Targeting regulatory molecule for intracellular target protein conformation and regulation method

Abstract

The present application relates to a kind of target protein conformation targeting regulatory molecules and regulatory methods in living cells.The protein conformation targeting regulatory molecules include: 1) with the targeting group of selectively binding target protein as skeleton, 2) two active functional groups are modified on the skeleton, c) rigid connecting arm introduced between targeting group and active functional group.Based on the above characteristics, after the protein conformation targeting regulatory molecules of the cell are incubated with cell, they are rapidly combined with target protein in cell specifically through cell membrane, and covalent reaction occurs with amino acid near binding pocket, to target the conformation of target protein is regulated.According to the difference of protein conformation and reaction site, different active groups and rigid connecting arm are used, to realize the accurate regulation of target protein conformation in different functional states.The present application provides important technical support in the field of exploring protein conformation and function, expanding the library of druggable proteins, researching new use of old drugs, disease treatment or prevention.

Description

TECHNICAL FIELD

[0001] The present application relates to a targeting regulatory molecule and a regulatory method for the conformation of a target protein in living cells, which can target and regulate the conformation of the target protein, change the function of the target protein, such as activating or inhibiting its activity, and even induce the aggregation or degradation of the protein. BACKGROUND

[0002] Proteins with dynamic conformations play a role in all cellular processes from signal transduction to transcription, and by inducing or stabilizing the conformation of proteins, the activity of dynamic proteins can be regulated, the structure-function relationship of proteins and related biological mechanisms can be analyzed, and the development of new drugs can also be expanded (Trends in Biochemical Sciences, 2022, 47(12): 1023-1037). The regulation of the activity of dynamic proteins is usually achieved by masking the binding surface through intramolecular or intermolecular interactions, inducing changes in protein conformation, and a combination of the two mechanisms. The current methods for inducing changes in protein conformation include 1) post-translational modification (PTM): nature often uses PTM to covalently modify amino acid residues to regulate the conformation of proteins in a positive and negative manner, such as ubiquitination, acetylation, phosphorylation, and glycosylation, which have been found to be allosteric modulators. PTM occurs on many protein-protein interaction interfaces to promote or inhibit the binding of the two, and fine-tuning protein function through combinations of PTMs can enable a single protein to exert different cellular effects. 2) Allosteric regulation: allosteric small molecules bind specifically to sites other than the substrate active site of the protein, causing changes in the conformation of the protein molecule and thus changing the activity of the protein. Since the allosteric sites between protein subfamily members or homologues are often not conserved, allosteric modulators have better subtype selectivity, which is beneficial to reduce the side effects of drugs. In addition, allosteric drugs that do not depend on orthosteric sites also provide solutions to "undruggable target proteins" and drug resistance problems. However, the development of allosteric molecules still faces great challenges, such as the difficulty of finding effective allosteric sites, the fact that allosteric sites are more prone to drug resistance mutations than orthosteric sites, the fact that allosteric modulators have lower binding affinity than orthosteric modulators, and the difficulty of obtaining co-crystal complexes of allosteric molecules and proteins. These difficulties make the selection of molecules for regulating protein conformation with allosteric molecules limited and require high screening costs (Current Opinion in Structural Biology, 2010, 20(2): 142-147; Cell Chemical Biology, 2020, 27(8): 986-997).

[0003] Chemical cross-linking technology is a chemical cross-linking method that uses a chemical cross-linking agent to covalently cross-link proteins with a spatial distance close enough. By using mass spectrometry and bioinformatics processing, the chemical proteomics analysis tool can analyze the composition and interaction interface of protein complexes. In recent years, this technology has also been combined with theoretical calculations and cryo- electron microscopy to analyze the structure of dynamic proteins. Chemical cross-linking has flexible controllability. By using different reactive functional groups, adjusting the length of the connecting arm, hydrophilicity or hydrophobicity, and flexibility, the conformation of the protein can be comprehensively described. However, due to the lack of targeting of chemical cross-linking reaction and poor controllability, it is difficult to achieve precise reaction of amino acids in specific regions of the protein.

