Application of compound targeting DOCK1 protein in preparation of medicine for treating leukemia

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a compound targeting DOCK1 protein in preparation of a medicine for treating leukemia. The compound capable of being combined with the DOCK1 protein is obtained through virtual screening and a surface plasmon resonance experiment; cell experiments verify that the related compounds can inhibit the survival rate of acute myelogenous leukemia cells, induce apoptosis of the acute myelogenous leukemia cells and weaken the colony ability of the acute myelogenous leukemia cells. The compound provided by the invention inhibits proliferation and development of acute myelogenous leukemia cells, reduces damage to normal tissues and improves tolerance of patients by targeting DOCK1 protein and inhibiting the activity of the DOCK1 protein, and provides a new strategy for treatment of acute myelogenous leukemia.

Description

Technical Field

[0001] This application belongs to the field of pharmaceutical technology, specifically relating to the use of compounds targeting the DOCK1 protein in the preparation of drugs for treating leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is the most common type of acute leukemia, with high morbidity and mortality, posing a serious threat to human health. Currently, treatment options for AML mainly include standard chemotherapy, hematopoietic stem cell transplantation, CAR-T therapy, and targeted therapy. Among these, targeted therapy, based on molecular biology and immunophenotype research, seeks therapeutic targets within the molecular mechanisms of AML development. Compared to traditional chemotherapy, targeted therapy offers significant advantages such as marked efficacy and fewer side effects, bringing hope to patients with AML who cannot tolerate intensive chemotherapy or who have relapsed or are refractory to treatment.

[0003] In recent years, the first-line standard chemotherapy for acute myeloid leukemia has been the "DA 3+7" regimen (3 days of daunorubicin + 7 days of cytarabine), which has a clinical remission rate of 60-80%. However, standard chemotherapy damages normal tissues such as bone marrow and gastrointestinal tract, is poorly tolerated by elderly patients or those with poor physical condition, and most patients experience relapse or drug resistance after treatment, resulting in unsatisfactory efficacy.

[0004] Therefore, developing novel targeted therapies to reduce damage to normal tissues, improve patient tolerance, and overcome relapse or drug resistance has become an important research direction in the treatment of acute myeloid leukemia. Summary of the Invention

[0005] Based on this, one embodiment of this application provides the use of a compound targeting the DOCK1 protein in the preparation of a medicament for treating leukemia.

[0006] This application provides, on the one hand, the use of compounds targeting the DOCK1 protein in the preparation of drugs for treating leukemia.

[0007] In some embodiments, the compound targeting the DOCK1 protein includes any one of the compounds shown in Formula I, Formula II, and Formula III;

[0008]

[0009] Formula I

[0010]

[0011] Formula II

[0012]

[0013] Formula III.

[0014] In some embodiments, the leukemia treated with the drug includes acute myeloid leukemia.

[0015] In some embodiments, the drug induces apoptosis of leukemia cells and inhibits leukemia cell colonies.

[0016] In some embodiments, the compound targeting the DOCK1 protein inhibits the function of the DOCK1 protein.

[0017] In some embodiments, the drug comprises one or more compounds represented by Formula I, Formula II and Formula III, as well as pharmaceutically acceptable excipients.

[0018] In some embodiments, the dosage form of the drug is an oral formulation or an injectable formulation.

[0019] In some embodiments, the oral formulation is a solid dosage form or a liquid dosage form.

[0020] In some embodiments, the injectable formulation is a liquid dosage form.

[0021] In some embodiments, the oral formulation includes one or more of the following excipients: binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, and fragrances.

[0022] The injectable formulation includes one or more of the following excipients: preservatives, analgesics, solubilizers, isotonic agents, and stabilizers.

[0023] In some embodiments, the subject of the drug is a mammal.

[0024] In some of these embodiments, the subject of the drug is a human being.

[0025] This application also provides a drug for treating leukemia, including compounds that target the DOCK1 protein;

[0026] In some embodiments, the compound targeting the DOCK1 protein includes any one of the compounds shown in Formula I, Formula II, and Formula III;

[0027]

[0028] Formula I

[0029]

[0030] Formula II

[0031]

[0032] Formula III.

