Activator effect of (r)-4*-methylklavuzon on p38 and JNK3 kinase enzymes

Abstract

The invention relates to the use of the (R)-4'-methylklavuzon compound as an activator of p38 and JNK3 kinase enzymes.

Description

[0001] ACTIVATOR EFFECT OF (R)-4'-METHYLKLAVUZON ON p38 AND JNK3 KINASE ENZYMES

[0002] Technical Field of the Invention

[0003] The invention relates to the field of kinase activators, which include organic molecules that activate kinase enzymes. The present invention refers to the use of the (R)-4’-methylklavuzon compound as an activator of p38 and JNK3 kinase enzymes.

[0004] State of the Art of the Invention (Prior Art)

[0005] Kinases are drug targets for many diseases such as cardiovascular diseases, cancer, inflammatory diseases, central nervous system diseases, and diabetes diseases.

[0006] Although kinase inhibition is prominent in the field of pharmacology, there may also be diseases that develop due to deficiency of kinase enzyme function. These can be listed as follows:

[0007] • desensitization of insulin receptor kinase in diabetes,

[0008] • inactivation of death-associated protein kinase (DAPK) in cancer,

[0009] • inactivation of LKB1 tumor suppressor kinase in cancer,

[0010] • decreased activity of PINK1 in the early stages of Parkinson's disease. Kinase activation is targeted in the treatment of these diseases. In relevant research studies, after it is proven that any substance used as an activator activates p38 kinase, it is shown that the activation is reversed by applying SB203580 or BIRB-796 inhibitor molecules and it is emphasized that the response obtained due to the substance depends on p38 activation. One of the most important ways to prove that a physiological response is obtained as a result of the change in the activity of the target enzyme is to use inhibitor or activator molecules.

[0011] The most common problem in kinase research is that there are limited molecular structures and that existing molecules are not selective and affect many kinases.

[0012] In almost all of the kinase activation techniques used in the literature, the relevant activator molecule is applied to cells, but the direct interaction of this molecule with the kinase enzyme is not examined. Therefore, kinase activation may have occurred through other mechanisms due to thousands of pathways in the cell.

[0013] The invention in the patent document numbered WO 2011 / 099947 Al relates to apoptosis inducers consisting of 6-bicycloaryl substituted 5,6-dihydro-2H-pyran-2-one derivatives. The (R)-(+)-6-(4-methylnaphthalen-l-yl)-5,6-dihydro-2H-pyran-2-on mentioned in the invention is the (R)-4’-methylklavuzon compound and its effect against cancer cells has been shown.

[0014] In the study conducted by Ali agir et al., the cytotoxic effects of (R)-4’-methylklavuzon were investigated on hepatocellular carcinoma cells (HuH-7 and HepG2) and HuH-7 EpCAM+ / CD133+ cancer stem cells. It was shown that (R)-4’-methylklavuzon inhibits the CRM1 protein, which provides increased retention of p53 and RIOK2 protein in the nucleus.

[0015] Activation of some kinase enzymes is important for drug development for diseases and the field of kinase activation has shown limited development in the present state of the technique. Therefore, new compounds are needed to be used in in vitro research and development analyses where the activities of kinase enzymes are studied.

[0016] Brief Description and Objects of the Invention

[0017] The primary purpose of the invention is to demonstrate the activating effect of (R)-4’- methylklavuzon compound by interacting directly with the related kinase enzymes p38 (p38a, p38p, p38y) and JNK3 kinase enzymes. By using (R)-4’-methylklavuzon, p38a and p38p kinase enzymes were activated by 100%, p38y kinase enzyme by 50%, and JNK3 enzyme by 40%. In this respect, (R)-4’-methylklavuzon provides more efficient activation than molecules known in the literature. The reason for this is the possibility of the molecule binding directly to the enzyme itself. (R)-4’-methylklavuzon has the potential to be used in research studies for both basic science and clinical science for kinase activation.