[0004] The present application combines the reported targeting drugs or ligands of proteins with the in situ cross-linking idea to develop a new protein conformation regulation method, which can accurately and controllably regulate the conformation and activity of the target protein, analyze the protein conformation-function relationship and related biological mechanisms, and develop new disease treatment strategies. SUMMARY

[0005] The object of the present application is to provide a targeting regulation molecule and a regulation method for the conformation of a target protein in living cells. After the protein conformation targeting regulation molecule is incubated with cells, it quickly penetrates the membrane and binds to the target protein under the action of the targeting group of the molecule. The double-end active functional groups on the molecule covalently react with the amino acids near the binding domain of the target protein, thereby fixing the conformation of the protein and changing the functional activity of the protein. The method of the present application has the characteristics of in situ targeting and controllable reaction, and realizes the in situ targeting regulation of the conformation and activity of the target protein in the cell environment.

[0006] A targeting regulation molecule for the conformation of a target protein in living cells,

[0007] The molecule comprises: a) a targeting group that selectively binds to the target protein; b) two active functional groups modified on the skeleton, the double-end functional groups have a short-range reaction activity, and only when the regulation molecule binds to the target protein, the functional groups react with the specific amino acid reaction sites within a distance of about 5E-10 meters, and the functional groups can covalently bind to the adjacent amino acids after being excited by light; and c) a rigid connecting arm introduced between the targeting group and the active functional group. According to the differences in the protein conformation to be regulated and the covalent reaction sites, different active groups or different rigid connecting arms are introduced between the targeting group and the active group in the protein conformation targeting regulation molecule, so as to realize the targeting and site-specific protein conformation regulation, better distinguish structurally similar proteins, and reduce off-target effects.

[0008] The binding affinity of the targeting group to the target protein is 1E-6 to 1E-13 moles, and the targeting group is derived from the target protein, inhibitors, agonists, active ligand molecules, substrate molecules, etc. that have been reported or screened by structural biology, computational methods, including one or more of polypeptides, small molecules, nucleic acids, antibodies, nanoparticles, etc.

[0009] The active functional group includes a functional group that can covalently react when the distance from the amino acid is less than about 5E-10 meters, or a photo-reactive functional group that becomes covalently reactive after photoactivation; the active functional group can be: acrylamide, alpha-cyanocrylamide, alkyne, aldehyde, 2-formylphenylboronic acid, nitrile group connected to aromatic heterocycle, fumarate, propargylamide, propargyl nitrile, alkenyl-substituted heteroarene, alkyne-substituted heteroarene, propargylamide, arylsulfonyl, halogenated hydrocarbon, alpha-halogenated methyl amide / ester / ketone, epoxide, aziridine, nitroalkane, nitroheteroarene, alpha-cyanoketone, heteroaryl nitrile, cyanamide, isothiocyanate, vinyl sulfonamide, vinyl sulfone, sulfonyl fluoride, sulfonylimide fluoride, aryl fluorosulfate, N-acyl-N-alkyl sulfonamide, N-methylisoxazolium, oxaziridine, and one or more of the derivatives of the foregoing;

[0010] The photo-reactive functional group is selected from one or more of a benzophenone group, a phenyl azide group, a diazirine, or a 2-aryl-5-carboxy tetrazole, and the photoexcitation wavelength of the photo-reactive functional group is 200-800 nm.

[0011] Different active functional groups or rigid linking arms are used between the targeting group and the active functional group according to the conformation of the target protein to be regulated and different covalent reaction amino acid sites, and the composition of the rigid linking arm includes a double bond, a triple bond, a glycosidic bond, or a cyclic group, etc., as well as one or more of the derivatives of the foregoing groups.

[0012] A method for targeting and regulating the conformation of a target protein in a living cell using the above-mentioned targeting regulatory molecule,

[0013] Preparation of the mother liquor of the protein conformation targeting regulatory molecule: using one or more of water, buffer, or acetonitrile, organic alcohols, organic acids, DMF, or DMSO to prepare a mother liquor with a concentration of 100 nM to 1 M.