[0033] This application describes the use of compounds targeting the DOCK1 protein in the preparation of drugs for treating leukemia. Through virtual screening and surface plasmon resonance experiments, compounds capable of binding to the DOCK1 protein were obtained. Cellular experiments verified that these compounds can inhibit the survival rate of acute myeloid leukemia (AML) cells, induce AML cell apoptosis, and reduce the colony-forming ability of AML cells. The compounds provided in this application, by targeting the DOCK1 protein and inhibiting its activity, suppress the proliferation and development of AML cells, reduce damage to normal tissues, and improve patient tolerance, providing a new strategy for the treatment of AML. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 The effects of different concentrations of Lys-CoA (TFA), RTSPSSR, and Box5 (TFA) on the survival rate of acute myeloid leukemia cells;

[0036] Figure 2 Flow cytometry detection of apoptosis induced by Lys-CoA (TFA), RTSPSSR and Box5 (TFA) in acute myeloid leukemia cells;

[0037] Figure 3 The effects of Lys-CoA (TFA), RTSPSSR, and Box5 (TFA) on the colony-forming ability of acute myeloid leukemia cells. Detailed Implementation

[0038] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0039] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0040] the term

[0041] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0042] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0043] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0044] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0045] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0046] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0047] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0048] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0049] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0050] In this document, "drug" includes any agent, compound, composition, or mixture that provides physiological and / or pharmacological effects in vivo or in vitro, and often provides beneficial effects. The scope of the physiological and / or pharmacological effects produced by the "drug" in vivo is not particularly limited; it may have systemic effects or only local effects. The activity of the "drug" is not particularly limited; it may be an active substance that can interact with other substances or an inert substance that does not interact with other substances.

[0051] In this article, "composition" can refer to a combination of multiple substances, or more specifically, a combination of substances used together, or a mixture formed by combining substances.

[0052] In this article, "pharmaceutically acceptable" means those ligands, materials, compositions, and / or dosage forms that are suitable for administration to patients within the bounds of reasonable medical judgment and that are commensurate with a reasonable benefit / risk ratio.

[0053] In this article, “pharmaceutically acceptable carriers” include buffers compatible with drug administration, sterile water for injection, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delay agents, and the like. Each carrier must be “pharmaceutically acceptable” in the sense of being compatible with other components in the formulation and harmless to the patient. Suitable examples include, but are not limited to: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch, potato starch and substituted or unsubstituted β-cyclodextrins; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth gum; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and corn oil. Rice oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) phosphate buffer; and (21) other non-toxic compatible substances used in pharmaceutical formulations.

[0054] In this document, "subject" and "patient" refer to animals, preferably mammals, and more preferably humans. Subjects include, but are not limited to, patients with diseases, conditions, and / or symptoms. The term "mammal" primarily refers to warm-blooded vertebrate mammals, including, but not limited to, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats and mice), pigs, cattle, sheep, horses, and humans, preferably primates, and more preferably humans.

[0055] Acute myeloid leukemia (AML) is a serious threat to human health due to its high incidence and mortality rates. Current treatments for AML primarily include standard chemotherapy, hematopoietic stem cell transplantation, CAR-T therapy, and targeted therapy. Among these, targeted drugs can specifically target specific molecular sites on tumor cells without causing significant damage to normal cells. Compared to traditional chemotherapy, targeted therapy has fewer side effects and is better tolerated by patients. Studies have reported that enoxaparin, by inhibiting DOCK1 protein expression, vimentin phosphorylation, and Akt activation, can increase the sensitivity of non-small cell lung cancer to gefitinib, suggesting that DOCK1 protein may be a novel target for cancer treatment.

[0056] Currently, research on DOCK1 in acute myeloid leukemia (AML) is lacking. This application utilizes bioinformatics techniques, combined with analyses of the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases, to predict that DOCK1 protein is associated with the development and progression of AML and could serve as a prognostic predictor gene for AML. Knockdown of the DOCK1 gene resulted in AML cells exhibiting phenotypes such as inhibited proliferation, increased apoptosis, cell cycle arrest, and weakened colony aggregation, suggesting that DOCK1 protein is a potential therapeutic target for AML. Developing novel drugs targeting DOCK1 protein will contribute to advancing research on AML treatment.

[0057] This application provides, on the one hand, the use of compounds targeting the DOCK1 protein in the preparation of drugs for treating leukemia.

