The use of dynamin-related protein 1 - gtpase inhibitors in the field of mitochondrial fission inhibition

Description

[0001] THE USE OF DYNAMIN-RELATED PROTEIN 1 - GTPASE INHIBITORS IN THE FIELD OF MITOCHONDRIAL FISSION INHIBITION

[0002] Technical Field

[0003] The invention relates to a molecule 3-[3-[3-(lH-indol-6-yl)-l,2,4-oxadiazol-5-yl]-N-[5-(methoxymethyl)-l,3,4-thiadiazol-2-yl]propanamide (formula (I)) which targets Drpl-GTPase interaction induced by mitochondrial fission activation and can be used in the treatment of various diseases or disorders.

[0004]

[0005] Formula (I)

[0006] Prior Art

[0007] Mitochondria are a vital organelle that controls the life and death processes of cells, and cells need to maintain appropriate mitochondrial quality and quantity in order to maintain normal functional] Mitochondria in motion were found to have a displacement of 2-30 pm per minute in the direction of their long axis. This displacement leads to changes in the shape of the mitochondria in the form of slight thickening and contraction on the axis, reflecting the localised internal organisation of the cristae. These internal dynamical changes observed in mitochondria are termed mitochondrial fission (the splitting of mitochondria into smaller fragments) and fusion (fusion of fragmented mitochondria) [2, 3]. It is known that mitochondria in cells of many tissues undergo fission or fusion by constantlychanging their shape and size, thus ensuring both the continuity of mitochondrial quality and the fulfilment of cellular energy needs. Studies on the proteins involved in fission and fusion processes have revealed that changes in the activity of these proteins cause changes in mitochondrial structure [4-6]. In this context, alterations in the expression of mitochondrial fission and fusion proteins affect not only the shape of mitochondria but also their distribution and dynamics within the cell. In general, Fisl, Mff, MiD49, MiD51 and dynamin -related protein 1 (Drpl) are involved in mitochondrial fission. [7] In mammals, Drpl is mostly distributed throughout the cytosol, with a portion localized as punctate structures on the outer mitochondrial membrane. [8] In addition to forming dimer / tetramer structures, Drpl also organises into ring-shaped structures. [9] Many of the proteins involved in mitochondrial fission are members of the dynamin GTPase protein family and act similarly to their endocytic forms. During fission, dynamin-related protein Drpl is recruited to the constriction sites of the outer mitochondrial membrane by adaptor proteins such as Fisl, Mff, MiD49, and MiD51, where it assembles into large homomultimeric structures that wrap around the mitochondrion in a helical manner.

[0008]

[0010] Mitochondrial fission also plays an active role in the even distribution of mitochondria to daughter cells during cell division, as well as in the transport of the organelle to energy-demanding sites in the cell, such as neuronal axons and lamellipods. With the elucidation of the mechanisms involved in mitochondrial dynamic processes, the active roles of these processes in different diseases (such as cancer, cardiovascular, diabetes and neurodegenerative diseases) have begun to be revealed. [11-14] Increased abnormal mitochondrial fission has also been implicated as a pathogenic factor in different cancer models.

[0015] In addition, researchers have shown that Drpl protein, which plays a role in mitochondrial fission mechanism, plays a role in both the initiation, transformation and metastatic stages of cancer.

[0014] Increased expression of Drpl has been demonstrated in breast, lung, pancreatic, and thyroid cancers through studies conducted by various researchers. [16- 18] In a study with hepatocellular carcinoma patients, it was reported that increased Drpl levels in tumour tissues were associated with poor prognosis.

[0018] In the same study, it was demonstrated that tumour growth wassignificantly suppressed in in vivo mouse models treated with a mitochondrial fission inhibitor.

[0009] In addition to cancer, mitochondrial fission mechanism regulated through Drpl has been found to be increased in neurodegenerative diseases. Studies have shown that silencing PINK1 leads to increased Drpl protein levels, which in turn enhances mitochondrial fission and oxidative stress, while reducing ATP production.

