E3 ligase GID4 ligand compound and application thereof

Abstract

The invention belongs to the technical field of medical chemistry and biology, and particularly discloses an E3 ligase GID4 ligand compound and application thereof. The novel E3 ligase GID4 ligand which is high in selectivity, stable in metabolism and high in binding capacity, namely the compound shown in the formula (I), is researched and developed through computer-assisted drug screening and can be applied to targeted protein degradation, and a new choice is provided for research and development of new drugs based on a protein degradation targeted chimera in the future.

Description

Technical Field

[0001] The present invention relates to the technical field of medical chemistry and biology, and in particular to an E3 ligase GID4 ligand compound and application thereof. Background Art

[0002] Protein homeostasis in cells requires the participation of many systems to maintain. In addition to protein synthesis, folding and transport, protein degradation pathways are also essential for maintaining protein homeostasis. The ubiquitin-proteasome system is one of the important protein degradation pathways in human cells, which mainly includes two major processes: substrate protein ubiquitination and ubiquitin-tagged protein degradation by proteasome. Ubiquitin (Ub) is a polypeptide composed of 76 amino acids, which is ubiquitinated in all eukaryotic organisms. Under the cascade catalysis of ubiquitin activating enzyme E1, ubiquitin conjugating enzyme E2 and ubiquitin ligase E3, the process of covalent binding of ubiquitin to lysine residues of substrate proteins is called protein ubiquitination modification. Ubiquitin-tagged proteins can be recognized and degraded by proteasomes. At present, based on the characteristics of the ubiquitin-proteasome system, the redirection of the ubiquitination degradation mechanism of non-native new substrates is an emerging disease intervention pharmacological strategy, which mainly includes protein hydrolysis targeting chimeras and molecular glues.

[0003] Protein degradation targeting chimera (PROTACs) is a heterobifunctional small molecule in which the E3 ligase ligand and the target protein ligand are connected by a linker, and the target protein is degraded by the ubiquitin-proteasome system. It is the hottest research direction in the field of targeted protein degradation (TPD). Compared with traditional small molecule inhibitors, PROTAC has many advantages, such as not needing to bind to the active site of the target protein to work, being able to degrade difficult-to-drug targets, being event-driven and catalytic, and being able to work at lower doses. It is currently the hottest technology in new drug development and has great potential in the field of drug development.

[0004] However, the human genome currently encodes more than 600 E3 ligases, of which only a few (VHL, CRBN, MDM2, cIAP, etc.) have been applied to PROTAC. It is of great significance to discover new E3 ligase ligands and apply them to targeted protein degradation. Glucose-induced degradation protein 4 homolog (GID4) is a substrate receptor of the CTLH E3 ligase protein complex. It is expressed in most mammalian tissues and is mainly distributed in the cytoplasm and nucleus. In addition, recent literature has reported that GID4 can degrade target proteins through proximity dependence at the cellular level, so GID4 protein is an ideal choice for the development of new PROTACs. There are currently only two documents ([1] Discovery and Structural Characterization of Small MoleculeBinders of the Human CTLH E3 Ligase Subunit GID4.J Med Chem.2022Oct 13;65(19):12725-12746.doi:10.1021 / acs.jmedchem.2c00509.Epub 2022Sep 18.PMID:36117290; PMCID:PMC9574856.[2]A chemical probe to modulate human GID4 Pro / N-degron interactions.Nat Chem Biol.2024Sep;20(9):1164-1175.doi:10.1038 / s41589-024-01618-0.Epub 2024May 21.PMID:38773330.) reported the discovery of GID4 ligands, but these ligands have many defects, such as poor affinity with GID4 and difficulty in entering cells due to their polypeptide structure. The following are the chemical structures of the three GID4 ligands reported in the above literature, among which compound 16 and compound 67 are fragment structures, so they have poor affinity with GID4 and poor specificity, and compound 88 is a polypeptide structure, which may have defects such as difficulty in entering cells.

[0005]

[0006] Therefore, it is of great significance to develop new small molecule ligands of GID4 with higher affinity and better membrane permeability and use them for targeted protein degradation. Summary of the invention

[0007] In view of the shortcomings of the prior art described above, the object of the present invention is to provide an E3 ligase GID4 ligand compound and its application. The ligand compound exhibits the advantages of high selectivity, metabolic stability and strong binding ability to the E3 ligase GID4. Its application in targeted protein degradation can provide new options for the future development of new drugs based on PROTAC.

