Novel IDH1 mutant inhibitor and application thereof
By developing compound Y0404481, the problem of Ivosidenib resistance caused by the IDH1R132C/S280F mutation in the existing technology was solved, and effective inhibition and apoptosis induction of HT1080 cells were achieved, showing good inhibitory activity and low toxicity.
Patent Information
- Application Number
- CN202510945218.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-09
- Publication Date
- 2025-10-03
AI Technical Summary
Existing technologies have not yet effectively overcome the problem of ivosidenib resistance caused by IDH1R132C/S280F mutations, and new IDH1 mutant inhibitors need to be developed to combat AML.
Compound Y0404481 was developed and screened, and in vitro experiments evaluated its strong inhibitory activity against HT1080 cells with IDH1 R132C and S280F mutations. As an IDH1 inhibitor, it is used to prepare proliferation inhibitors, migration inhibitors and apoptosis inducers for IDH1 mutant cells.
Compound Y0404481 has strong inhibitory activity against IDH1R132C mutation and IDH1R132C/S280F mutation in HT1080 cells, and has low toxicity to LO2 cells, significantly inhibiting cell migration and inducing apoptosis, showing good targeting and low cytotoxicity.
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Figure CN120732844A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel IDH1 mutant inhibitor and application thereof. Background Art
[0002] Isocitrate dehydrogenase (IDH) is a cytoplasmic enzyme whose primary function is to convert isocitrate to α-ketoglutarate (α-KG) in the tricarboxylic acid cycle (TCA cycle). During this process, nicotinamide adenine dinucleotide phosphate (NADP) is reduced to nicotinamide adenine dinucleotide hydrogen phosphate (NADPH). This reaction is crucial for regulating intracellular free radical levels and reactive oxygen species production, enhancing cellular antimicrobial capacity. Furthermore, α-KG, as a key metabolic intermediate, participates in the synthesis of adenosine triphosphate (ATP) in mitochondria, maintaining cellular energy homeostasis. Wild-type IDH (wtIDH) is ubiquitous in various organisms and is responsible for carrying out these metabolic processes. Mutations in IDH1 were first discovered in a whole-exome sequencing study of 22 glioblastoma cells. Subsequent studies have shown that mutations in both IDH1 and its homolog IDH2 (collectively referred to as IDHs) are widely present in various cancer types, with gliomas and acute myeloid leukemia (AML) being the most common cancer types associated with IDH1 and IDH2 mutations, respectively. IDH1 is mainly located in the cytoplasm, while IDH2 is located in the mitochondria. In addition to the ability of IDH to oxidize isocitrate to α-KG through NADP+ coupling, mutated IDH1 or IDH2 also acquires a new catalytic function to convert α-KG into 2-hydroxyglutarate (2-HG). The accumulated 2-HG occupies the same space as α-KG in the active sites of various α-KG-dependent dioxygenases and competitively inhibits their biological activity, affecting the occurrence and development of tumors in many ways. In 2018, the U.S. Food and Drug Administration (FDA) approved a molecular entity, Ivosidenib, that targets IDH1 with the R132 mutation as a treatment option for AML with mIDH1. It is worth noting that in some patients treated with Ivosidenib in the same year, the expression of IDH1 in the AML was observed to be related to the oxidative stress response. R132C / S280F Second site mutations can lead to drug resistance or recurrence of the disease. Therefore, there is an urgent need to develop drugs targeting IDH1. R132C / S280F New inhibitors against IDH1 in AML R132C / S280F Ivosidenib resistance caused by mutations.
[0003] Currently, reports indicate that DS1001b, a drug currently in Phase II clinical trials, may be able to overcome ivosidenib resistance caused by second-site mutations. However, there is still no IDH1 mutant inhibitor that can overcome the resistance caused by second-site mutations. Summary of the Invention
[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a novel IDH1 mutant inhibitor and its application.
[0005] This study successfully discovered novel inhibitors of IDH1 mutants and then evaluated their biological activity in vitro. Compound Y0404481 demonstrated potent inhibitory activity against HT1080 cells (human fibrosarcoma cells) harboring the IDH1 R132C and S280F mutations. IDH1 R132C represents a mutation from arginine (R) to cysteine (C) at amino acid position 132 of IDH1, while IDH1 S280F represents a mutation from serine (S) to phenylalanine (F) at amino acid position 280 of IDH1. Human IDH1 (NCBI Gene Accession No.: 3417; Reference Sequence No.: NM_005896.3) was used as a reference sequence for these mutations.
