CN114681446A - Application of acetamide compound as glutamate dehydrogenase inhibitor - Google Patents
Application of acetamide compound as glutamate dehydrogenase inhibitor Download PDFInfo
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- CN114681446A CN114681446A CN202011625226.2A CN202011625226A CN114681446A CN 114681446 A CN114681446 A CN 114681446A CN 202011625226 A CN202011625226 A CN 202011625226A CN 114681446 A CN114681446 A CN 114681446A
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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Abstract
The invention provides an application of acetamide compound (S) -2- (2- (1-H-indole-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide as a Glutamate Dehydrogenase (GDH) inhibitor; the compound has effective inhibition effect on the activity of Glutamate Dehydrogenase (GDH) under the condition of micromolar concentration. Glutamate dehydrogenase is a mitochondrial enzyme and is a key enzyme for entry of glutamate into the tricarboxylic acid cycle to produce ATP. One of the reasons why the function gain mutation of GDH is responsible for the Congenital Hyperinsulinemia (CHI) in newborn infants. With the development of research, there is increasing evidence that GDH is closely related to the activities of tumor cell proliferation, migration, invasion, etc., and there are abnormal expression phenomena in many tumor cells. The GDH inhibitor provided by the invention can well inhibit the activity of GDH, and further achieves the effects of treating CHI caused by GDH mutation and searching for effective cancer treatment drugs.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to application of an acetamide compound (S) -2- (2- (1-H-indole-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide as a glutamate dehydrogenase inhibitor.
Background
Congenital Hyperinsulinemia (CHI) is persistent hypoglycemia caused by excessive insulin secretion and increased blood insulin concentration, and is difficult to cure. The causes of hypoglycemia are complex (at least 11 pathogenic genes have been found), and the major pathogenic genes include:
1) a key enzyme for amino acid metabolism, namely Glutamate Dehydrogenase (GDH), obtains functional mutation;
2) a key enzyme of glucose metabolism, Glucokinase (GCK) gain of function mutation;
3) loss of function mutations in the ATP-dependent potassium channel (SUR1 and kir 6.2);
4) loss of function mutation of fatty acid metabolism key enzyme (SCHAD), etc.
The functional mutation of Glutamate Dehydrogenase (GDH) is a subtype of CHI, the mutant site of GDH has enhanced function if it interferes with the sensitivity to endogenous inhibitors, and the functional enhancement of GDH in islet beta cells directly results in inappropriate secretion of insulin, which in turn leads to excessive insulin release and hypoglycemia. Patients not only suffer from hypoglycemia, but also hyperammonemia caused by liver and kidney function damage, epilepsy caused by brain function damage, delayed brain development, learning ability disorder and the like, are systemic diseases, and urgently need a systemic inhibitor aiming at mutant GDH.
At present, the treatment means and medicines for CHI are very limited, the first-line medicine diazoxide only aims at one third of the children with hypoglycemia, no treatment method is available for brain dysfunction and liver and kidney damage, side effects such as water and sodium retention and hyperglycemia can be caused, and the treatment of the children with hypoglycemia caused by non-genetic diseases is greatly limited. For patients with diazoxide ineffectiveness, pancreas needs to be surgically removed, the pancreas is difficult to be surgically removed in China, diabetes is directly caused, and insulin needs to be injected for life, so that the patients urgently need new therapeutic drugs, and the development of GDH inhibitors provides a new approach for treating CHI.
Increased transcription of the GDH gene is currently found in many types of cancers, such as breast, glioma, colorectal, and ovarian cancers. The tumor cell gene mutation leads to the obvious change of the glucose aerobic sugar metabolic pathway, and under the condition of sufficient oxygen, most of glucose is subjected to anaerobic glycolysis to generate a large amount of ATP to maintain the self metabolic demand. In order to meet the requirement of biological macromolecule synthesis, a tumor cell takes in a large amount of glutamine, glutamic acid is generated in a mitochondria through the catalysis of the glutamine enzyme, alpha-ketoglutarate (alpha-KG) generated by the catalysis of the GDH on the glutamic acid participates in tricarboxylic acid (TCA) cycle, and provides an energy source for the tumor cell, so that the aim of inhibiting the growth of the tumor cell can be achieved by inhibiting the activity of the GDH in the pathway. GDH is becoming a potential target for cancer therapy, and screening for GDH inhibitors has become a focus of research.