[0008] To achieve the above-mentioned object and other related objects, the present invention provides a compound, the chemical structure of which is shown in the following formula (I):

[0009]

[0010] In formula (I), R1 is independently selected from hydrogen atom, alkyl, alkoxy, halogen atom or Any one of, wherein R' is independently selected from any one of a hydrogen atom, an alkyl group, an alkoxy group or a halogen atom; R2 and R3 are each independently selected from any one of a hydrogen atom, an alkyl group, an alkoxy group or a halogen atom.

[0011] In some embodiments, in formula (I), R1 is selected from Wherein, R' is independently selected from any one of a hydrogen atom, an alkyl group, an alkoxy group or a halogen atom.

[0012] In some embodiments, the alkyl group has 1 to 6 carbon atoms.

[0013] In some embodiments, the halogen atom is selected from any one of fluorine, chlorine, bromine or iodine.

[0014] In a specific embodiment, the chemical structure of the compound is as follows:

[0015]

[0016] The present invention also provides a small molecule compound, which is a protein degradation targeting chimera, comprising an E3 ligase GID4 ligand and a target protein ligand connected to the E3 ligase GID4 ligand, wherein the E3 ligase GID4 ligand comprises a compound or a salt thereof having a chemical structure as shown in the above formula (I).

[0017] In some embodiments, the salt of the compound as shown in formula (I) is a pharmaceutically acceptable salt, including but not limited to hydrohalide (including hydrochloride, hydrobromide), sulfate, citrate, maleate, methanesulfonate, citrate, lactate, L-tartrate, fumarate, L-malate, phosphate, dihydrogenphosphate, pyrophosphate, metaphosphate, oxalate, malonate, benzoate, mandelate, succinate, trifluoroacetate, glycolate or p-toluenesulfonate, etc.

[0018] In some embodiments, the E3 ligase GID4 ligand and the target protein ligand are connected together via a linker arm, and the linker arm includes but is not limited to polyethylene glycol (PEG) and the like.

[0019] The present invention also provides a use of a compound as shown in formula (I) or a salt thereof as a ligand of E3 ligase GID4.

[0020] In some embodiments, the compound represented by formula (I) or a salt thereof is used as a ligand of the E3 ligase GID4 for targeted protein degradation, or for preparing or screening agents for preventing or treating diseases by targeted protein degradation.

[0021] In some embodiments, the targeted protein degradation comprises: using a compound as shown in formula (I) or a salt thereof as a ligand of the E3 ligase GID4, connecting the target protein ligand to form a protein degradation targeting chimera (PROTACs), and using the ubiquitin-proteasome system to degrade the target protein.

[0022] In some embodiments, the diseases include but are not limited to tumors, infectious diseases, autoinflammatory diseases, inflammatory diseases, autoimmune diseases, neurological diseases, respiratory diseases, anemia, hemorrhagic shock, transplant rejection, multiple organ dysfunction syndrome (MODS), sarcoidosis, adult respiratory distress syndrome, cardiovascular disease, Ongwericht syndrome, Richter syndrome (RS), acute liver failure and diabetes, etc.

[0023] The present invention also provides a use of a compound as shown in formula (I) or a salt thereof in drug preparation or screening, wherein the drug prevents or treats the above-mentioned diseases by targeted protein degradation.

[0024] The present invention also provides a pharmaceutical composition, comprising a first active ingredient and at least one pharmaceutically acceptable carrier, wherein the first active ingredient is a compound represented by formula (I) or a pharmaceutically acceptable salt thereof.

[0025] In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, a filler, a stabilizer, a dispersant, a suspending agent, a diluent, an excipient, a thickener, a colorant, a solvent, or an encapsulating material. The carrier must be "acceptable" if it is compatible with the other ingredients in the pharmaceutical composition and is harmless to the patient. Pharmaceutically acceptable carriers include, for example: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository wax; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; buffers, such as phosphate buffer; polyoxyethylene, polyvinyl pyrrolidone, polyacrylamide, poloxamer; and other non-toxic compatible substances frequently used in pharmaceutical preparations.

[0026] In some embodiments, the pharmaceutical composition further comprises a second active ingredient, and the second active ingredient is combined with the first active ingredient to treat the above-mentioned disease. Combined treatment means that there is a synergistic effect between the second active ingredient and the first active ingredient, and the combined use of the two can synergistically enhance the effect of disease prevention or treatment.

