invention discloses a use of the compound shown in formula 3 in the production of a medicament for the treatment of Alzheimer's disease and diabetes mellitus.
  • the aim of the invention is to develop a precursor molecule which can be medicament for Alzheimer's disease and diabetes mellitus.
  • the molecule of the invention accelerates cleavage of amyloid- ⁇ by binding to the exosite of insulin degrading enzyme.
  • Figure 1 Measurement of IDE activity increase for amyloid- ⁇ degradation in the presence of the compound shown in the formula 3 in concentrations changing between 0.02 and 40 ⁇ .
  • Figure 2 Measurement of the effect of the compound in the formula 3 on the initial rate of IDE in amyloid- ⁇ catabolism.
  • the 1 -[(4-chloro-3-nitrophenyl)methyl]-2-(4-chloro-3-nitrophenyl)-1 H-benzimidazole compound shown in formula 3 is effective on amyloid- ⁇ and increases amyloid- ⁇ degradation by %20.
  • the structures and conformation of formula 3 in the IDE exosite region, and their interactions with IDE residues and distances between interacting atoms are determined.
  • the compound in formula 3 interacts with exocite residues Gly 361 and Gin 363 . Also there is an interaction with His 332 (Table 1 ).
  • the interaction with Gly 339 and Gly 361 is significant as similar interactions were also significant when bradykinin interacted with the IDE exosite; therefore the interaction of the compound formula 3 with Gly 361 is significant.
  • IDE proteolysis activity of said compound was determined using different substrates.
  • the compounds were prepared at fixed 20 ⁇ concentrations and it was researched that whether the effect of the IDE was inhibited.
  • IDE activity was determined by monitoring the amount of hydrolysis of fluorogenic-substrate V (7-methoxycoumarin-4-y-acetyl-NPPGFSAFK-2, 4-dinitrophenyl), insulin-FITC, and FA3B by using recombinant human IDE purified from E.coli BL21.
  • Degradation of fluorogenic-substrate V, insulin-FITC, and FA3B is measured by pre-developed method based on homogenous fluorescent.
  • the compound in formula 3 enhances amyloid- ⁇ degradation (Fa3B) by approximately 20 %. It was observed that the compound is not effective on insulin degradation.
  • the compound enhances substrate V degradation by approximately 15 %.
  • the effective concentrations of the compound i.e. EC 50 values were determined with a dose response assay.
  • the effective concentration (EC 50 ) of the compound in formula 3 in amyloid- ⁇ degradation was calculated as 1.91 ⁇ ( Figure 1 ).
  • Figure 1 In order to explore the effect of the compound in formula 3 of the invention on the initial activity of the IDE, a series of experiments both in the presence and absence of the compound were performed.
  • the compound in formula 3 accelerates the initial rate of amyloid- ⁇ proteolysis by IDE by a factor of 1.3 (Fig. 2) and it does not affect proteolysis activity of IDE for other substrates..
  • the activities of the compounds are characterized with a cellular assay.
  • An established MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide or thiazolyl blue) method using HELA cells to determine whether the small molecule affects cell viability was used.
  • MTT 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide or thiazolyl blue
  • a purple formazan dye forms as a result of the cleavage of the yellow tetrazolium MTT within metabolically active cell.
  • the resulting precipitate of the intracellular formazan can be dissolved in a detergent solution and quantified spectrophotometrically at 595 nm.
  • the cytotoxicity was determined by incubating HELA cells in the presence of the compound in the formula 3 at concentrations ranging from 1 to 100 ⁇ for 24 h. After 12 h culture, cell viability was measured using the MTT method. The extent of cell death was expressed relative to a control containing DMSO. Although the compound in formula 3 demonstrate toxicity at high concentrations, it is not toxic at EC 50 concentration; 1 ,91 ⁇ (Fig. 3). These results indicate that the compound in the formula 3 is not toxic to mammalian cells.
  • the compound shown in the formula 3 is effective in the Alzheimer's disease and type 2 diabetes mellitus.
  • the compound in the formula 3 binding the exosite of the IDE with high connection affinity improves cleavage rate of amyloid- ⁇ .
  • the compound shown in the Formula 3 can be used for the production of a medicament which is suitable for the treatment of the Alzheimer's disease and type 2 diabetes mellitus. Said compound can be administered in a pharmaceutically efficient amount to a patient, who is in need of a treatment, in a drug form suitable for therapeutic use.
  • the formulations to be prepared for said purpose can comprise the compound of the invention or pharmaceutically acceptable salt thereof and different excipients known in the art.