Disclosure of Invention
In order to overcome the defects of the existing medicines for treating congenital hyperinsulinemia, and develop the function of GDH as a cancer treatment target, the invention provides a GDH inhibitor which shows a certain inhibition effect on GDH activity in an enzyme kinetic test of GDH under the condition of micromolar concentration.
The invention is realized by adopting the following modes:
use of the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide as glutamate dehydrogenase inhibitor, characterized in that the above compound components are used for inhibiting the activity of glutamate dehydrogenase;
the chemical formula of the compound component is as follows:
further, the above GDH inhibitor was used to inhibit the activity of GDH, and the half inhibitory concentration IC50 of the fraction was 17.23 μ M.
In a second aspect of the present invention, there is provided a method for using a GDH inhibitor, characterized in that a compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide is used as a glutamate dehydrogenase inhibitor, and the GDH inhibitor is used to inhibit the activity of GDH;
further, the application method applies the inhibitor to reduce insulin release, raise blood sugar and relieve islet pressure.
Further, the application method applies the inhibitor to congenital hyperinsulinemia and cancer treatment.
Further, the application method applies the inhibitor to the treatment of hyperinsulinemia-hyperammonemia syndrome.
In a third aspect of the present invention, there is provided a therapeutic agent for congenital hyperinsulinemia, which comprises the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide, which is used as an inhibitor of glutamate dehydrogenase.
In a fourth aspect of the present invention, there is provided a therapeutic agent for hyperinsulinemia-hyperammonemia syndrome, which comprises the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide, which is used as an inhibitor of glutamate dehydrogenase.
The invention has the beneficial effects that:
the invention provides application of a compound (S) -2- (2- (1-H-indole-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide in improvement of hypoglycemia in treatment of functional mutation obtained by glutamate dehydrogenase.
The micromolar concentration of (S) -2- (2- (1-H-indole-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide can effectively inhibit the activity of GDH, so that effective attempts are provided for solving the problem of rare medicines for rare CHIs, the current situation of CHI treatment can be obviously improved, the passive situation that GDH cannot be used by patients with functional mutation is broken, the life quality of the patients is improved, and meanwhile, the medicines capable of effectively inhibiting the activity of GDH in tumor cells are screened and developed, and the application prospect is wide.
Drawings
FIG. 1 is a graph showing the results of the GDH enzyme kinetic assay; the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide (represented by AR-Z-005) has an inhibitory effect curve on GDH activity under micromolar conditions.
FIG. 2 shows the results of islet incubation experiments; bar graphs of the amount of insulin released by adding the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide (denoted as AR-Z-005) at various concentrations to the glutamine and leucine groups.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained by combining the specific embodiments.
Example one
The use of the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide as an inhibitor of glutamate dehydrogenase, for the inhibition of glutamate dehydrogenase activity, of the following compound components:
the chemical formula of the compound component is as follows:
the GDH inhibitor described above was used to inhibit GDH activity with a half inhibitory concentration IC50 of 17.23 μ M.
Example two
A method for using a GDH inhibitor, characterized in that (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide is used as a glutamate dehydrogenase inhibitor, and the GDH inhibitor is used for inhibiting the activity of GDH;
the inhibitor is used for reducing insulin release, increasing blood sugar, and relieving pancreatic islet pressure.
The inhibitor can be used for treating congenital hyperinsulinemia and cancer.
The inhibitor is used for treating hyperinsulinemia-hyperammonemia syndrome.
EXAMPLE III
A therapeutic agent for congenital hyperinsulinemia, which comprises (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide, which is the compound described in example one, and which is used as a GDH inhibitor.
Example four
A therapeutic agent for hyperinsulinemia-hyperammonemia syndrome, comprising (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide, which is the compound described in example one, and which is used as a GDH inhibitor.
The following detailed description is of the invention in order to further illustrate the principles, features and advantages of the invention.
1. Virtual high-throughput drug screening:
(1) the 3D protein structure of GDH was selected. In this patent, a crystal of bovine wild-type GDH was selected as the 3D structure to be screened, and code number of PDB was 6 DHL.
(2) And processing the protein target points and establishing a docking grid.
And (3) carrying out hydrogenation, moisture removal and energy optimization treatment on the GDH protein by using a protein prepaparationwizard module in Maestro software. ADP has a stimulating effect on GDH, but ECG has an inhibitory effect on GDH; it can be concluded that compounds that bind to the pocket of the ADP (or ECG) site in GDH may have either stimulatory or inhibitory effects. In the present invention, ECG sites were used as sites for high throughput screening and docking grids were produced by receptorgrid generation in Maestro software.