[0058] In some embodiments, the compound targeting the DOCK1 protein includes one or more compounds shown in Formula I, Formula II, and Formula III;

[0059] In some embodiments, the molecular formula of the compound Lys-CoA (TFA) represented by Formula I is C 33 H 54 F3N 10 O 21 P3S has a molecular weight of 1108.82. Lys-CoA (TFA) is a selective p300 histone acetyltransferase (HAT) inhibitor that can significantly inhibit the survival rate of acute myeloid leukemia (AML) cells, induce AML cell apoptosis, and weaken the colony-forming ability of AML cells at a concentration of 1 μM.

[0060]

[0061] Formula I

[0062] In some embodiments, the molecular formula of the compound RTSPSSR represented by Formula II is C 30 H 55 N 13 O 12 With a molecular weight of 789.84, RTSPSSR is a synthetic peptide that binds to claudin-1. At a concentration of 0.1 μM, it can significantly inhibit the survival rate of acute myeloid leukemia (AML) cells, and at a concentration of 1 μM, it can induce apoptosis of AML cells and weaken their colony-forming ability.

[0063]

[0064] Formula II

[0065] In some embodiments, the compound Box5 (TFA) represented by Formula III has the molecular formula C 32 H 51 F3N6O 15 S2, with a molecular weight of 880.90. Box5 (TFA) is a potent Wnt5a antagonist that inhibits Wnt5a signaling and Wnt5a-mediated Ca2+. 2+ Release of the drug significantly inhibited the survival rate of acute myeloid leukemia (AML) cells at a concentration of 0.1 μM, induced apoptosis of AML cells at a concentration of 1 μM, and weakened the colony-forming ability of AML cells.

[0066]

[0067] Formula III

[0068] This application obtained several compounds that target and bind to the DOCK1 protein through virtual screening and surface plasmon resonance experiments. Then, through cell experiments, three compounds that can inhibit acute myeloid leukemia cells were screened: Lys-CoA (TFA), RTSPSSR, and Box5 (TFA). Lys-CoA (TFA) is a selective p300 histone acetyltransferase (HAT) inhibitor that induces a senescence phenotype in normal human melanocytes. RTSPSSR is a synthetic peptide that binds to claudin-1 and was visualized in mouse colorectal tumors using near-infrared fluorescence imaging. Box5 (TFA) is a potent Wnt5a antagonist that inhibits Wnt5a signaling and Wnt5a-mediated Ca2+. 2+ Release. Box5 (TFA) can inhibit cell migration.

[0069] In some embodiments, the leukemia includes acute myeloid leukemia.

[0070] In some embodiments, the treatment of acute myeloid leukemia refers to inducing apoptosis of leukemia cells and inhibiting leukemia cell colonies.

[0071] In some embodiments, the compound targeting the DOCK1 protein treats the leukemia by inhibiting the function of the DOCK1 protein.

[0072] In some embodiments, the leukemia includes acute myeloid leukemia.

[0073] In some embodiments, the medicament comprises one or more compounds represented by Formula I, Formula II and Formula III, as well as a pharmaceutically acceptable carrier and / or excipient.

[0074] In some embodiments, the dosage form of the drug includes an oral formulation or an injectable formulation.

[0075] In some embodiments, the oral formulation is a solid dosage form or a liquid dosage form, and the injectable formulation is a liquid dosage form.

[0076] In some embodiments, the oral formulation includes at least one of the following carriers and / or excipients: binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, colorant, and fragrance;

[0077] The injectable formulation includes at least one of the following carriers and / or excipients: preservatives, analgesics, solubilizers, isotonic agents, and stabilizers.

[0078] This application also provides a drug for treating leukemia, including compounds that target the DOCK1 protein.

[0079] In some embodiments, the compound targeting the DOCK1 protein includes any one of the compounds shown in Formula I, Formula II, and Formula III.

[0080] In some embodiments, the pharmaceutical composition and pharmaceutically acceptable excipients are used together to formulate the drug. Pharmaceutically acceptable excipients, as used herein, include, but are not limited to, diluents, wetting agents, binders, disintegrants, lubricants, flavor and color modifiers, solvents, solubilizers, co-solvents, emulsifiers, antioxidants, metal complexing agents, inert gases, preservatives, local analgesics, pH adjusters, isotonic or isotropic modifiers, etc.