[0019] These effects seen as a result of PINK 1 silencing were reversed by increasing Mfn 1 , Mfn2 and 0PA1 protein expressions as well as by using a Drpl inhibitor called Mdivi-1.

[0020] In addition to Parkinson’s disease, increased expression of the Drpl protein involved in mitochondrial fission has also been identified in Alzheimer’s disease. In Alzheimer's disease, mitochondrial dysfunctions such as impaired lipid metabolism and calcium homeostasis, decreased energy metabolism and increased oxidative damage are among the markers seen in the early stage of disease pathology. [21, 22] In addition, abnormal mitochondrial shape and dynamics reported in recent studies also reveal a clear relationship between mitochondrial form and function in Alzheimer's disease. In 2006, Baloyannis et al. showed that abnormally small mitochondria with damaged cristae structures were found in neurons of patients with Alzheimer's disease as a result of electron microscopy studies.

[0023] Finally, it has been reported that increased mitochondrial fragmentation associated with effects such as increased reactive oxygen species (ROS) production, decreased mitochondrial function and increased susceptibility to apoptosis were detected in studies conducted in mice and rat models with type 1 and type 2 diabetes. [24, 25] In the study by Yu et al. it was determined that the increase in ROS was the result of mitochondrial fragmentation and it was found that the increase in mitochondrial fission was directly related to the pathology of the disease.

[0025]

[0010] The first Drpl-GTPase inhibitor is Dynasore, a molecule developed by Macia et al. in 2006, which targets Drpl-GTPase activity and is still commercially available today.

[0026] Researchers claim that this inhibitor elicits a rapid response within 1-2 minutes after treatment. However, in 2015 Preta et al. found that Dynasore is not only a Drpl inhibitor, but also reduces labile cholesterol in the plasma membraneand disrupts the organization of the lipid stack. The second commercial inhibitor on the market, obtained as a result of chemical screening by Cassidy -Stone et al. and suggested to have an inhibitory effect on the GTPase activity of Drpl, is the molecule mdivi-l.

[0027] However, in 2017, Bordt et al. revealed that mdivi-1 causes mitochondrial complex I inhibition in addition to Drpl inhibition.

[0028] In 2012 and 2018, mitochondrial fission inhibitor development studies conducted by the same groups focused on the relationship between Drpl and the proteins that transport Drpl to the outer membrane of mitochondria rather than Drpl -GTPase activity.

[0011] Researchers first developed a molecule called Pl 10 on the inhibition of Drpl and Fisl protein interaction, and then developed a second molecule called P259 on the inhibition of Drp and Mff protein interaction in 2018. However, it has been reported that the Pl 10 molecule that inhibits Drpl -Fisl protein interaction only works under cellular stress conditions.

[0029] In addition, both Pl 10 and P259 inhibitors have been studied in the neuroblastoma cell model and the effects of these inhibitors on cancer and diabetes models in which increased mitochondrial fission is observed are unknown.

[0012] Patent document WO2020241638A1 describes compounds of Formula (I), or their pharmaceutically acceptable salts, which may be useful for the inhibition of Drpl and for the treatment of various Drpl -mediated conditions or diseases.

[0013] When the existing studies were examined, there is a need for the development of a 3-[3-(lH-indol-6-yl)- 1,2, 4-oxadiazol-5-yl]-N-[5-(methoxymethyl)- 1,3,4-thiadiazol-2-yl]propanamide molecule that targets mitochondrial fission activation-induced Drpl-GTPase interaction in lung cancer cells (A549), pancreatic P cells (1.1B4) and neuroblastoma (SHSY-5Y) cells and can be used in the treatment of various diseases or disorders.