(3) And (5) sorting the small molecule database and establishing a docking database. And importing the database file into Maestro software, and performing conformation search and energy optimization on the small molecules through a LigPrep plate. And finally establishing a docking Ligands database.
(4) And carrying out high-throughput virtual screening by using docking software. High-throughput screening is performed by using an HTVS mode in a LigandDocking plate in Maestro software. And (4) carrying out sequencing analysis on the scoring results, and carrying out SP docking analysis on the compounds before ranking, so as to select the compounds of the invention.
GDH enzyme kinetic assay:
(1) preparation of analysis buffer and premix:
configuration of assay buffer: 1212mg of Trisma and 820mg of Sodium Acetate were weighed out and added to 500mL of distilled water, and after completely dissolved, 20. mu.L of 500mM EDTA was added to make a volume of 1L, and finally the pH was adjusted to 8.0.
Preparing a premixed liquid: 10mg NADH are dissolved in 6.4ml NH4Cl, diluted 4-fold with assay buffer to give a premix.
(2) Preparing a tissue homogenate buffer solution: 5mM Na2HPO4, 5mM K2HPO4，1mM EDTA，1％Triton X-100。
(3) Preparing tissues: a small amount of tissue was homogenized in tissue homogenization buffer (about 200. mu.L) in a 1.5mL centrifuge tube on ice, and then allowed to stand on ice for 20 minutes.
(4) 10L of the premix, 5. mu.L DEM (20. mu.M), 5. mu.L of the sample (0.1 to 0.2. mu.g/. mu.L protein in liver tissue) and 5. mu.L GTP were added to 384-well plates, respectively, and 5. mu.L of alpha-KG was added to the pre-read (5 minutes), mixed, and read again (5 minutes).
3. And (3) separating, purifying and culturing the islets of langerhans of the mice:
after mouse anesthesia, injecting collagenase (concentration: 2mg/mL) into pancreatic duct, separating pancreas from abdominal cavity, digesting tissue (shaking at 37 deg.C), purifying to obtain pancreatic tissue with purity over 95%, placing pancreatic tissue in cell culture box, heating at 37 deg.C and 5% CO2The culture medium is RPMI1640, and 10% fetal calf serum and 10mM glucose are added.
4. Examination of high throughput islet incubation insulin secretion:
(1) preparation of incubation solution Krebs-Ringer bicarbonate buffer (KRBB):
buffer A: 107g of NaCl were dissolved in 1000mL of distilled water.
Buffer B: 5.96g of KCl, 32.256g of NaHCO3 and 3.25g of MgCl2-6H2O were dissolved together in 1000mL of distilled water.
Buffer C: 5.168g of CaCl2-2H2O was dissolved in 1000mL of distilled water.
Krebs buffer: 50mL of each of the buffers A, B and C was taken, dissolved in distilled water, and added with 2.0g of BSA and 1.906g of HEPES, the pH was adjusted to 7.4, and the volume was adjusted to 800mL to obtain KRBB.
(2) Preparing an irritant liquid: different stimulating solutions were prepared with KRBB according to experimental requirements.
(3) Manually selecting islets with similar sizes, placing the islets in a V-shaped bottom 96-well plate, placing 1-5 islets in each well, and adding 150 mu L of KRBB.
(4) The plate centrifuge was centrifuged (500r, 10s) and the supernatant removed and repeated once.
(5) Adding 150. mu.L KRBB, and standing at 37 deg.C and 5% CO2The supernatant was removed after centrifugation (500r, 10s) by gentle shaking for 30 minutes on a shaker in an incubator.
(6) Adding different stimulating solutions, and standing at 37 deg.C and 5% CO2The supernatant was collected by centrifugation (500r, 10s) after gentle shaking for 30 minutes on a shaker of an incubator, stored in a refrigerator at-20 ℃ until hormone measurement, and the effect of the stimulant or drug on insulin secretion was confirmed by measuring insulin secretion of the supernatant.
(7) Taking 10 mu L of supernatant after the islet stimulation experiment, transferring the supernatant into a 384-pore plate, adding an antibody according to the kit instructions, shaking, uniformly mixing, placing at room temperature, incubating for 4 hours, reading by using a Clariostar microplate reader HTRF program of BMG, and calculating a hormone secretion value according to a standard curve. Insulin was detected using the insulin detection kit from Cisbio.