[0014] The mother liquor of the protein conformation targeting regulatory molecule is diluted with one of cell culture medium DMEM or MEM or R1640, and is added to the cells, which are incubated at 37℃ in a 5% CO2 environment for 0.5-120 hours; if the double-end active group comprises a photo-reactive group, after the regulatory molecule and the cells are incubated under the above conditions, light irradiation is performed to initiate covalent reaction with the target protein. The light irradiation conditions include: the light excitation wavelength is 200-800 nm, the light irradiation treatment time is 10 s-60 min, and the binding selectivity of the targeting regulatory molecule to the target protein and the appropriate reaction site are predicted and analyzed.

[0015] The prediction and analysis is performed by one or more of X-ray crystal diffraction, cryo-EM, molecular docking, molecular dynamics simulation, drug virtual screening, conformation search and optimization, and the binding selectivity of the targeting regulatory molecule to the target protein and the appropriate reaction site are predicted and analyzed.

[0016] The in-situ cross-linking mass spectrometry technology, nuclear magnetic resonance technology, X-ray crystal diffraction, cryo-EM and a series of functional experiments (such as CCK-8 experiment, clonogenic assay, immunofluorescence, co-immunoprecipitation, post-translational modification site identification, etc.) are used to verify the regulatory effect of the protein conformation targeting regulatory molecule on the conformation and functional activity of the target protein. The experimental operation in the present application is usually performed according to the conventional experimental conditions, or can be performed according to the experimental conditions in the examples, and the reagents can be used according to the manufacturer's instructions.

[0017] The present application has the following advantages:

[0018] (1) The present application develops a method for targeting and regulating the conformation of the target protein in cells by combining the reported targeting drugs or ligands with the in-situ cross-linking idea, which has high specificity and reaction site selectivity, and realizes the active regulation of the activity and function of the target protein in cells.

[0019] (2) The protein conformation regulatory molecule in the present application can be modularized and flexibly modified. Different targeting groups and active groups are selected according to the relationship between the conformation and function of the target protein and the structure, and the regulation degree is controllable by combining with the calculation method.

[0020] (3) The protein conformation targeting regulatory molecule developed in the present application has good discrimination for the target protein and its conformationally similar or sequence similar proteins, because it simultaneously introduces a targeting group specifically binding to the target protein and an active group developed for the reaction site of the target protein and a rigid connecting arm. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The chemical structural formula of the PIN1 protein targeting conformation regulatory molecule KP1 prepared in Example 1.

[0022] Figure 2 Synthetic route of KP1 in Example 1.

[0023] Figure 3 Crystal structure of KP1 complexed with PIN1.

[0024] Figure 4 Inhibition of Hela cell proliferation by conformational targeting regulator KP1 of PIN1.

[0025] Figure 5 Immunoblotting of PIN1 degradation induced by conformational targeting regulator KP1 of PIN1 in MCF-7 cells. DETAILED DESCRIPTION

[0026] Example 1

[0027] Synthesis of PIN1 protein targeting conformational regulator KP1

[0028] (1) A PIN1 protein targeting conformational regulator KP1 was developed, and its structural formula is shown in Figure 1 ZL-PIN13, a targeting inhibitor of PIN1, has an active group, chloroacetamide, which can covalently bind to Cys113. A vinyl sulfone fluorophore was modified on the biphenyl group of the targeting group to react with Lys132, thereby regulating the conformation of the PPIase domain catalytic enzyme activity pocket of PIN1.

[0029] (2) The synthetic route is shown in Figure 2

[0030] Step a: To a solution of 2-furan-amine (1) (1 mmol) and 1-Boc-4-piperidone (1.1 mmol) in toluene (15 mL) solution, add mercaptoacetic acid (1.2 mmol), mix at room temperature. Stir the reaction mixture at room temperature for 5 minutes and reflux for 12 hours. Cool the reaction mixture to room temperature, dilute with water (10 mL), and extract with ethyl acetate (15 mL x 2). Wash the combined organic phase with brine, dry over anhydrous Na2SO4, and evaporate. Purify the residue by flash chromatography to obtain compound 2. 1 H NMR (500 MHz, CDCl3) δ 7.55 (s, 3H), 6.38 (s, 2H), 6.28 (s, 3H), 4.06 (s, 6H), 1.53 (s, 6H).