[0081] Furthermore: diluents, such as starch, sucrose, cellulose, inorganic salts, etc.; wetting agents, such as water, ethanol, etc.; binders, such as starch paste, dextrin, sugar, cellulose derivatives, gelatin, povidone, polyethylene glycol, etc.; disintegrants, such as starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, sodium bicarboxymethyl cellulose, crospovidone, surfactants, effervescent disintegrants, etc.; lubricants, such as talc, calcium stearate, magnesium stearate, magnesium dodecyl sulfate, micronized silica gel, polyethylene glycol, etc.; color, flavor, and aroma modifiers, such as pigments, fragrances, sweeteners, etc. Flavoring agents, adhesives, and deodorants, such as fuchsin and xylitol; solvents, such as water, oil, ethanol, glycerin, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oils, and ethyl acetate; solubilizers, such as Tween compounds, maltose compounds, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates; cosolvents, such as organic acids (e.g., citric acid) and their salts, amides and amines, inorganic salts, polyethylene glycol, povidone-iodine, and glycerin; emulsifiers, such as Span compounds, Tween compounds, maltose compounds, benzyl esters, glyceryl fatty acid esters, and high-methylene glycol. Fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth gum, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxides, silica, bentonite, etc.; suspending agents, such as glycerin, syrup, gum arabic, tragacanth gum, agar, sodium alginate, cellulose derivatives, povidone, carboplatin, polyvinyl alcohol, thixotropic gum, etc.; antioxidants, such as sulfites, metabisulfites, bisulfites, ascorbic acid, gallic acid and its esters, etc.; metal complexing agents, such as disodium ethylenediaminetetraacetate, polycarboxylic acid compounds, etc.; inert gases, such as... Nitrogen, carbon dioxide, etc.; preservatives, such as parabens, organic acids and their salts (e.g., sodium benzoate), quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils; local analgesics, such as benzyl alcohol, chlorobutanol, lidocaine, and procaine; pH adjusters, such as hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphates, acetates, citric acid, and citrates; isotonic or isotonic regulators, such as glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0082] It is understood that the diluent described in the embodiments of this application can also be called a filler, and it plays the same role in pharmaceutical preparations; the water described in the embodiments of this application is water that meets the requirements of pharmaceutical preparations, such as water for injection, purified water, etc., and the oil is oil for injection; the preservative described in the embodiments of this application can also be called an antibacterial agent, and it plays a role in inhibiting microbial growth and extending shelf life in the preparation; the lubricant in the embodiments of this application contains flow aids, anti-adhesion agents, etc.; the sugar described in the embodiments of this application can be sugar powder or syrup, and the type of sugar is not limited to glucose; the fragrance described in the embodiments of this application includes, but is not limited to, flavorings.

[0083] In some embodiments, the subject of the drug is a mammal.

[0084] In some of these embodiments, the subject of the drug is a human being.

[0085] The pharmaceutical composition of this application contains an effective amount of the compound represented by Formula I, Formula II, or Formula III. In this document, "effective amount" means the dose by which the corresponding component achieves therapeutic, preventative, alleviating, and / or remission of a specific disease, condition, and / or symptom in a subject; unless otherwise specified, it refers to the dose that achieves therapeutic, preventative, alleviating, and / or remission of symptoms of acute myeloid leukemia infection. In this document, the subject may be a mammal. Further, the subject may be a mouse or a human.

[0086] Based on the above application, this application also provides a method for the prevention or treatment of acute myeloid leukemia, comprising administering a therapeutically effective amount of the compound represented by Formula I, Formula II, or Formula III to the patient. That is, administering a therapeutically effective amount of the compound represented by Formula I, Formula II, or Formula III to the patient.

[0087] In this article, "patient" refers to an animal, preferably a mammal, and more preferably a human. The term "mammal" mainly refers to warm-blooded vertebrate mammals, including but not limited to: such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, rats, pigs, cattle, sheep, horses, and humans, with primates being the preferred species, and humans being the most preferred.

[0088] Method of administration: There are no particular limitations on the dosage form and method of administration of the compounds or pharmaceutical compositions thereof represented by Formula I, Formula II or Formula III of this application.