[0018] While the increase in enzyme activity continued at increasing concentrations in p38a and p38p enzymes, the activity remained constant and did not increase at high doses in p38y. The affinity of the (R)-4’-methylklavuzon molecule for p38a and p38p is higher than its affinity for p38y and it can be used as a reference molecule in the development of new active substances by allowing other researchers to test their own inhibitor or activator molecules in order to provide selective targeting between p38a, p38p, p38y and JNK3, which have highly conserved amino acid sequences in evolutionary terms.

[0019] (R)-4’-methylklavuzon molecule can be used as a reference molecule in research on developing treatments for diseases such as NRASQ61K mutant melanoma due to its statistically significant activation of p38a, p38p, p38y kinase enzymes.

[0020] (R)-4’-methylklavuzon molecule can be used as a reference molecule in research on developing neoadjuvant chemotherapy for breast cancer due to its statistically significant activation of JNK3 kinase enzyme.

[0021] (R)-4’-methylklavuzon molecule can be used as a positive activator molecule for in vitro enzyme activity assays including the direct interaction between purified p38a, p38p, p38y, JNK3 enzymes and the substance. In basic science research experiments, the greatest evidence of whether the experiment itself, i.e. the enzyme, substrate and solutions are working or not is the positive controls used in the experiment itself. Many different enzyme activity assay kits contain activating or inhibiting positive / negative controls. (R)-4’-methylklavuzon can be used as an activating positive control in p38 and JNK3 enzyme activity assays in basic science research.

[0022] In addition to being a positive control, it can be used directly in “kinase enzyme kinetics” studies in basic science research. In enzyme kinetics studies, groups working on kinase inhibitors will be able to test whether their molecules directly interact with the kinases and then show a permanent or decreasing profile by adding (R)-4’-methylklavuzon as an activator when investigating the inhibition of p38 or JNK3. In contrast to this experimental design, they will be able to investigate the kinetics of the “activated kinase” inhibition of their inhibitor molecules by first directly interacting p38 and JNK3 with the activator molecule (R)-4’- methylklavuzon and then adding their inhibitor molecules to the reaction. As a third experimental design, they will be able to investigate whether their molecules have a competitive or non-competitive effect on the kinase enzyme activity according to the enzyme kinetics experiment results in which they directly interact with the kinase enzyme with (R)-4’- methylklavuzon and their own inhibitor molecules at the same time.

[0023] Definitions of Figures Describing the Invention

[0024] Figure 1: In vitro effect of (R)-4’-methylklavuzon on the p38a enzyme.

[0025] Figure 2: In vitro effect of (R)-4’-methylklavuzon on the p38p enzyme.

[0026] Fgure 3: In vitro effect of (R)-4’-methylklavuzon on the p38y enzyme.

[0027] Fgure 4: In vitro effect of (R)-4’-methylklavuzon on the JNK3 enzyme.

[0028] Detailed Description of the Invention

[0029] The invention relates to the use of a compound represented by Formula (I), (R)-4’- methylklavuzon, or its salt or solvate, in an in vitro assay measuring p38 or JNK3 kinase enzyme activity.

[0030] Formula I

[0031] The p38a, p38p, p38y and JNK3 kinase enzymes are shown for the first time as targets for activation, and the molecular structure of (R)-4’-methylklavuzon, which can activate p38 and JNK3 by “interacting directly with the kinase enzyme”, which is almost unheard of in the literature among the studies conducted for activation, is presented.

[0032] Within the scope of the invention, there is an in vitro analysis method for measuring p38 or JNK3 kinase enzyme activity and it includes the steps of preparing (R)-4’-methylklavuzon and p38 or JNK3 kinase enzyme mixture, adding kinase substrate, ATP, kinase reaction buffer, kinase detection reagent and cofactor to the mixture, and measuring the luminescence of the phosphorylated kinase substrate formed after the kinase reaction.

[0033] In the preferred application of the invention, the commercial kits “Kinase Selectivity Profiling System” and “ADP-Glo™ Kinase Assay Kit” which are readily available in the market were used. The experimental steps performed by using these kits and results of the experiments are explained in detail below.