[0027] In some embodiments, the second active ingredient includes but is not limited to anti-tumor agents, chemotherapeutic agents, immunotherapeutic agents, gene therapy agents, etc. The anti-tumor agents, chemotherapeutic agents, immunotherapeutic agents and gene therapy agents can all be selected from known substances.

[0028] The administration routes of the above-mentioned drugs and pharmaceutical compositions can be prepared into suitable preparation forms, such as spray preparations, patches, tablets, capsules, dragees, lozenges, powders, granules, powder injections, suppositories, liquid preparations, injections or lyophilized compositions, according to appropriate administration routes (including but not limited to nasal administration, inhalation administration, topical administration, oral administration, oral mucosal administration, rectal administration, pleural cavity administration, peritoneal administration, vaginal administration, intramuscular administration, subcutaneous administration, transdermal administration, epidural cavity administration, intrathecal administration and intravenous administration, etc.).

[0029] As described above, the E3 ligase GID4 ligand compound and its application of the present invention have the following beneficial effects:

[0030] The present invention has developed a novel ligand for E3 ligase GID4 through computer-assisted drug screening, which is highly selective, metabolically stable and has strong binding ability, namely, a compound as shown in formula (I), which can be applied to targeted protein degradation and provide new options for the future development of new drugs based on PROTAC. The core group of the compound is a secondary amine This structure can mimic the binding mode of Pro / N-degron and play a key role in the interaction with the protein GID4. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the virtual screening of the Pro / N-degron pocket of the GID4 protein. By setting multiple rounds of screening with different precisions, 28 GID4 candidate ligand compounds were finally determined.

[0032] Figure 2 The chemical structure of compound 4110 is shown.

[0033] Figure 3 Shown are the SPR screening verification results of the candidate ligand compounds obtained by virtual screening of the GID4 protein, where the red dot is compound 4110, and the other two black dots above the red line are the positive controls before and after the start of the experiment.

[0034] Figure 4 The results of SPR binding analysis of compound 4110 obtained by actual screening are shown.

[0035] Figure 5 Shown are the results of MST binding assay of GID4 protein and compound 4110.

[0036] Figure 6 Shown as wild-type GID4 124-289 Protein, E237A, Y258F mutant GID4 124-28 MST binding assay results of 9 proteins with compound 4110. DETAILED DESCRIPTION

[0037] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0038] Example 1

[0039] 1. Virtual Screening

[0040] like Figure 1As shown, targeting the Pro / N-degron pocket of the GID4 protein, about 1.8 million small molecule compounds were preliminarily identified using virtual screening based on a computer model. By setting up multiple rounds of screening with different accuracies, 28 new candidate ligands for GID4 were finally screened out based on comprehensive considerations from all aspects.

[0041] 2. GID4 protein purification

[0042] Truncated gene GID4 124-289 (commercially available) cloned into pET28a (His-tag) expression plasmid (commercially available), GID4 124-289 The E237A and Y258F mutations in were generated using site-directed mutagenesis. The protein was expressed by BL21 (DE3) Escherichia coli competent cells (commercially available) grown at 37°C and induced by 0.2 mM isopropyl β-D-Thiogalactoside (IPTG) at 16°C. After 18 hours, the cells were harvested by centrifugation at 4500 rpm and resuspended in lysis buffer (PBS pH = 8.0, 150 mM NaCl, 20 mM imidazole, 1 mM PMSF). The mixture was then incubated with Ni NTA Beads (Tian Di Ren He) and eluted by buffer A (PBS, pH = 8.0, 150 mM NaCl, 100 mM imidazole). Subsequently, a size exclusion chromatography step was performed in PBS, pH = 8.0, 150 mM NaCl, 1 mM DTT buffer (SEC buffer).

[0043] 3. SPR Binding Analysis

[0044] In vitro binding analysis was performed by surface plasmon resonance (SPR) at 25°C using a Biacore 8K (Cytiva) instrument. All experiments used PBS buffer (10mM PBS PH=7.4, 2.7mM KCl, 150mM NaCl, 0.05% Tween-20). According to the manufacturer's instructions, GID4 (amino acids 124-289) was immobilized on the flow cell of a carboxylated dextran-coated sensor chip (CM5), while another flow cell was fixed and left blank for reference subtraction and monitoring any nonspecific binding. The compound was diluted with PBS buffer to obtain a drug solution with a final dimethyl sulfoxide (DMSO) concentration of 2%, and serially diluted (two-fold dilution) in PBS buffer containing 2% DMSO. The compound was then passed through the sensor chip at a flow rate of 30μl / min using the Binding Screen drug screening mode and the MultiCycle Kinetics multi-cycle kinetics mode. The association time was 200 seconds, the dissociation time was 300 seconds, followed by a 120 second stabilization period of flow. PBS buffer alone (containing 2% DMSO) was used for blank injections, and PBS buffer containing 1.5% to 3.5% DMSO was used for solvent correction injections. The binding constant (Kd) was obtained using Biacore 8K Evaluation Software v.3.1.