[0089] Representative administration methods include, but are not limited to, oral, rectal, parenteral (intravenous, intramuscular or subcutaneous) injection and local administration.

[0090] Solid dosage forms for oral administration include tablets, capsules, granules, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following components: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. In capsules, tablets, and pills, the dosage form may also contain a buffer. Solid dosage forms such as tablets, sugar pills, capsules, pills, and granules can be prepared using coatings and shells, such as casings and other materials known in the art. They may contain opacifying agents, and the release of the active compound or compound in such compositions may be delayed in a portion of the digestive tract. Examples of encapsulating components that may be used are polymeric substances and waxes. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0091] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers, and emulsifiers, specifically, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butanediol, dimethylformamide, and oils, particularly cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil, and sesame oil, or mixtures thereof. Besides these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. For example, suspensions may contain suspending agents, specifically, for example, ethoxylated isooctadecyl alcohol, polyoxyethylene sorbitol and dehydrated sorbitan esters, microcrystalline cellulose, aluminum methoxide, and agar, or mixtures thereof.

[0092] Compositions intended for rectal or parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous or non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0093] Dosage forms for topical administration include ointments, powders, patches, sprays, and inhalers. They are prepared by mixing the active ingredient under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants as needed.

[0094] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0095] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0096] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0097] Example 1

[0098] I. Virtual screening to predict compounds targeting DOCK1 protein

[0099] The DOCK1 protein has three domains: SH3, DHR-1, and DHR-2. The SH3 domain mediates the formation of a functional complex with the phagocytic and cellular motility protein ELMO1; the DHR-1 domain targets membranes rich in PtdIns (3,4,5)P3 and enhances protein activity through interaction with GTPases such as Rac1; the DHR-2 domain contains Rho-GTPase exchange factor activity. Based on the importance of the DHR-1 domain for the cellular function of the DOCK1 protein, this application performs virtual screening on the DHR-1 domain of DOCK1 (PDB ID: 3L4C), selecting the phospholipid-binding pocket of the protein (key amino acids: LYS439 / LYS442 / LYS446 / LYS524) to obtain compounds with strong binding affinity to the target protein.

[0100] 1. Prepare protein

[0101] The three-dimensional structure of the Human DOCK1 protein (PDBID: 3L4C) was downloaded from the PDB website (https: / / www.pdbus.org / ). The protein was processed using the Protein Preparation Wizard module, including solvent removal, B-chain deletion, hydrogen addition, and subsequent energy optimization (OPLS2005 force field, RMSD of 0.3 Å). A grid file was created using the Receptor Grid Generation module, centered on amino acid residues LYS439 / LYS442 / LYS446 / LYS524, with a box size of 20 Å × 20 Å × 20 Å.

[0102] 2. Preparation of compounds

[0103] The 2D format of compounds in the database is processed using the LigPrepModule in the Schrödinger software, undergoing hydrogenation and energy optimization to output 3D structures for virtual screening. Next, molecular docking is performed. The prepared compounds are imported, and molecular docking is conducted using the Glide module, where acceptor and ligand molecules dock with each other through geometric and energy matching.

[0104] 3. The binding force between the target and the compound, the compound structure, etc. were manually verified, and the top 13 compounds with the highest absolute values ​​of molecular docking scores (Table 1) were selected for subsequent experiments.

[0105] Table 1: The top 13 compounds obtained from virtual screening

[0106]

[0107] II. Recombinant Protein Production

[0108] Using molecular cloning technology, the coding sequence of the DOCK1 amino acid sequence (G422-L619) contained in the aforementioned Receptor Grid Generation file was cloned into the pET28a vector, with a 6×histidine tag designed at the N-terminus of the cloned sequence. The pET28a vector was transfected into *E. coli* BL21(DE3)pLySS, and after induction of expression, the *E. coli* cells were disrupted by sonication, and the recombinant protein was purified by nickel column affinity chromatography. Subsequently, the 6×histidine tag was removed using the ULP1 enzyme, and purification was performed again to obtain a qualified recombinant protein.

[0109] III. Surface plasmon resonance experiments yielded three compounds targeting DOCK1.

[0110] In this embodiment, Biacore was used to determine the affinity of the compound for interaction with the DOCK1 protein.