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  • Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Diabetes (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Neurosurgery (AREA)
  • Epidemiology (AREA)
  • Obesity (AREA)
  • Hospice & Palliative Care (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Psychiatry (AREA)
  • Emergency Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The present invention relates to the treatment of Alzheimer's disease and type 2 diabetes mellitus by a small molecule changing the activity of IDE. The molecule binding to the exosite of insulin degrading enzyme accelerates amyloid-β cleavage.

Description

DESCRIPTION
A MOLECULE REGULATING ACTIVITY OF INSULIN DEGRADING ENZYME (IDE) Technical Field
The present invention relates to the treatment of Alzheimer's disease and type 2 diabetes mellitus by a small molecule changing activity of IDE. The molecule of the invention binds to the exosite of insulin degrading enzyme. Therefore, the molecule accelerates amyloid-β cleavage.
Prior Art Insulin degrading enzyme (IDE) is an allosteric Zn+2 metalloprotease involved in the degradation of peptides including amyloid-β, and insulin. Said enzyme plays a key role in Alzheimer's disease and type 2 diabetes mellitus. Crystal structure of IDE revealed that N- terminal has an exosite. Said exosite plays a role as a regulation site by orientation of the substrates of IDE to the catalytic site. It is possible to find small molecules which bind to the exosite of IDE. Thus, its proteolytic activity towards different substrates can be enhanced. Amyloid-β protein and insulin are substrates of the insulin degrading enzyme.
IDE consists of two 56 kDa N- and C-terminal domains. These domains have four structurally homologous αβ roll domains. These two N- and C-terminal domains are connected by 28 amino acid residues loop and constitute a large catalytic chamber where peptides smaller than 70 residues can fit.
Ensuring the regulation of IDE by using molecules such as medicament is very important approach for the treatment of Alzheimer's disease and type 2 diabetes mellitus. Amyloid-β protein and insulin, which are two substrates of insulin degrading enzyme is known to be important in the pathogenesis of Alzheimer's disease and type 2 diabetes mellitus. Type 2 diabetes mellitus has a strong relationship with insulin resistance. Recent studies in the state of the art attract attention to the relationship of insulin resistance and the disorder glucose tolerance with Alzheimer's disease. It is believed that elimination of insulin resistance decreases the risk of Alzheimer's disease.
Cabrol et al. introduced two novel compounds that stimulated the proteolysis of short peptides of IDE using high-throughput screening. These two molecules are the precursor molecules that enhance the catalytic activity of IDE in the presence of ATP; however said molecules accelerate cleavage of small substrates and amyloid-β. The molecules found to be efficient are shown in formula 1 and formula 2:
Figure imgf000004_0001
Formula 1
Figure imgf000004_0002
Formula 2
The present invention discloses a compound which accelerates cleavage of amyloid-β. Brief Description of the Invention
The present invention relates to high binding affinity of the compound shown in formula 3 to exosite of the insulin degrading enzyme.
I
Figure imgf000005_0001
Formula 3
The present invention discloses a use of the compound shown in formula 3 in the production of a medicament for the treatment of Alzheimer's disease and diabetes mellitus.
Objectives of the Invention
The aim of the invention is to develop a precursor molecule which can be medicament for Alzheimer's disease and diabetes mellitus. The molecule of the invention accelerates cleavage of amyloid-β by binding to the exosite of insulin degrading enzyme. Brief Description of the Drawings
Figure 1 : Measurement of IDE activity increase for amyloid-β degradation in the presence of the compound shown in the formula 3 in concentrations changing between 0.02 and 40 μΜ. Figure 2: Measurement of the effect of the compound in the formula 3 on the initial rate of IDE in amyloid-β catabolism.
Figure 3: Measurement of the viability of HeLa cells in the presence of certain concentrations of the compound in the formula 3. Description of the Invention
Increase in elderly population, more frequent occurrence of systemic diseases such as hypertension, cardiac disease, Alzheimer's disease and diabetes, etc. and complications related to them pave the way for studies on activity of insulin degrading enzyme for treating Alzheimer's disease and diabetes. In the present invention, approximately 800.000 chemical molecules are screened with computer for said reason and the molecules which are expected to be efficient are analyzed experimentally. It is seen that the molecules regulate the activity of IDE enzyme and as a result accelerate the cleavage of insulin and amyloid-β substrates. These are precursor molecules which can be medicament for Alzheimer's disease and diabetes mellitus potentially.
