Targeted regulation molecule and regulation method for intracellular target protein conformation

Abstract

The invention relates to a targeted regulation molecule and a regulation method for target protein conformation in living cells. The protein conformation targeting regulation molecule comprises: 1) a targeting group selectively combined with a target protein as a skeleton, 2) two active functional groups modified on the skeleton, and c) a rigid connecting arm introduced between the targeting group and the active functional groups. After being co-incubated with cells, the protein conformation targeted regulation molecule based on the characteristics can be rapidly and specifically combined with target protein in the cells through cell membranes, and is subjected to covalent reaction with amino acid near a binding pocket, so that the conformation of the target protein is regulated in a targeted manner. According to different protein conformations and different reaction sites, different active groups and rigid connecting arms are adopted to realize accurate regulation and control of target protein conformations in different functional states. The invention provides important technical support in the fields of exploration of protein conformation and functions, expansion of patent medicine protein libraries, research of new use of old medicines and disease treatment or prevention.

Description

Technical Field

[0001] The present invention relates to a targeted regulatory molecule and a regulatory method for the conformation of a target protein in a living cell, which can regulate the conformation of the target protein in a targeted manner, change the function of the target protein, such as activating or inhibiting its activity, and even induce protein aggregation or degradation. Background Art

[0002] Proteins with dynamic conformations play a role in all cellular processes from signal transduction to transcription. The activity of dynamic proteins can be regulated by inducing or stabilizing the conformation of proteins, which can dissect the structure-function relationship of proteins and their related biological mechanisms, and can also expand the development of new drugs (Trends in Biochemical Sciences, 2022, 47(12): 1023–1037). The activity regulation of dynamic proteins is usually achieved by masking the binding surface through intramolecular or intermolecular interactions, inducing protein conformational changes, and a combination of the two mechanisms. The methods currently developed to induce protein conformational changes include 1) post-translational modification (PTM): Nature often uses PTMs to covalently modify amino acid residues to regulate protein conformation in both positive and allosteric ways, such as ubiquitination, acetylation, phosphorylation, and glycosylation, which have been found to be allosteric regulators. PTMs appear at many protein-protein interaction interfaces to promote or inhibit the binding of the two. Fine-tuning protein functions through a combination of PTMs can enable a single protein to play different cellular roles. 2) Allosteric regulation: Allosteric molecules specifically bind to other sites other than the active site of the protein substrate, causing conformational changes in the protein molecule and thus changing the activity of the protein. Since allosteric sites between protein subfamily members or homologs 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 rely on orthosteric sites also provide solutions to the problems of "undruggable target proteins" and drug resistance. However, the development of allosteric molecules still faces great challenges, such as the difficulty in discovering effective allosteric sites, the fact that allosteric sites are more prone to drug resistance mutations than orthosteric sites, the lower binding affinity of allosteric modulators than orthosteric modulators, and the difficulty in obtaining co-crystal complexes between allosteric molecules and proteins. These difficulties limit the molecules that can be selected for regulating protein conformation with allosteric molecules 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 proteomics analysis tool that uses chemical cross-linking agents to achieve covalent cross-linking of proteins that are close enough in space, and uses mass spectrometry technology and bioinformatics processing to analyze the composition of protein complexes and interaction interfaces. In recent years, this technology has also been combined with theoretical calculations, cryo-electron microscopy and other methods to analyze the structure of dynamic proteins. Chemical cross-linking has flexible controllability and can comprehensively depict the conformation of proteins by using different reactive functional groups, adjusting the length of the connecting arm, hydrophilicity, and flexibility. However, due to the lack of targeting of chemical cross-linking reactions and poor controllability of the reactions, it is impossible to perform in situ cross-linking on specific proteins, and it is difficult to achieve precise reactions to amino acids in specific regions of the protein.

