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 insulin or 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.

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:

Formula 1

Formula 2

The present invention discloses a compound which accelerates cleavage of insulin or amyloid-β without using ATP. 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 compound accelerates IDE related insulin and amyloid-β degradation. The compound accelerates insulin degradation by approximately 72 %.

The present invention discloses a use of the compound shown in formula 3 in the production of a medicament for treating Alzheimer's disease and type 2 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 type 2 diabetes mellitus. The molecule of the invention accelerates cleavage of insulin or amyloid-β by binding to the exosite of insulin degrading enzyme.

A compound shown in the following formula characterized in that the compound increases degradation of IDE related insulin.

Formula 3

Brief Description of the Drawings

Figure 1 : Measurement of IDE activity increase for insulin degradation in the presence of the compound shown in the formula 3 in concentrations changing between 0.02 and 40 μΜ. Figure 2: Effect of the compound in the formula 3 on the initial rate of insulin catabolism. Figure 3: Measurement of the viability of HeLa cells in the presence of certain concentrations of the compound in the formula 3.

Figure 4: Effect of IDE activators on insulin degradations in HeLa cells.

Figure 5: Representative images of live HeLa cells loaded with FITC-ins.

Figure 6: ATP effect on IDE related hydrolysis of insulin in the presence 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. Chemical structure of the compound, which is considered to be efficient as a result of the computer screening, is shown in Formula 3.

The compound in Formula 3 is 2-[(2,5-dimethyl-1 H-pyrazole-4-yl)methylidene]-5-(4- methoxyphenyl)-7-methyl-3-oxo-N-phenyl-2,3-dihydro-5H-[1 ,3]thiazoloi[3,2-a]pyrimidine-6- carboxamide. Said compound shows high binding affinity to the exosite of insulin degrading enzyme. It was found that the compound increases the cleavage of the insulin cleavage by 70 to 80 %, approximately 72 %. Interactions of Formula 3 with IDE residues and distances between interacting atoms are determined. It was found that the compound interacts with exocite residues His ³³⁶ and Gly ³⁶ , and also creates interactions with Glu ⁴⁵³ and with Tyr ⁶⁰⁹ (Table 1 ). Binding

Docking

Energy Interacting

Compound Chemical Structure energy

Free residues

(kcal/mol)

(kcal/mol)

His ³³⁶ , Gly ³⁶¹ ,

Formula 3 -1 1.13 -13.1 1

Glu ⁴⁵³ , Tyr ⁶⁰⁹

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 . It was found that the compound in formula 3 enhances the substrate V degradation by 10 to 20 %, approximately 15 %. The compound in formula 3 enhances IDE related insulin degradation by approximately 72 % (Figure 1 ). It was observed that the compound also enhances FA3B hydrolysis by 3 to 8 %. It was observed that the formula 3 is relatively effective particularly on insulin degradation. Formula 3 enhances insulin degradation in the range of 70-80 %, by approximately 72 %. After the effects were provided, 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 the formula 3 in insulin degradation was determined as 20.04 μΜ. 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. D3 increases the initial rate of proteolysis activity of IDE on insulin substrate by about 17-fold (Fig. 2). D3 does not have any effect on the proteolysis activity of IDE for amyloid-β.

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. Consequently, it was found that D3 compound did not demonstrate any toxicity even at high concentrations (Fig. 3). These results indicate that the compound in the formula 3 is not toxic to mammalian cells.

The effect of the compound shown in the formula 3 on insulin catabolism in cultured cells was explored. In HeLa cells, it is seen that the degradation of insulin with comparable to that obtained in vitro was enhanced (Fig. 4). The changes in fluorescence in the presence of compound in the formula 3 were monitored. In order to translocate FITC-labeled insulin within the cells, and show that cytoplasmic IDE can degrade FITC-labeled insulin, live cell imaging of HeLa cells was conducted. Changes in the fluorescent were monitored (Fig. 5).

In order to determine ATP effect on the insulin degradation, an experiment was conducted by the compound in the formula 3; and it was determined that IDE activity was not changed significantly during the proteolysis in the presence of 0.1 mM ATP (Fig. 6). As a result of conducted experiments, it is expected that the compound shown in the formula 3 will be 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 insulin or amyloid-β cleavage rate of the enzyme.

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.