As a result of the computer screening, these compounds show high binding affinity to the exosite of insulin degrading enzyme.
The 1 -[(4-chloro-3-nitrophenyl)methyl]-2-(4-chloro-3-nitrophenyl)-1 H-benzimidazole compound shown in formula 3 is effective on amyloid-β and increases amyloid-β degradation by %20. The structures and conformation of formula 3 in the IDE exosite region, and their interactions with IDE residues and distances between interacting atoms are determined. The compound in formula 3 interacts with exocite residues Gly361 and Gin363. Also there is an interaction with His332 (Table 1 ). The interaction with Gly339 and Gly361 is significant as similar interactions were also significant when bradykinin interacted with the IDE exosite; therefore the interaction of the compound formula 3 with Gly361 is significant.
Binding
Docking
Energy Interacting
Compound Chemical Structure energy
Free residues
(kcal/mol)
(kcal/mol)
Compound of His332, Formula 3 -1 1.77 -14.23 Gly361 ,
Gin363 a N '—Cr
//
0
Table 1 In order to detect efficiency of the compound in the formula 3 of the invention, the effect of the IDE on the enzymatic activity was researched. For this reason, IDE proteolysis activity of said compound was determined using different substrates. The compounds were prepared at fixed 20 μΜ concentrations and it was researched that whether the effect of the IDE was inhibited. IDE activity was determined by monitoring the amount of hydrolysis of fluorogenic-substrate V (7-methoxycoumarin-4-y-acetyl-NPPGFSAFK-2, 4-dinitrophenyl), insulin-FITC, and FA3B by using recombinant human IDE purified from E.coli BL21. Degradation of fluorogenic-substrate V, insulin-FITC, and FA3B is measured by pre-developed method based on homogenous fluorescent. The compound in formula 3 enhances amyloid-β degradation (Fa3B) by approximately 20 %. It was observed that the compound is not effective on insulin degradation. The compound enhances substrate V degradation by approximately 15 %.
After it was demonstrated that the compound is effective, the effective concentrations of the compound i.e. EC50 values were determined with a dose response assay. The effective concentration (EC50) of the compound in formula 3 in amyloid-β degradation was calculated as 1.91 μΜ (Figure 1 ). In order to explore the effect of the compound in formula 3 of the invention on the initial activity of the IDE, a series of experiments both in the presence and absence of the compound were performed. The compound in formula 3 accelerates the initial rate of amyloid-β proteolysis by IDE by a factor of 1.3 (Fig. 2) and it does not affect proteolysis activity of IDE for other substrates..
In order to explore whether they have any cytotoxic effects of compound in the formula 3 of the invention, the activities of the compounds are characterized with a cellular assay. An established MTT (3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide or thiazolyl blue) method using HELA cells to determine whether the small molecule affects cell viability was used. In said method, a purple formazan dye forms as a result of the cleavage of the yellow tetrazolium MTT within metabolically active cell. The resulting precipitate of the intracellular formazan can be dissolved in a detergent solution and quantified spectrophotometrically at 595 nm. The cytotoxicity was determined by incubating HELA cells in the presence of the compound in the formula 3 at concentrations ranging from 1 to 100 μηι for 24 h. After 12 h culture, cell viability was measured using the MTT method. The extent of cell death was expressed relative to a control containing DMSO. Although the compound in formula 3 demonstrate toxicity at high concentrations, it is not toxic at EC50 concentration; 1 ,91 μΜ (Fig. 3). These results indicate that the compound in the formula 3 is not toxic to mammalian cells.
As a result of the conducted experiments, it is expected that the compound shown in the formula 3 is effective in the Alzheimer's disease and type 2 diabetes mellitus. The compound in the formula 3 binding the exosite of the IDE with high connection affinity improves cleavage rate of amyloid-β .
The compound shown in the Formula 3 can be used for the production of a medicament which is suitable for the treatment of the Alzheimer's disease and type 2 diabetes mellitus. Said compound can be administered in a pharmaceutically efficient amount to a patient, who is in need of a treatment, in a drug form suitable for therapeutic use. The formulations to be prepared for said purpose can comprise the compound of the invention or pharmaceutically acceptable salt thereof and different excipients known in the art.