[0014] Objective of the Invention

[0015] The object of the present invention is to develop a molecule 3-[3-[3-(lH-indol-6-y 1) - 1 ,2,4-oxadiazol-5-yl] -N-[5 -(methoxymethyl)- 1 ,3 ,4-thiadiazol-2-yl]propanamide that targets Drpl-GTPase interaction caused by mitochondrial fission activation and can be used in the treatment of various diseases or disorders.

[0016] Detailed Description of the Invention

[0017] The results of the evaluation of the molecule used within the scope of the invention are indicated in the attached figures.

[0018] Figures;

[0019] Figure 1: Graphical representation of the RMSD value for 1000 ns simulation of the 4hlu3 molecule.

[0020] Figure 2: Confocal microscope image of mitochondria dynamic changes in 1.1B4 cells treated with 1 pM Drpl-GTPase inhibitor 4hlu3 for 72 hours, visualized using Mitotracker Red under 40x magnification.

[0021] Figure 3: Confocal microscope image of mitochondria dynamic changes in A549 cells treated with 1 pM Drpl-GTPase inhibitor 4hlu3 for 72 hours, visualized using Mitotracker Red under 40x magnification.

[0022] Figure 4: Confocal microscope image of mitochondrial dynamic changes in SHSY-5Y cells treated with 1 pM Drpl-GTPase inhibitor 4hlu3 for 72 hours, visualized using Mitotracker Red under 40x magnification.

[0023] Figure 5A: Figure of A m analyses of BEAS-2B cells treated with 1 pM 4hlu-3 and Mdivi-1 for 72 hours.

[0024] Figure 5B: Figure of A m analyses of 1.1B4 cells treated with 1 pM 4hlu-3 and Mdivi-1 for 72 hours.

[0025] Figure 5C: Figure of A m analyses of A549 cells treated with 1 pM 4hlu-3 and Mdivi-1 for 72 hours.Figure 5D: Figure of A m analyses of SHSY-5Y cells treated with 1 .M 4hlu-3 and Mdivi-1 for 72 hours.

[0026] (For Figure 5, p<0.05 (*) and p<0.005 (**) were considered statistically significant. Columns show the mean values of three independent experiments (± SD))

[0027] Figure 6: Graphical representation of the thermal kinetics of the Drp-1 - 4hlu3 interaction by differential colorimetric screening.

[0028] Figure 7: Graphical representation of the binding affinity of the 4hlu3 inhibitor to the Drpl protein based on Tm values.

[0029] The invention relates to the molecule 3-[3-[3-(lH-indol-6-yl)-l,2,4-oxadiazol-5-yl]-N-[5-(methoxymethyl)-l,3,4-thiadiazol-2-yl]propanamide, which targets Drpl-GTPase interaction induced by mitochondrial fission activation and can be used in the treatment of various diseases or disorders.

[0030] At the stage of obtaining the molecule, firstly, predictions of candidate molecules that could bind to the GTPase region of Drpl protein were carried out. The structure of Drpl - GTPase region combined with the C-terminal GTPase effector (GED) region was obtained from the protein data bank. (PDB code: 4H1U) The "protein-preparation-wizard" module of the Schrodinger program was used for the docking calculations of the protein.

[0030]

[0031] The steps for virtual scanning calculations are described below.

[0032] Step 1:

[0033] The two-dimensional structures of 3.5 million small molecules, which are ready for purchase and show lead molecule properties, were first obtained from the ZINC database in smiles format with AutoDock Vina.

[0034] During the preparation of the molecules for bonding calculations, their ionisation and tautomerisation were performed with the Epic module of the Schrodinger program.

[0031] For AutoDock Vina calculations, the file formats of proteins andsmall molecules were converted to PDBQT file format with AutoDock Tools and open babel programmes. The similarities of these molecules with Mdivi-1 and Dynasore molecules were calculated with RDKIT python library before docking. The centre of the grid box was set as 38.59, 68.13, -103.94 coordinates and the dimensions of the grid box were chosen as 30A in all axes. Tanimoto scoring was used for similarity calculations, and as a result, 33 molecules were found to have over 70% similarity to Mdivi-1. There was no molecule with more than 70% similarity with Dynasore. Therefore, docking of 33 molecules was not performed.