[0045] Figure 3 The results showed that compound 4110 (whose chemical structure is as follows Figure 2 The binding response value of the PCR product with GID4 was the highest, indicating that the binding effect with the GID4 protein was the best.

[0046] Figure 4 The results showed that the binding of GID4 protein to compound 4110 was significantly concentration-dependent, with Kd = 68 μM.

[0047] 4. MST Assay for Protein Binding

[0048] Microscale thermophoresis (MST) was used to evaluate the binding affinity between wild-type GID4 protein, E237A and Y258F mutant GID4 proteins (commercially available) and their compound ligand 4110. In brief, a GID4 protein with N-terminal His tag at a final concentration of 600nM was used in the MST assay. The compound was diluted with MST buffer (containing PBS pH 7.4, 1mM DTT, 0.05% Tween 20) to obtain a drug solution with a final DMSO concentration of 1%, and the compound was serially diluted (two-fold dilution) starting from the highest concentration. The E3 ligase (commercially available) and its ligand were mixed and incubated at room temperature for 30 minutes for equilibrium and then transferred to the MST capillary. The MST assay was performed using the Monolith NT.115 system (NanoTemper) using the Nano-Red mode. The laser power was set to Auto and the MST power was set to medium. The determination was repeated at least three times on different occasions, and the Kd values ​​were calculated using MO.analysis (v3.2) software and the quadratic equation combined with the Kd model.

[0049] Figure 5 The results showed that GID4 protein significantly bound to compound 4110 with Kd = 2.43 μM.

[0050] Figure 6 The results showed that wild-type GID4 124-289 The protein binds well to 4110, E237A mutant GID4 124-289 The Y258F mutant GID4 124-289 Although the protein still binds to 4110, its binding affinity is far lower than that of the wild type.

[0051] In summary, compound 4110, as a new ligand for E3 ligase GID4, exhibits the advantages of high selectivity, metabolic stability and strong binding ability, and can be applied to targeted protein degradation, providing a new option for the future development of new PROTAC-based drugs. The core group of compound 4110 is a secondary amine This structure can mimic the binding mode of Pro / N-degron and play a key role in the interaction with the protein GID4.

[0052] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A compound, characterized in that The chemical structural formula of the compound is shown in the following formula (I): In formula (I), R1 is independently selected from hydrogen atom, alkyl, alkoxy, halogen atom or Any one of, wherein R' is independently selected from any one of a hydrogen atom, an alkyl group, an alkoxy group or a halogen atom; R2 and R3 are each independently selected from any one of a hydrogen atom, an alkyl group, an alkoxy group or a halogen atom.

2. The compound according to claim 1, characterized in that: The alkyl group has 1 to 6 carbon atoms.

3. The compound according to claim 1 or 2, characterized in that: The halogen atom is selected from any one of fluorine, chlorine, bromine or iodine.

4. The compound according to claim 1, characterized in that The chemical structural formula of the compound is shown below:

5. A small molecule compound, characterized in that: The small molecule compound is a protein degradation targeting chimera, including an E3 ligase GID4 ligand and a target protein ligand connected to the E3 ligase GID4 ligand, and the E3 ligase GID4 ligand includes a compound or a salt thereof having a chemical formula as described in any one of claims 1 to 4.

6. Use of the compound or salt thereof according to any one of claims 1 to 4 as a ligand for E3 ligase GID4.

7. The use according to claim 6, characterized in that: The compound or the salt thereof is used as a ligand of E3 ligase GID4 for targeted protein degradation, or for preparing or screening agents for preventing or treating diseases by targeted protein degradation.

8. Use of the compound or salt thereof according to any one of claims 1 to 4 in drug preparation or screening, characterized in that: The drug prevents or treats diseases by targeting protein degradation.

9. A pharmaceutical composition, characterized in that: It comprises a first active ingredient and at least one pharmaceutically acceptable carrier, wherein the first active ingredient is the compound according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof.

10. The pharmaceutical composition according to claim 9, characterized in that: The pharmaceutical composition further comprises a second active ingredient, which is used in combination with the first active ingredient to treat a disease.

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