Claims

A compound having the following formula characterized in that the compound shows high binding affinity to the exosite of insulin degrading enzyme.
Figure imgf000010_0001
2. A compound according to claim 1 characterized in that the insulin degrading enzyme interacts with exocite residues His332 ,Gly361, and Gin363
3. A compound according to claim 1 characterized by accelerating amyloid-β degradation.
4. A compound according to claim 1 characterized by increasing amyloid-β degradation by approximately 20 %.
5. A compound according to claim 1 characterized by being used in the treatment of Alzheimer's disease and type 2 diabetes mellitus.
6. A pharmaceutical formulation characterized by comprising a compound according to claim 1 or pharmaceutically acceptable salt thereof and different excipients.
7. Use of a compound having the following formula in the production of a medicament for the treatment of Alzheimer's disease and type 2 diabetes mellitus.
Figure imgf000011_0001
8. The use according to claim 7 characterized in that said compound increases amyloid-β degradation.
9. The use according to claim 7 characterized in that said compound has high binding affinity to exocite of insulin degrading enzyme.
10. The use according to claim 7 characterized in that said compound increases amyloid-β degradation by approximately 20 %.
11. The use according to claim 7 characterized in that said compound interacts with exocite residues His332 ,Gly361 , and Gin363 of the insulin degrading enzyme .
12. A compound having the following formula characterized in that said compound accelerates amyloid-β degradation.
Figure imgf000011_0002
13. A compound according to claim 12 characterized in that the compound increases amyloid-β degradation by approximately 20 %.
PCT/EP2012/062404 2011-07-01 2012-06-27 A molecule regulating activity of insulin degrading enzyme (ide) WO2013004556A1 (en)

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TRA2011/06573 2011-07-01
TR2011/06573A TR201106573A2 (en) 2011-07-01 2011-07-01 A molecule that regulates the activity of the insulin-degrading enzyme (IDE).

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002069965A1 (en) * 2001-03-05 2002-09-12 Transtech Pharma, Inc. Benzimidazole derivatives as therapeutic agents

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002069965A1 (en) * 2001-03-05 2002-09-12 Transtech Pharma, Inc. Benzimidazole derivatives as therapeutic agents

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ÇAKIR BILAL ET AL: "Structure based discovery of small molecules to regulate the activity of human insulin degrading enzyme.", PLOS ONE 2012 LNKD- PUBMED:22355395, vol. 7, no. 2, February 2012 (2012-02-01), pages e31787, XP009161586, ISSN: 1932-6203 *
DATABASE REGISTRY [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; 24 August 2006 (2006-08-24), XP002681020, Database accession no. 904267-37-4 *
DATABASE REGISTRY [Online] CHEMICAL ABSTRACTS SERVICE, COLUMBUS, OHIO, US; 30 December 2003 (2003-12-30), XP002683880, retrieved from STN Database accession no. 632296-24-3 *

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