[0006] To achieve the above object, the technical solution adopted by the present invention is: In a first aspect, the present invention provides the use of compound Y0404481 in the preparation of an IDH1 mutant inhibitor, wherein the chemical structure of compound Y0404481 is shown in the following formula (I):
[0007] Formula (I).
[0008] The present invention has found that compound Y0404481 has a relatively good inhibitory effect on the activity of HT1080 cells and IDH1 R132C mutation and R132C and S280F mutations of HT1080 cells, and can be used as an IDH1 inhibitor.
[0009] Preferably, the IDH1 mutant comprises the R132C mutation.
[0010] Preferably, the IDH1 mutant comprises R132C and S280F mutations.
[0011] In a second aspect, the present invention provides use of compound Y0404481 in preparing a proliferation inhibitor of IDH1 mutated cells.
[0012] In a third aspect, the present invention provides use of compound Y0404481 in preparing a migration inhibitor for IDH1 mutated cells.
[0013] In a fourth aspect, the present invention provides use of compound Y0404481 in preparing an apoptosis inducer for IDH1 mutated cells.
[0014] Preferably, the IDH1 mutation includes the R132C mutation.
[0015] Preferably, the IDH1 mutations include R132C and S280F mutations.
[0016] Preferably, the cells comprise tumor cells.
[0017] Preferably, the tumor comprises a fibrosarcoma.
[0018] In a fifth aspect, the present invention provides the use of compound Y0404481 in the preparation of anti-tumor drugs, wherein the chemical structure of compound Y0404481 is shown in the following formula (I):
[0019] Formula (I).
[0020] Preferably, the tumor comprises a fibrosarcoma.
[0021] Preferably, the compound Y0404481 inhibits IDH1 mutation in tumor cells.
[0022] Preferably, the compound Y0404481 inhibits the R132C mutation of IDH1 in tumor cells.
[0023] Preferably, the compound Y0404481 inhibits R132C and S280F mutations in tumor cells.
[0024] Compared with the prior art, the present invention has the following beneficial effects: The present invention successfully screened and obtained a novel IDH1 mutant inhibitor, and then evaluated its biological activity in vitro. The screened compound Y0404481 has an inhibitory effect on HT1080 cell IDH1 R132C Mutation and IDH1 in HT1080 cells R132C / S280F The mutants all had strong inhibitory activity and low toxicity to LO2 cells. As a specific experiment: MTT results showed that the compound could inhibit IDH1 in HT1080 cells at a concentration of 1.58 μM. R132C The mutation produced a strong inhibitory effect. At the same time, it could inhibit the expression of IDH1 in HT1080 cells at a concentration of 4.18 μM. R132C / S280F Compound Y0404481 has a strong inhibitory effect on IDH1 in HT1080 cells. R132C Mutation and IDH1 in HT1080 cells R132C / S280FThe inhibitory activity of the mutant was 50 and 24 times higher than that of the positive drug DS1001b. Compound Y0404481 showed lower inhibitory ability against HT1080 cells with IDH1 knockdown, proving that compound Y0404481 has good targeting. R132C / S280F Among the mutants, compound Y0404481 showed stronger inhibitory activity. Comprehensive MTT study results showed that compound Y0404481 has good activity and low toxicity at the cellular level and is a novel IDH1 mutant inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] Figure 1 The process of screening compounds.
[0026] Figure 2 Y0404481 for HT1080 and HT080 IDH1 R132C / S280F Effects of Y0404481 on wound healing of HT1080 cells; A is an inverted microscope image showing the effects of Y0404481 on wound healing of HT1080 cells; B is an image showing the effects of Y0404481 on HT080IDH1 R132C / S280F Inverted microscope pictures of the effects of Y0404481 on cell wound healing; C is the wound healing rate of HT1080 cell wound healing; D is the effect of Y0404481 on HT080 IDH1 R132C / S280F Wound healing rate of cellular wound healing.