[0031] ​Step b: Compound 2 (1 mmol) was dissolved in dry DMF (10 mL) followed by the addition of N-bromosuccinimide (NBS, 1.2 mmol) at 0 °C. After stirring at room temperature overnight, the reaction mixture was quenched with water (15 mL) and extracted with ethyl acetate (EtOAc, 20 mL x 3). The combined organic phase was washed with brine, dried over anhydrous Na2S04and evaporated. The residue was purified by flash chromatography to give compound 3. 1 H NMR (500 MHz, CDC13) δ 7.55 (s, 3H), 6.38 (s, 2H), 6.28 (s, 3H), 5.30 (s, 3H), 4.80 (s, 3H), 3.63 (s, 7H), 3.59 (s, 3H), 3.49 (s, 4H), 2.43 (s, 2H), 2.15 (s, 2H), 1.42 (s, 26H).

[0032] Step c: Compound 3 (1 mmol) and 1,3-naphthalene diboronic acid (1.1 mmol) were dissolved in a solution of isopropyl alcohol (IP A) / H20 in a volume ratio of 3: 1 (24 mL), followed by the addition of Pd(PPh3)2Cl2(0.05 mmol) and Na2C03(5 mmol). Under nitrogen protection, the reaction mixture was stirred at 85 °C for 12 hours, and then cooled to room temperature. The reaction mixture was diluted with water (15 mL) and extracted with EtOAc (30 mL x 3). The combined organic phase was washed with brine, dried over anhydrous Na2S04and evaporated. The residue was purified by flash chromatography to give compound 4. 1 H NMR (500 MHz, CDC13) δ 6.48 (s, 2H), 6.15 (s, 2H), 5.34 (s, 2H), 4.84 (s, 2H), 3.61 (d, J = 18.1 Hz, 7H), 3.49 (s, 3H), 2.43 (s, 1H), 2.15 (s, 1H), 1.42 (s, 18H).

[0033] Step d: A sealed tube under nitrogen protection was charged with ethylenesulfonyl fluoride (0.108 mmol), Pd(OAc)2(10 mol), Cu(OAc)2(0.072 mmol), LiOAc (0.043 mmol) and 2.0 mL of anhydrous tetrahydrofuran. Subsequently, compound 4 (0.036 mmol) was dissolved in anhydrous tetrahydrofuran (2.0 mL) and added dropwise to the reaction mixture in the above-mentioned sealed tube using a syringe within about 30 minutes. The reaction mixture was stirred at room temperature for 4 hours. Upon completion, 10 mL of a methylamine solution was immediately added, stirred for 10 minutes, and then filtered through a plug of celite. The filtrate was concentrated in vacuo and purified by column chromatography on neutral alumina using 30% ethyl acetate in hexane as the eluent to give compound 5. 1H NMR (500 MHz, CDC13) δ 8.40 (s, 5H), 7.89 (s, 5H), 7.87-7.81 (m, 17H), 7.79 (s, 3H), 7.71 (s, 5H), 7.16 (s, 3H), 6.97 (s, 7H), 6.12 (s, 5H), 5.04 (s, 5H), 4.92 (s, 5H), 3.66 (s, 10H), 3.59 (s, 5H), 3.49 (s, 7H), 2.41 (s, 3H), 2.16 (s, 6H), 1.42 (s, 43H).