[0111] The specific method was as follows: First, the purified DOCK1 recombinant protein was immobilized onto a CM5 chip. The chip was activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide at a flow rate of 10 μL / min. Then, the DOCK1 recombinant protein was diluted with sodium acetate to 50 μg / mL and immobilized on the chip at a flow rate of 10 μL / min. Finally, the channels were blocked with ethanolamine at a flow rate of 10 μL / min. Next, the interaction between the compounds and the DOCK1 recombinant protein was detected.

[0112] The specific method was as follows: The compounds were diluted to 100 μM with PBS solution and flowed at a rate of 10 μL / min for 150 s during each run. At the end of each flow, the chip was regenerated with 10 mM glycine hydrochloride (pH 2.0) solution for 5 min. This process was repeated until all compounds were analyzed. Finally, the Biacore instrument (model: 8K) provided the response unit (RU) value for each compound.

[0113] The results are as follows: Through virtual screening, the docking results of digitally simulated compounds with DOCK1 molecules were obtained. Specifically, the higher the absolute value of the docking score, the stronger the binding force between the compound and the protein. The top 13 compounds with the highest absolute docking scores were selected for surface plasmon resonance experiments to obtain the response unit (RU) value of each compound's interaction with the DOCK1 recombinant protein. Compounds with a response unit value > 20 were considered to have the ability to bind to the DOCK1 recombinant protein.

[0114] Through virtual screening and surface plasmon resonance experiments, three compounds targeting the DOCK1 protein were obtained: Lys-CoA (TFA), RTSPSSR, and Box5 (TFA). The results are shown in Table 2.

[0115] Table 2: Three compounds targeting the DOCK1 protein

[0116]

[0117] IV. Cell Viability Experiment

[0118] Cell viability assays were performed using the Cell Counting Kit-8 (CCK-8). The specific procedures are as follows:

[0119] (1) Acute myeloid leukemia cells in the logarithmic growth phase were seeded into 96-well plates, with 2 × 10⁶ cells seeded per well. 4Each well was filled with culture medium containing different compound concentrations to a final volume of 100µL. Each experimental group had 3 replicates and was incubated at 37℃ in a 5% CO2 incubator for 48h.

[0120] (2) Take out the 96-well plate after culture, add 10µL CCK-8 solution to the well, and then incubate in a 37℃, 5% CO2 incubator for 4h.

[0121] (3) Take out the 96-well plate, gently mix it on a track shaker for 1 min, and measure the absorbance at 450 nm using an ELISA reader.

[0122] (4) Calculate cell viability based on absorbance:

[0123] Cell viability (%) = [(As-Ab) / (Ac-Ab)] × 100%.

[0124] As = Absorbance of experimental wells (absorbance of wells containing cells, culture medium, CCK-8 and the compound to be tested).

[0125] Ab = Absorbance of blank wells (absorbance of wells containing culture medium and CCK-8).

[0126] Ac = Absorbance of control wells (absorbance of wells containing cells, culture medium, and CCK-8).

[0127] Acute myeloid leukemia cells were treated with 0.01 μM, 0.1 μM, 1 μM and 10 μM Lys-CoA (TFA), RTSPSSR and Box5 (TFA) for 48 h, respectively, and then the survival rate of acute myeloid leukemia cells was detected by CCK8 reagent.

[0128] The results showed that compounds RTSPSSR and Box5 (TFA) significantly inhibited the survival rate of acute myeloid leukemia cells at a concentration of 0.1 μM, and compound Lys-CoA (TFA) also significantly inhibited the survival rate of acute myeloid leukemia cells at a concentration of 1 μM.

[0129] The results are as follows Figure 1 As shown in the figure, the concentration gradients were 0.01 μM, 0.1 μM, 1 μM and 10 μM, the treatment time was 48 h, the acute myeloid leukemia cell line used was MV4-11, and each group had 3 replicates.

[0130] V. Apoptosis Experiment

[0131] Apoptosis experiments were performed using Annexin V-APC / PIApoptosis Kit (catalog number: E-CK-A217). The specific procedures are as follows:

[0132] (1) Acute myeloid leukemia cells in the logarithmic growth phase were seeded into 6-well plates, with approximately 5 × 10⁶ cells seeded per well. 5 Each well was filled with culture medium containing different compound concentrations to a final volume of 3 mL. Each experimental group had 3 replicates. The wells were incubated at 37°C in a 5% CO2 incubator for 48 h.