[0034] KIBase SelectSiffly ProfTIBg System: CMGC-1 Enzyme Family Panel

[0035] During the drug development process, the fact that kinase inhibitor candidate molecules do not show activity against a large number of kinases is an element that increases drug safety. Promega's "Kinase Selectivity Profiling System" kit contains kinase and substrate pairs in 8- tube strips and allows a rapid study on kinase profiling reactions. The "Kinase Selectivity Profiling System" kit allows a study on a strip containing a single kinase family or with a general kinase panel representing the human kinome. Each kit contains all the reagents needed to screen the candidate molecule. These are kinase reaction buffer, 8 different kinases on each strip, and 8 substrates and cofactors for these kinases on another strip. Kinase stock solutions are standardized and when diluted, they provide optimal ATP-ADP conversion with appropriate signal / background ratio in a 5 pl reaction volume (in a 384-well microplate) using the ADP- Glo™ Kinase Assay kit.

[0036] All analyses were performed using the Kinase Selectivity Profiling System: CMGC-1 (Promega catalog number: V6854) and the ADP-Glo™ Kinase Assay kit (ADP-Glo™ Kinase Assay, Promega catalog number: V6930) according to the manufacturer's recommended protocol.

[0037] Kinases are provided in 25X or 50X concentrations, substrates and cofactors in 3.3X concentrations in 8-tube strips. After a single-step dilution, sufficient kinase and ATP / Substrate solution for 25 reactions in the 384-well microplate are provided. In the preparation of all enzyme reactions, the reagents are added to the 384-well microplate, then centrifuged, mixed in an orbital shaker and incubated.

[0038] ADP-Glo™ KQiase Assay Kt (ADP-Glo™ KBase Assay)

[0039] ADP-Glo™ Kinase Assay kit (Promega catalog number: V6930) is a luminescence ADP (Adenine diphosphate) detection assay and is a kinase activity measurement method based on determining the amount of ADP produced as a result of the kinase reaction for multiple screenings. The ADP-Glo™ Kinase Assay kit can be used to measure the activity of many enzymes that can produce ADP. The assay is performed in multi-well plates and can determine reactions even in 5 pl volumes. The assay consists of two parts; in the first part, after the kinase reaction, an equal volume of ADP-Glo™ Reagent is added to terminate the kinase reaction and the remaining ATP (Adenine triphosphate) molecules are degraded. In the second part, Kinase Detection Reagent is added and the ADP in the medium is converted to ATP and the newly synthesized ATP is measured by the luciferase / luciferin reaction. The resulting luminescence is measured by a luminometer. (Auld, D.S. et al., 2009; Jia, Y. et al., 2008; Walsh, D.A. and Glass, D.B., 1991; Hidaka, H., Watanabe, M. and Kobayashi, K., 1991; Zhang, J.H. et al., 1999; Alli, C. et al., 2009; Auld, D.S. et al., 2009). Luciferase is an enzyme of the oxidoreductase class. It increases the reaction rate in converting reactants into products. In this reaction, the substrate of the luciferase enzyme, which is an oxidoreductase, is luciferin and it is converted to the product oxyluciferin by the luciferase enzyme.

[0040] Kinase reactions are initiated by mixing 1 pl of the test molecule (R)-4’-methylklavuzon and 2 pl of Kinase enzyme and incubating them in an incubator at room temperature for 10 minutes without adding substrate. In this way, it is aimed for the molecule to bind to the enzyme easily in a substrate-free environment. After this incubation, 2 pl of ATP / Substrate (IPTTPITTTYFFFKKK peptide, SignalChem, P03-58) solution is added and incubated in an incubator at room temperature for 1 hour. After 1 hour of incubation at room temperature, kinase activity is measured with the ADP-Glo™ Kinase Assay kit. For this, 5 pl of the ADP- Glo Reagent provided with the kit is added to each well and incubated in an incubator at room temperature for 40 minutes. As the last step, 10 pl of Kinase Detection Reagent is added to all wells and incubated for 30 minutes in an incubator at room temperature. After all incubations, luminescence measurements are performed using a microplate reader (Thermo, Varioskan). The luminescence values obtained are analyzed with the Graphpad Prism 9 program. In this analysis, the enzyme activity without (R)-4’-methylklavuzon is accepted as 100% and the enzyme activity of other samples is compared to the control enzyme.