[0034] Steps e and f: To a solution of HC1 / EtOAc (2 mol / L, 20 mL) was added compound 5 (1 mmol) at room temperature. After stirring overnight, the reaction was concentrated in vacuo and basified with 1 M NaOH aqueous solution to pH 8-9. The mixture was extracted with EtOAc (25 mL x 3), washed with brine, dried over anhydrous Na2S04, filtered and evaporated to give the crude product. The crude product was dissolved in anhydrous dichloromethane (25 mL) under nitrogen protection, followed by the addition of potassium carbonate (1.1 mmol) and chloroacetyl chloride (1.1 mmol). The reaction mixture was stirred at room temperature for 1 hour and evaporated. The residue was purified by flash chromatography to give KP1. 1 H NMR (500 MHz, CDC13) δ 8.40 (s, 5H), 7.89 (s, 5H), 7.87-7.81 (m, 17H), 7.79 (s, 3H), 7.71 (s, 5H), 7.16 (s, 3H), 6.97 (s, 7H), 6.12 (s, 5H), 5.04 (s, 5H), 4.92 (s, 5H), 3.66 (s, 10H), 3.59 (s, 5H), 3.49 (s, 7H), 2.41 (s, 3H), 2.16 (s, 6H), 1.42 (s, 43H).

[0035] The chemical structure of KP1 is shown below:

[0036]

[0037] Example 2

[0038] Targeting the conformation and activity of PIN1 protein in HeLa cells

[0039] (1) The developed PIN1 protein conformation targeting regulatory molecule KP1 regulates the conformation of the PPIase domain catalytic enzyme activity pocket of PIN1, hinders the recognition and binding of the PPIase domain with the substrate, and inhibits the biological activity of PIN1 protein promoting cancer cell proliferation.

[0040] (2) Regulating molecules binding target proteins: Hela cells were inoculated in 96-well plates at 5000 cells per well. After the cells adhered and grew well, 100 microliters of cell culture solution was added with a final concentration of 0, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM, 750 nM, and 1 micromole of KP1, mixed, and incubated at 37°C in a cell incubator containing 5% CO2 and 95% air for 24 hours.

[0041] (3) CCK8 was added to the cells at 10 microliters, and the cells were incubated at 37°C in a cell incubator containing 5% CO2 and 95% air for 1 hour.

[0042] (4) The absorbance at 450 nm was measured by an enzyme marker. Three repeats were taken for each concentration point. The specific results are shown in Table 1. Figure 4

[0043] (5) Figure 4 The results of the influence of the conformational targeting regulatory molecule KP1 on the proliferation of Hela cells using PIN1 showed that KP1 could effectively inhibit the proliferation of Hela cells, and the IC50 was 450 nM.

[0044] Example 3

[0045] Targeting the conformation and activity of PIN1 protein in MCF-7 cells

[0046] (1) Regulating molecules KP1 binding PIN1: KP1 (prepared with dimethyl sulfoxide) was added to the well-grown MCF-7 cells at a final concentration of 0, 10 nM, 100 nM, and 1 micromole, and incubated at 37°C in a 5% CO2 cell incubator for 24 hours.

[0047] (2) Protein extraction: The cell culture medium was discarded, and the cells were washed twice with 4-degree pre-cooled PBS. One milliliter of RIPA (strong) lysis solution containing 1% protease inhibitor was added, and the cells and proteins were collected on ice for 30 minutes.

[0048] (3) The BCA protein concentration determination kit was used to determine the protein concentration, and the protein concentration was adjusted to 2 mg / mL using PBS.

[0049] (4) 50 micrograms of whole protein 2 mg / mL protein solution was taken for each experimental group, 6xLoadingBuffer was added, heated at 95 degrees for 5 minutes. The heated protein was subjected to SDS-PAGE gel electrophoresis.

[0050] ​(5) The bands on the gel were transferred to PVDF membrane using wet transfer method. After transfer, the membrane was blocked with 5% skim milk (dissolved in TBST) at room temperature for 1 hour, and then the membrane was soaked in a TBST (Tris-buffered saline containing 0.1% Tween 20) solution containing 2 μg / mL PIN1 antibody at 4 degrees overnight. The membrane was washed with TBST for 3 times, and a TBST solution containing 2 μg / mL goat anti-rabbit secondary antibody was added, and incubated at room temperature for 30 minutes. The membrane was washed with TBST for 3 times.