5. The experimental results are as follows:
(1) screening out a compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide with the butt joint fraction of-8.893 from a database by using a computer high-throughput screening mode.
(2) GDH enzyme kinetic assay the results of the assay are shown in FIG. 1 and show that (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide can inhibit GDH activity in the liver of wild type mice with an IC50 value of 17.23. mu.M. The results show that the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide (marked by AR-Z-005) has an inhibitory effect on the activity of GDH under the condition of micromolar content.
(3) The results of the islet incubation experiment are shown in FIG. 2, from which it can be seen that the insulin secretion level increases after addition of 10mM leucine (Leu) compared to the 2mM Glutamine (Glutamine, Q) group; meanwhile, the results show that the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide added into the group of glutamine and leucine can effectively reduce the release of insulin at both 10 mu M and 25 mu M, thereby playing the role of increasing blood sugar and relieving the pressure of pancreatic islets.
The foregoing illustrates and describes the principles and features and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention as defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (8)
1. Use of the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide as an inhibitor of glutamate dehydrogenase, characterized in that the above compound components are used for inhibiting the activity of glutamate dehydrogenase;
the chemical formula of the compound component is as follows:
2. the use of the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide according to claim 1 as glutamate dehydrogenase inhibitor for the inhibition of GDH activity, with a half maximal inhibitory concentration IC50 of 17.23 μ M of the component.
3. A method for using the glutamate dehydrogenase inhibitor, which comprises using the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide according to claim 1 as a glutamate dehydrogenase inhibitor for inhibiting the activity of glutamate dehydrogenase.
4. The method of claim 3, wherein the inhibitor of glutamate dehydrogenase comprises: the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide according to claim 1 as glutamate dehydrogenase inhibitor for use in lowering insulin release, increasing blood glucose, relieving pancreatic islet stress.
5. The method of claim 3, wherein the inhibitor of glutamate dehydrogenase comprises: the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide according to claim 1 as glutamate dehydrogenase inhibitor for use in the treatment of congenital hyperinsulinemia and cancer.
6. The method of claim 3, wherein the inhibitor of glutamate dehydrogenase comprises: the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide according to claim 1 as glutamate dehydrogenase inhibitor for use in the treatment of hyperinsulinemic-hyperammonemic syndrome.
7. A therapeutic agent for congenital hyperinsulinemia, which comprises the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide, which is used as an inhibitor of glutamate dehydrogenase.
8. A therapeutic agent for hyperinsulinemia-hyperammonemia syndrome, comprising the compound (S) -2- (2- (1-H-indol-3-yl) acetamido) -N, 2-bis (4-methoxyphenyl) acetamide, which is used as an inhibitor of glutamate dehydrogenase.
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| US20080319008A1 (en) * | 2004-06-04 | 2008-12-25 | Alan Verkman | Compounds Having Activity in Increasing Ion Transport by Mutant-Cftr and Uses Thereof |
| CN103356617A (en) * | 2013-07-17 | 2013-10-23 | 上海交通大学医学院附属瑞金医院 | Application of deacetylase inhibitors in preparation of medicaments for promoting insulin secretion |
| CN109988124A (en) * | 2017-12-29 | 2019-07-09 | 杭州健昵福生物科技有限公司 | A kind of inhibitor of the kidney type glutaminase allosteric site of diazole containing oxygen group elements or tetrazole structure |
| CN111166742A (en) * | 2018-11-12 | 2020-05-19 | 南京盛德生物科技研究院有限公司 | Application of vitamin E in treatment of functional mutation of glutamate dehydrogenase |
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| US20080319008A1 (en) * | 2004-06-04 | 2008-12-25 | Alan Verkman | Compounds Having Activity in Increasing Ion Transport by Mutant-Cftr and Uses Thereof |
| CN103356617A (en) * | 2013-07-17 | 2013-10-23 | 上海交通大学医学院附属瑞金医院 | Application of deacetylase inhibitors in preparation of medicaments for promoting insulin secretion |
| CN109988124A (en) * | 2017-12-29 | 2019-07-09 | 杭州健昵福生物科技有限公司 | A kind of inhibitor of the kidney type glutaminase allosteric site of diazole containing oxygen group elements or tetrazole structure |
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