[0027] Figure 3 Y0404481 for HT1080 and HT080 IDH1 R132C / S280F Effects of Y0404481 on cell apoptosis; A is the flow cytometric graph of the effect of Y0404481 on HT1080 cell apoptosis; B is the percentage of apoptotic cells in HT080 cells treated with Y0404481; C is the effect of Y0404481 on HT080 IDH1 R132C / S280F Flow cytometry diagram of the effect of Y0404481 on HT080IDH1 R132C / S280F The percentage of apoptotic cells; Figure 4 Y0404481 for HT1080 (A, B) and HT080 IDH1 R132C / S280F (C, D) Effects of cell cycle. Flow cytometry graph of the effect of Y0404481 on the cell cycle of HT1080 cells; B is the effect of Y0404481 on the cell cycle ratio of HT080 cells; C is the effect of Y0404481 on the IDH1 of HT080 cells R132C / S280F Flow cytometry diagram of cell cycle effects; D is the effect of Y0404481 on HT080IDH1 R132C / S280F The influence of cell cycle ratio. DETAILED DESCRIPTION
[0028] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.
[0029] Unless otherwise specified, other materials and reagents used in the examples can be obtained from commercial sources.
[0030] Example 1 Screening of compounds like Figure 1 As shown, the X-ray crystal structure of IDH1R132C / S280F in complex with NADPH and the inhibitor DS1001b was obtained from the PDB database (PDB ID: 7PJN; resolution: 2.45 Å). The structure was preprocessed using the Protein Preparation Wizard module in Schrödinger 2015, which included adding hydrogen atoms, removing all water molecules, assigning charges and protonation states at pH 7.0, and optimizing the structure using the OPLS-2005 force field. Subsequently, the receptor box was constructed using the Glide Receptor Grid Generation tool in Schrödinger 2015 based on the OPLS-2005 force field. The grid center was defined using DS1001b as the reference point, and the grid box was set to accommodate a ligand of similar size to the ligand.
[0031] This study used the Chemdiv and Specs databases (2022 edition) as screening sources, which contain approximately 1,130,807 compounds. Chemical structures were preprocessed using the LigPrep module in Schrödinger 2015 software and the OPLS-2005 force field. Epik was used to generate all possible states of the molecule at a pH of 7.0 ± 2.0, and tautomers were generated, ultimately generating up to eight low-energy conformations for each ligand.
[0032] The generated grid and prepared ligands underwent structure-based virtual screening using the virtual screening workflow in the Glide module. First, the prepared ligands were pre-filtered using QikProp to ensure they possessed the desired physicochemical properties and adhered to Lipinski's rule of five. Subsequently, ligands containing potentially reactive functional groups were eliminated. The Glide screening process consists of three progressive docking stages. The first stage is dedicated to high-throughput virtual screening (HTVS), designed to rapidly process a large number of molecules. Molecules that pass this stage enter the second stage, standard precision (SP) docking. In each stage, only the top 20% of molecules are retained for further evaluation as potential inhibitor candidates. Finally, molecules that have passed the first two stages enter the third stage, extra precision (XP) docking, from which 2,000 molecules are selected as final candidates.
[0033] The Phase module (Schrödinger 2015) was used to construct an electronic pharmacophore hypothesis. This hypothesis is based on the complementarity between receptor and ligand features and utilizes the crystal structure of mIDH1 in complex with DS1001b (PDB ID: 7PJN). When screening ligands from the database, a PhaseScreenScore threshold of greater than 1.0 was set to assess the degree of match between the ligands and the proposed pharmacophore features. A total of 835 ligands were retained.
[0034] Shape Screening identifies other molecules that are most similar in shape to a given molecule. During this process, each conformation of a given molecule is superimposed on a positive drug in multiple ways, and shape similarity is calculated based on the volume of the overlapping spheres. For each molecule, the conformation with the highest similarity and its optimal alignment are recorded in the Maestro file and displayed as a "Shape Sim" attribute in the project table. To assess the degree of match between the ligand and the DS1001b features, a shape similarity (Shape Sim) threshold of greater than 0.45 was set. 357 small molecules passed the shape-based virtual screening.
[0035] In Maestro, the Prime MM-GBSA method was used to calculate the binding free energy between potential inhibitors and the IDH1 R132C / S280F crystal structure. Specifically, the Prime MM-GBSA method was used to estimate the binding free energy of candidate compounds to the receptor DS1001b. The 10 molecules that performed best in the Prime MM-GBSA analysis were selected as the focus of subsequent research.