[0133] (2) Collect cells, wash twice with PBS solution, add 500 μL of diluted 1×AnnexinV Binding Buffer to resuspend cells, add 5 μL of Annexin V-A PCReagent and 5 μL of PIReagent (50 μg / mL) to the cell suspension, gently vortex to mix, and incubate at room temperature in the dark for 20 min.

[0134] (3) Flow cytometry detection of apoptosis.

[0135] Acute myeloid leukemia cells were treated with 1 μM Lys-CoA (TFA), RTSPSSR and Box5 (TFA) for 48 h, and then the apoptosis of acute myeloid leukemia cells was detected by flow cytometry.

[0136] The results showed that all three compounds significantly induced apoptosis in acute myeloid leukemia cells, as indicated by the results below. Figure 2 As shown in the figure. Caption: All compounds used were at a concentration of 1 μM, the treatment time was 48 h, the acute myeloid leukemia cell line used was MV4-11, Early represents early apoptosis rate, Late represents late apoptosis rate, and Total represents total apoptosis rate. Each group had three replicates.

[0137] VI. Colony Formation Experiment

[0138] The colony formation experiment was conducted using the MethoCult™ H4434 Classic, and the specific procedures are as follows:

[0139] (1) Acute myeloid leukemia cells in the logarithmic growth phase were seeded into 12-well plates, with approximately 3 × 10⁻⁶ cells seeded per well. 3 Each well was filled with methylcellulose medium containing different compounds to a final volume of 1.5 mL. Each experimental group had 3 replicates and was incubated in a 37°C, 5% CO2 incubator for 2 weeks.

[0140] (2) Take out the 12-well plate and count the number of cell colonies in each well.

[0141] The effects of three compounds on the colony-forming ability of acute myeloid leukemia (AML) cells were examined using methylcellulose medium. The final concentration of each compound in the medium was 1 μM, and cell colonies were counted after two weeks of culture. The results showed that all three compounds significantly reduced the number of AML cell colonies, and the area of ​​AML cell colonies treated with these compounds was also reduced.

[0142] The results are as follows Figure 3 As shown in the figure caption: the concentration of all compounds used was 1 μM, the culture time was 2 weeks, the acute myeloid leukemia cell line used was MV4-11, and each group had 3 replicates.

[0143] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. Use of a compound targeting DOCK1 protein in the preparation of a medicament for treating leukemia.

2. Use according to claim 1, characterized in that, The compound targeting DOCK1 protein includes any one of the compounds shown in Formula I, Formula II and Formula III; Formula I Formula II Formula III.

3. Use according to claim 1, characterized in that, The leukemia treated by the medicament includes acute myeloid leukemia.

4. Use according to claim 3, characterized in that, The medicament induces apoptosis of leukemia cells and inhibits leukemia cell colony.

5. Use according to claim 3 or 4, characterized in that, The compound targeting DOCK1 protein inhibits DOCK1 protein from functioning.

6. Use according to any one of claims 1 to 4, characterized in that, The medicament includes one or more of the compounds shown in Formula I, Formula II and Formula III and pharmaceutically acceptable adjuvants.

7. Use according to claim 6, characterized in that, The dosage form of the medicament is oral preparation or injection preparation; Optionally, the oral preparation is solid dosage form or liquid dosage form; Optionally, the injection preparation is liquid dosage form.

8. Use according to claim 7, characterized in that, The oral preparation includes one or more of the following adjuvants: binding agent, lubricant, disintegrating agent, excipient, solubilizing agent, dispersing agent, stabilizing agent, suspending agent, coloring agent and aromatic agent; The injection preparation includes one or more of the following adjuvants: preservative, analgesic, solubilizing agent, isotonic agent and stabilizing agent.

9. The use according to any one of claims 1 to 4 and 7 to 8, characterized in that, The subject of the medicament is mammal; Optionally, the subject of the medicament is human.

10. A medicament for treating leukemia, characterized by comprising the compound of claim 1. The compound targeting DOCK1 protein; Optionally, the compound targeting DOCK1 protein includes any one of the compounds shown in Formula I, Formula II and Formula III; Formula I Formula II Formula III.

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