[0035] 3.5 million molecules were docked with AutoDock Vina with an "Exhaustiveness" parameter of 8.

[0036] Step 2:

[0037] After the first step, the molecules were ranked according to their binding scores and 200,000 molecules with the highest binding scores were selected. In the second step of the virtual screening, detailed screening of these 200,000 molecules was performed with AutoDock Vina and LeDock programmes. With AutoDock VINA programme, this time the "exhaustivenees" parameter was set to 24. In LeDock, the RMSD parameter was used as 1 A.

[0038] Step 3:

[0039] As a result of the second step, 321 molecules in the top 5000 in LeDock and AutoDock VINA results were identified. The binding calculations of 321 molecules were then performed again with the glide XP protocol.

[0040] In the glide XP results of 321 molecules, molecular clustering and ADME / T analyses were performed. Accordingly, 70 molecules with high oral drug scores were identified. The binding affinities of these 70 molecules were then examined by classical MD simulations of 50 ns length and then MM / GBSA analyses. In these simulations, the 4hlu3 molecule, which remained stable in the binding site and had a high MM / GBSA energy value, was selected for experimental tests. The chemical structure of the compound used in the invention is given below.o

[0041]

[0042] Formula (II)

[0043] Ri contains the 1 ,3,4-thiadiazolc ring bonded to the amide group and the methoxymethyl group (-OCH3) bonded to it.

[0044]

[0045] Ri. hydrogen (H) that bonded on the nitrogen of the amide group;

[0046] R3 consists of the indole ring and the 1,2,4-oxadiazole ring bonded to this ring.

[0047]

[0048] The chemical structure of the compound is given in formula (I).

[0049]

[0050] Formula (I)The compound has selective binding to the Drpl-GTPase binding site. It contains pharmaceutically acceptable salts.

[0051] The compound has a structure modified to include derivatives of groups Ri, R2 and R3. In this context;

[0052] Ri can convert to a functional group containing alkyl or aryl groups.

[0053] R2 may contain small alkyl groups such as methyl or ethyl.

[0054] R3 may contain additional functional groups such as hydroxyl, halogen, alkyl, carboxylic acid, amine, nitro and sulphonate attached to the indole ring.

[0055] When the hydrogen in the R2 group is cleaved with a strong base, an alkyl group, acyl group, halogen atom, aromatic ring, phosphonate or sulphate group can be attached to the double bonded oxygen atom. The negatively charged oxygen atom in group R2 forms a complex with a metal ion.

[0056] The inventive molecule is used in the treatment of cancer, cardiovascular diseases, diabetes and neurodegenerative diseases. It provides inhibition of mitochondrial fission in cellular conditions where Drpl activity is excessively increased.

[0057] The molecule ensures the maintenance of mitochondrial membrane potential (A m) in cells.

[0058] Application of the molecule of the invention in an exemplary embodiment comprises inhibition of Drpl-GTPase activity under conditions of increased mitochondrial fission, and the steps of applying 3-[3-[3-(lH-indol-6-yl)-l,2,4-oxadiazol-5-yl]-N-[5-(methoxymethyl)-l,3,4-thiadiazol-2-yl]propanamide, suppressing Drpl protein activity and inhibiting mitochondrial fission in target cells.

[0059] The method of application is applied to disrupt cellular energy metabolism by suppressing mitochondrial fission in cancer cells. It is applied to restore mitochondrial dysfunction in neurodegenerative diseases. It is applied to reduceoxidative stress and improve mitochondrial functions in cardiovascular diseases. It is applied to reduce the effect of increased reactive oxygen species (ROS) and inhibit mitochondrial fission in diabetes models.