[0036] Molecular dynamics (MD) simulations were performed on the complex formed by mIDH1 and DS1001b, along with selected representative active compounds. The initial structure of the mIDH1-DS1001b complex was obtained from the PDB database. Bond charge corrections (BCCs) were applied to fit the inhibitor's partial charges. The general AMBER force field (GAFF) was used to parameterize the compounds, while the AMBERff14SB force field was used to parameterize the mIDH1 structure. The complex was solvated at a distance of 12 Å from the periodic boundary using the TIP3PBOX water model. To ensure electroneutrality, appropriate amounts of chloride and sodium ions were added. The system was first minimized using the steepest descent method for 2500 steps, followed by further optimization using the conjugate gradient method. Next, the system was heated from 0 K to 310 K in the NVT ensemble, with positional restraints applied to the backbone atoms. The restraining force was gradually reduced from 10 kcal / (mol·Å²) to 0.1 kcal / (mol·Å²) over 0.9 ns. The system was simulated for 500 ns in the NPT ensemble at 1.0 atm and 310 K without any constraints. Trajectory analysis was performed using the Cpptraj module in the AMBER18 software.
[0037] After completing the molecular dynamics (MD) simulation, the trajectory was analyzed in detail using the Cpptraj module in AMBER 18. First, the RMSD and RMSF were calculated based on the MD simulation trajectory to assess system stability and conformational changes. Subsequently, the last 5,000 frames of the trajectory were selected from a total of 25,000 frames, and the binding free energy was estimated using the MM / GBSA method. Ultimately, six hit compounds were screened out with binding free energies that were favorable compared to the positive drug: compounds V0135975, Y0404481, F1440410, G6410306, AK918, and V0236946.
[0038] (1) The chemical formula of compound V0135975 is as follows: .
[0039] (2) The chemical formula of compound Y0404481 is as follows: .
[0040] (3) The chemical formula of compound F1440410 is as follows: .
[0041] (4) The chemical formula of compound G6410306 is as follows: .
[0042] (5) The chemical formula of compound AK918 is as follows: .
[0043] (6) The chemical formula of compound V0236946 is as follows: .
[0044] Example 2 Study on the inhibitory activity of the target compound against IDH1 R132C / S280F in vitro The compounds AK918, V0135975, Y0404481, F1440410, G6410306, and V0236946 screened in Example 1 were tested for their inhibitory activity on HT1080 shIDH1, HT1080 IDH1 R132C / S280F, and LO2 cell proliferation (24 and 48 h), respectively. DS1001b was used as a positive control.
[0045] The results are shown in Table 1. Y0404481, F1440410, G6410306, and V0236946 can inhibit HT1080 to a certain extent, and have similar or even better IC values than DS1001b in the 48 h HT1080 cell inhibition experiment. 50 value, and the IC between HT1080 shIDH1 50 The IC values of HT1080 cells and LO2 were all greater than 1.0, indicating that these compounds have targeting activity against IDH1R132C / S280F. 50 As can be seen from the ratio of values, Y0404481 and F1440410 have no obvious cytotoxicity to the non-malignant human normal stem cell line LO2. Among them, Y0404481 has less toxicity than DS1001b. When IDH1 is mutated, although Y0404481 has an IC 50 The values increased, and the IC values at 24 h and 48 h 50 The values were 4.18 μM and 2.47 μM, but for HT1080 IDH1 R132C / S280F It still has a better inhibitory effect than the positive drug DS1001b.
[0046] Table 1 Effects of compounds on HT1080 shIDH1 and HT1080 IDH1 R132C / S280F and LO2 cell proliferation (24, 48 h).
[0047]
[0048] In Table 1, sh refers to HT1080 shIDH1 cells, LO2 refers to normal human liver cell line LO2 cells, R132C refers to HT1080IDH1 R132C cells, and R132CS280F refers to HT1080 IDH1 R132C / S280F cells, sh / R132C is HT1080 shIDH1R132C cells, sh / R132CS280F is HT1080 shIDH1 R132C / S280F LO2 / R132C is LO2 IDH1 R132C cells, LO2 / R132CS280F is LO2 IDH1 R132C / S280F cell.
[0049] 3×10 5 Cells were seeded on a 6-well plate and cultured until the cells adhered to the dish. A "wound" was created on the dish using a short 200 μL pipette. After three washes with PBS, complete culture medium containing different concentrations (1 μM, 2 μM, 4 μM, and 8 μM) of drugs (Y0404481 and 50 μM of the positive control DS1001b) was added. After 24 hours, wound healing was photographed using an inverted microscope. The wound healing rate (*) was calculated by measuring the healing area. p <0.05, ** p <0.01, *** p <0.001).
[0050] The results are as follows Figure 2 As shown, Y0404481 treatment can significantly reduce HT1080 and HT080 IDH1 R132C / S280F Cell healing area and migration distance. Y0404481 at a concentration of 4 μM had similar inhibitory activity to 50 μM DS1001b, and at a concentration of 8 μM, it exhibited a stronger inhibitory effect than 50 μM DS1001b.