[0041] In the experiments within the scope of the invention, a substrate called "p38 substrate" was used. The substrate is a synthetic peptide and has serine / threonine protein kinase phosphorylation sites. It is also used as a substrate for p38 family kinases. Its amino acid sequence is Ile-Pro-Thr-Thr-Pro-Ile-Thr-Thr-Thr-Tyr-Phe-Phe-Phe-Lys-Lys-Lys (IPTTPITTTYFFFKKK). Reference: https signalchem.com / shared, product heetsZEi298-

[0042] PreparatBn of the Dose-Response Exper nient

[0043] The dose-response experiment was performed to investigate the kinase enzyme activity of the drug candidate (R)-4’-methylklavuzon molecule used in different concentrations by following the steps below.

[0044] • 2 pl of kinase solutions and (R)-4’-methylklavuzon prepared at different doses are placed in wells 1 to 22. Centrifuged to collect all the content at the bottom. Mixed for 2 minutes on a microplate mixer and centrifuged again, then incubated at room temperature for 10 minutes.

[0045] • 2 pl of ATP / Substrate solution is added to all wells. Centrifuged, mixed for 2 minutes on a microplate mixer and centrifuged again, then incubated at room temperature for 60 minutes.

[0046] • After incubation, 5 pl of ADP-Glo™ Reagent is added to all wells, centrifuged, mixed for 2 minutes, and centrifuged again, then incubated at room temperature for 40 minutes.

[0047] • 10 pl of kinase detection solution is added to all wells, centrifuged, mixed for 2 minutes, and centrifuged again, then incubated at room temperature for 30 minutes.

[0048] • Luminescence is measured by a microplate reader (Thermo, VarioSkan).

[0049] • For data analysis, the luminescence from the wells without enzyme is subtracted from the luminescence of all wells with or without (R)-4’-methylklavuzon. The kinase activity of the control wells without (R)-4’-methylklavuzon is considered as 100% and the percentage of kinase activity in the wells containing the drug candidate is calculated. Control ExperOients for ATP Degradation and ATP Synthes 8

[0050] The experimental design given in Table 1 below was performed to understand whether (R)-4’- methylklavuzon inhibits enzymes such as ATPase and Luciferase that provide luminescence production in the analysis kits. Table 1. Control Experiments

[0051] As a result of control experiments, it was shown that (R)-4’-methylklavuzon did not interfere with the ATP degradation reaction or ongoing reactions and that the high measured values of (R)-4’-methylklavuzon in kinase activation experiments were mostly due to kinase activation. The increase in p38a, p38p, p38y and JNK3 enzyme activities by (R)-4’-methylklavuzon is shown in Figures 1-4.

Claims

CLAIMS1. Use of a compound of Formula (I), (R)-4’-methylklavuzon, or its salt or solvate, in an in vitro assay measuring p38 or JNK3 kinase enzyme activity.Formula (I)2. The use according to Claim 1, using of p38a, p38p or p38y as the p38 kinase enzyme.

3. An in vitro analysis method to measure p38 or JNK3 kinase enzyme activity, the method comprising;• Preparation of (R)-4’-methylklavuzon and p38 or JNK3 kinase enzyme mixture,•Addition of kinase substrate, ATP, kinase reaction buffer, kinase detection reagent, oxidoreductase enzyme, oxidoreductase enzyme substrate and cofactor to the mixture,•Measurement of the luminescence of the phosphorylated kinase substrate formed after the kinase reaction.

4. An in vitro analysis method according to Claim 3, the method comprising; mixing of (R)-4’- methylklavuzon with at least one enzyme selected from the group comprising p38a, p38p and p38y.

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