[0060] The interaction of the molecule 4hlu3 (ZINC000079493999), which was determined as active in the experiments, with the Drpl-GTPase site was reexamined by MD simulation with a length of 1000ns. In this simulation, the molecule remained quite stable in the binding site (Figure 1).

[0061] A549 (lung cancer), 1.1B4 (pancreatic beta) and SH-SY5Y (neuroblastoma) cells were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS (fetal bovine serum) and 1% antibiotic (100 pg / ml streptomycin and 100 U / ml penicillin) under standard culture conditions and grown in an incubator at 37°C with 5% CO2. The cells were checked every 2-3 days under inverted microscope and when the cells became semi-confluent, they were removed from the surface with cell scraper or trypsin-EDTA and transferred to 15 ml sterile centrifuge tubes. After centrifugation at 3000 rpm for 5 minutes, the supernatant was discarded and the pellet was dissolved in 1-2 ml fresh medium. Cells were added to culture flasks containing fresh medium at the rate of 1-2 x 105cells / ml for each new passage and allowed to proliferate. In this way, the cells were checked every 2-3 days and new passages were made at certain intervals.

[0062] After different cell lines (A549, 1.1B4 and SHSY-5Y) were treated with different concentrations (5 nM, 500 nM, 1 pM and 10 pM) of Drpl-GTPase inhibitor, mitochondria were labelled with Mitotracker Red and the inhibitor concentration (IC50 values) that inhibited mitochondrial fission by half was determined by confocal microscopy studies. For confocal microscopy studies, BEAS-2B, A549, 1.1B4 and SHSY-5Y cells were seeded on 6-well micro-slides (ibidi) at 3 x 105cells / ml per well. Cells were treated with different concentrations of Drpl-GTPase inhibitors (5 nM, 500 nM, 1 pM and 10 pM), washed twice with cold DPBS and then incubated in 25 nM Mitotracker Red staining solution for 20 min at 37°C in an incubator with 5% CO2. Changes in the mitochondrial morphology of the cells were observed under a Leica TCS-SPE confocal microscope at 40x magnification.Changes in mitochondrial morphology were analysed using filtering (median), thresholding, binarisation and particle analysis in ImageJ,

[0063] (number of particles x 10000) / total mitochondrial pixels)

[0064] According to the formula, the images obtained from three different experiments were randomly selected and evaluated.

[0032] (Figure 2-4).

[0065] In order to determine the mitochondrial membrane potential (A m) of A549, 1.1B4 and SHSY-5Y cells treated with Drpl-GTPase inhibitor, the cells were collected from the wells at the end of the incubation periods and taken into flow cytometry tubes. After centrifugation at 3000 rpm for 5 minutes, 2 ml serum-free medium was added to the pellet and mixed. 1 pl / ml Rhodamine 123 was added and incubated at 37°C in the dark for 15 minutes. At the end of incubation, the cells were centrifuged at 3000 rpm for 5 minutes and 2.5 ml PBS was added to each tube after removing the supernatants. The cells were centrifuged at 3000 rpm for 5 minutes and 500 pl PBS was added to each tube after the supernatants were removed. The samples were analyzed by flow cytometry (Figure 5).

[0066] During the determination of Drpl-GTPase protein-ligand interactions of the 4hlu3 inhibitor, 96-well PCR plates were used, and samples containing only the dye were considered as negative controls. For Drp-1, 10 nM HEPES (p.H: 7.4), 2.5 pl 50X SYPRO Orange were prepared with a total reaction volume of 25 pl, with 200 ng / ml protein in each well. The well to which no inhibitor was added was considered as control. After the application of increasing concentrations of 4hlu3 (5, 50, 200, 500 and 1000 nM), the fluorescence intensity was measured in Real-time PCR (Bio-RAD) device at a wavelength of 580 nm for 10 seconds (0.5°C / 15 s) at 0.5°C between 20°C and 95°C. (Figure 6,7)