[0051] HT1080 and HT1080 IDH1 R132C / S280F Cells were seeded in 6-well plates at a density of 1×10 5Cells were cultured overnight to allow attachment to the culture dish. Different concentrations (1 μM, 2 μM, 4 μM, and 8 μM) of drugs (Y0404481 and 50 μM positive control DS1001b) were added and treated for 24 hours. After treatment, cells were harvested with trypsin, washed with PBS, and collected by centrifugation. Apoptotic and necrotic cells were identified using the Annexin V / PI assay after 24 hours of incubation with Y0404481 (1-8 μM) and DS1001b (50 μM). Live cells were collected and stained in the dark for 15 minutes at room temperature. Flow cytometry was then used to quantify the percentage of apoptotic cells. (* p <0.05, ** p <0.01, *** p <0.001).
[0052] Annexin V / PI double staining was used to investigate the mechanism of cell death induced by Y0404481 (1-8 μM) and DS1001b (50 μM). Phosphatidylserine is exposed on the outer membrane during early apoptosis. Annexin V binds to phosphatidylserine, while PI acts as a nucleic acid dye to stain the DNA of dead cells. Figure 3 As shown, after 24 h of treatment with Y0404481 (1-8 μM) and DS1001b, HT1080 and HT080 IDH1 R132C / S280F The apoptosis rate of cells was significantly increased. In HT1080, the apoptosis rates of compound Y0404481 at concentrations of 1, 2, 4, and 8 μM and DS1001b at a concentration of 50 μM were 4.38%, 7.37%, 10.94%, 13.36%, and 8.67%, respectively. R132C / S280F In the study, the apoptotic rates of compound Y0404481 at concentrations of 1, 2, 4, and 8 μM, and DS1001b at a concentration of 50 μM, were 3.31%, 7.30%, 9.81%, 10.47%, and 5.12%, respectively. At a concentration of 4 μM, Y0404481 had a similar apoptotic rate as 50 μM DS1001b, and at a concentration of 4 μM, it exhibited a higher apoptotic rate than 50 μM DS1001b.
[0053] Propidium iodide is a fluorescent dye for double-stranded DNA. It can generate fluorescence after binding to double-stranded DNA, and the fluorescence intensity is proportional to the content of double-stranded DNA. After the DNA in the cell is stained with propidium iodide, the DNA content of the cell can be measured by flow cytometry, and then the cell cycle analysis can be performed based on the distribution of DNA content. Figure 4As shown in the figure, after 24 h of treatment with Y0404481 (1~8 μM) and DS1001b (50 μM), Y0404481 had a significant effect on the expression of IDH1 in HT1080 and HT080 cells. R132C / S280F The cell cycle ratio did not change significantly, while the cell distribution of DS1001b shifted significantly from the S phase to the G1 phase, indicating that DS1001b arrested the cell cycle in the G1 phase.
[0054] In summary, a series of novel IDH1 mutant inhibitors were screened out based on virtual screening combined with kinetic simulation, and in vitro experiments showed that among these compounds, compound Y0404481 had a good inhibitory effect on both HT1080 and HT1080 IDH1R132C / S280F cells.
[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.
Claims
1. Use of compound Y0404481 in the preparation of IDH1 mutant inhibitors, wherein the chemical structure of compound Y0404481 is shown in the following formula (I): ； Formula (I).
2. The use according to claim 1, characterized in that The IDH1 mutant includes the R132C mutation.
3. The use according to claim 1, characterized in that The IDH1 mutants include R132C and S280F mutations.
4. Use of compound Y0404481 in the preparation of a proliferation inhibitor of IDH1 mutated cells.
5. Use of compound Y0404481 in the preparation of a migration inhibitor for IDH1 mutated cells.
6. Use of compound Y0404481 in the preparation of an apoptosis inducer for IDH1 mutated cells.
7. The use according to any one of claims 4 to 6, characterized in that The IDH1 mutation includes the R132C mutation.
8. The use according to any one of claims 4 to 6, characterized in that The IDH1 mutations include R132C and S280F mutations.
9. Use of compound Y0404481 in the preparation of anti-tumor drugs, wherein the chemical structure of compound Y0404481 is shown in the following formula (I): ； Formula (I).
10. The use according to claim 9, characterized in that Such tumors include fibrosarcomas.