[0051] (6) The luminescent solution was added dropwise to the membrane, and the reaction was carried out in the dark for 2 minutes, and then exposed. The exposure results are shown in Figure 5

[0052] (7) Figure 5 The results of inducing PIN1 degradation in MCF-7 cells using the conformational targeting molecule KP1 of PIN1. As shown in the results, under the premise that the protein loading amount is the same as indicated by the internal reference protein β-Actin, after the application of KP1, the PIN1 protein in MCF-7 is degraded, and the degradation of PIN1 has a concentration-dependent effect.​

Claims

1. An intracellular target protein conformation targeting regulatory molecule, characterized in that, The structure of the target protein conformation targeting regulatory molecule is as follows: 。 2. A preparation method of the target protein conformation targeting regulatory molecule of claim 1, characterized in that, The preparation, synthesis route of the target protein conformation targeting regulatory molecule is as follows: ， The specific preparation method comprises the following steps: Step a: 1.2 mmol of mercaptoacetic acid is added to a toluene solution containing 1 mmol of 2-furan-amine, i.e. compound 1, and 1.1 mmol of 1-Boc-4-piperidone, the mixture is mixed at room temperature, the reaction mixture is stirred at room temperature for 5 minutes and refluxed for 12 hours, the reaction mixture is cooled to room temperature, diluted with 10 mL of water, and extracted with 15 mL of ethyl acetate twice, the combined organic phase is washed with brine, dried over anhydrous Na2SO4, and evaporated, the residue is purified by flash chromatography to obtain compound 2; Step b: 1.2 mmol of N-bromosuccinimide is added to 1 mmol of compound 2 dissolved in 10 mL of anhydrous DMF at 0°C, the mixture is stirred at room temperature overnight, the reaction mixture is quenched with 15 mL of water and extracted with 20 mL of ethyl acetate three times, the combined organic phase is washed with brine, dried over anhydrous Na2SO4, and evaporated, the residue is purified by flash chromatography to obtain compound 3; Step c: 1 mmol of compound 3 and 1.1 mmol of 1,3-naphthalene diboronic acid are dissolved in 24 mL of a solution of isopropyl alcohol / H2O with a volume ratio of 3:1, 0.05 mmol of Pd(PPh3)2Cl2 and 5 mmol of Na2CO3 are further added, the reaction mixture is stirred at 85°C for 12 hours under nitrogen protection, and then cooled to room temperature, the reaction mixture is diluted with 15 mL of water and extracted with 30 mL of EtOAc three times, the combined organic phase is washed with brine, dried over anhydrous Na2SO4, and evaporated, the residue is purified by flash chromatography to obtain compound 4; Step d: a sealed tube under nitrogen protection is charged with 0.108 mmol of ethenesulfonyl fluoride, 10 mol of Pd(OAc)2, 0.072 mmol of Cu(OAc)2, 0.043 mmol of LiOAc, and 2.0 mL of anhydrous tetrahydrofuran, then 0.036 mmol of compound 4 is dissolved in 2.0 mL of anhydrous tetrahydrofuran, and the reaction mixture in the sealed tube is added dropwise to the above reaction mixture using a syringe within 30 minutes, the reaction mixture is stirred at room temperature for 4 hours, after completion, 10 mL of a methylamine solution is immediately added, stirred for 10 minutes, then filtered through a diatomite plug, the filtrate is concentrated under vacuum, and purified by neutral alumina column chromatography using 30% ethyl acetate in hexane as the eluent to obtain compound 5; Step e: To a 20 mL solution of 2 mol / L HCl in EtOAc was added 1 mmol of compound 5 at room temperature, after stirring overnight, the reaction was concentrated in vacuo and basified with 1 M aqueous NaOH to pH 8-9, the mixture was extracted with 25 mL EtOAc three times, washed with brine, dried over anhydrous Na2SO4, filtered and evaporated to give the crude product, under nitrogen protection, the crude product was dissolved in 25 mL anhydrous dichloromethane solution, then 1.1 mmol of potassium carbonate and 1.1 mmol of chloroacetyl chloride were added, the reaction mixture was stirred at room temperature for 1 hour and evaporated, the residue was purified by flash chromatography to give the protein conformation targeting regulator, i.e., compound 6.

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