[0067] With the present invention, 3-[3-[3-(lH-indol-6-yl)-l,2,4-oxadiazol-5-yl]-N-[5-(methoxymethyl)-l,3,4-thiadiazol-2-yl]propanamide molecule has significant advantages over Mdivi-1, one of the mitochondrial fission inhibitors in the current market, and has been shown to provide more mitochondrial fission inhibition in different cell lines (A549, 1.1B4 and SHSY-5Y). Mitochondrial imaging studiesusing confocal microscope in different cell lines showed that molecule 3-[3-[3-(lH-indol-6-yl)- 1 ,2,4-oxadiazol-5-yl] -N-[5 -(methoxymethyl)- 1 ,3 ,4-thiadiazol-2-yl]propanamide effectively suppressed mitochondrial fission.

[0068] Different studies have reported that the commercial inhibitor Mdivi-1 causes mitochondrial complex I inhibition other than Drpl-GTPase interaction and therefore it is not a specific Drpl-GTPase inhibitor.

[0041] Although the cells used in these studies were neuronal cells, it has been reported in different literature studies that complex I inhibition in neuronal cells causes mitochondrial membrane depolarization.

[0033] It was observed that Mdivi-1 decreased the mitochondrial membrane potential by 40% in SHSY-5Y cells, whereas 3-[3-[3-(lH-indol-6-yl)-l,2,4-oxadiazol-5-yl]-N-[5-(methoxymethyl)-l,3,4-thiadiazol-2-yl]propanamide molecule disclosed within the scope of the invention decreased the membrane potential by 19%. (Figure 5)

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Claims

CLAIMS1. A compound of general formula (II),0^2wherein Ri is alkyl or aryl or amide group derivatives of the 1,3,4- thiadiazole ring and the methoxymethyl group (-OCH3) alkoxy group or alkyl or aryl groups bonded to the amide group,R2 is hydrogen (H) or methyl or ethyl,R3 is hydroxyl, halogen, alkyl, carboxylic acid, amine, nitro and sulphonate or indole ring or derivatives thereof,R3 is an oxadiazole, hydroxyl, halogen, alkyl, carboxylic acid, amine, nitro or sulphonate, or derivatives thereof, bonded to the indole ring.

2. The compound according to claim 1, characterized in that Ri is an alkoxy group bonded to the 1,3,4-thiadiazole ring.

3. The compound according to claim 1, characterized in that Ri is a 1,3,4- thiadiazole ring containing a methoxymethyl group at position 2.

4. The compound according to claim 1, characterized in that R3 is an oxadiazole ring attached to the indole ring.

5. The compound according to claim 1, characterized in that it is 3-[3-[3-(lH- indol-6-yl)- 1 ,2,4-oxadiazol-5-yl] -N-[5 -(methoxymethyl)- 1 ,3 ,4-thiadiazol- 2-yl]propanamide.

6. The compound according to claim 1, characterized in that it has selective binding to the Drpl-GTPase binding site.

7. The compound according to claim 5, characterized in that it is a pharmaceutically acceptable salt of 3-[3-[3-(lH-indol-6-yl)-l,2,4- oxadiazol-5-yl]-N-[5-(methoxymethyl)-l,3,4-thiadiazol-2-yl]propanamide.

8. The compound according to claim 1 or 5, characterized in that it is an inhibitor of Drpl-GTPase activity mediating increased mitochondrial fission.

9. The compound according to claim 1 or 5, characterized in that the compound is for use in the treatment of cancer, cardiovascular diseases, diabetes and neurodegenerative diseases.

10. The compound according to claim 1, characterized in that it suppresses mitochondrial fission by inhibiting Drpl and GTP protein interactions.

11. The compound according to claim 5, characterized in that it suppresses mitochondrial fission by inhibiting GTP protein interactions with Drpl.

12. The compound according to claim 1, characterized in that it maintains mitochondrial membrane potential (A m) in cells.

13. The compound according to claim 5, characterized in that it is capable of maintaining mitochondrial membrane potential (A m) in cells.

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