[0004] This patent integrates the reported targeted drugs or ligands of proteins with the in situ cross-linking concept to develop a new method for protein conformation regulation, which can achieve precise and controllable conformation and activity regulation of target proteins, analyze the protein conformation-function relationship and its related biological mechanisms, and develop new disease treatment strategies. Summary of the invention

[0005] The purpose of the present invention is to provide a targeted regulatory molecule and a regulatory method for the conformation of a target protein in a living cell, wherein the protein conformation targeted regulatory molecule is incubated with the cell and then quickly permeates the membrane, and under the action of the targeting group of the molecule, the molecule binds to the target protein, and the two-terminal active functional groups on the molecule react covalently 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 invention has the characteristics of in situ targeting and controllable reaction, and realizes the targeted regulation of the conformation and activity of the target protein in the in situ environment of the cell.

[0006] A targeted regulatory molecule for target protein conformation in living cells.

[0007] The molecule includes: a) a targeting group that selectively binds to the target protein as a skeleton; b) two active functional groups are modified on the skeleton, and the double-end functional groups have short-range reaction activity. Only when the regulatory molecule binds to the target protein, the distance between the functional group and the adjacent specific amino acid reaction site is less than about 5E-10 meters, can a covalent reaction occur or the functional group can be covalently bound to the adjacent amino acid after being excited by light; 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 different covalent reaction sites, the protein conformation targeted regulatory molecule uses different active groups or introduces different rigid connecting arms between the targeting group and the active group to achieve both targeted and site-specific protein conformation regulation, better distinguish proteins with similar structures, and reduce off-target effects.

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

[0009] The reactive functional groups include functional groups that can covalently react when the distance from the amino acid is less than about 5E-10 meters, or photoreactive functional groups that obtain covalent reaction activity after photoactivation; the reactive functional groups can be: acrylamide, α-cyanoacrylamide, alkynyl, aldehyde, 2-formylphenylboronic acid, nitrile group connected to aromatic heterocycle, fumarate, allenamide, propiolenitrile, alkenyl substituted heteroaromatic hydrocarbons, alkynyl substituted heteroaromatic hydrocarbons, propargylamide, arylsulfonyl, halogenated hydrocarbons, α-halomethylamide / ester / ketone, epoxide, aziridine, nitroalkane, nitroheteroaromatic hydrocarbons, α-cyanenone, heteroaryl nitrile, cyanamide, isothiocyanate, vinylsulfonamide, vinyl sulfone, sulfonyl fluoride, sulfonimidoyl fluoride, aryl fluorosulfate, N-acyl-N-alkylsulfonamide, N-methylisoxazolium, oxaziridine, and one or two of the foregoing derivatives;

[0010] The photoreactive functional group is selected from one or two of a benzophenone group, a phenylazide group, a diaziridine group or a 2-aryl-5-carboxytetrazole group, and the light excitation wavelength of the photoreactive functional group is 200-800 nm.

[0011] Depending on the conformation of the target protein to be regulated and the different covalent reaction amino acid sites, different active functional groups are used or a rigid connecting arm is introduced between the targeting group and the active functional group. The composition of the rigid connecting arm includes double bonds, triple bonds, glycosidic bonds or cyclic groups, as well as one or more of the derivatives of the aforementioned groups.

[0012] A method for targeted regulation of the conformation of a target protein in a living cell using the above-mentioned targeted regulation molecule,

[0013] Preparation of the mother solution of protein conformation targeted regulatory molecules: use one or more organic solvents selected from water, buffer or acetonitrile, organic alcohols, organic acids, DMF or DMSO to prepare a mother solution with a concentration of 100nM-1M.

[0014] The mother solution of protein conformation targeted regulatory molecules is diluted with one of the cell culture mediums DMEM, MEM or R1640, etc., added to the cells, and incubated at 37°C, 5% CO2 for 0.5-120h; if the double-end active group contains a photoreactive group, after the regulatory molecules and cells are incubated under the above conditions, light is irradiated to induce a covalent reaction with the target protein. The light initiation conditions include: light excitation wavelength of 200-800nm, light treatment time of 10s-60min, prediction and analysis of the binding selectivity of the targeted regulatory molecules and the target protein and the appropriate reaction site.

[0015] The predictive analysis uses one or more of the following methods: X-ray crystal diffraction, cryo-electron microscopy, molecular docking, molecular dynamics simulation, drug virtual screening, conformational search and optimization, to predict and analyze the binding selectivity of the targeted regulatory molecules to the target protein and the appropriate reaction sites.

[0016] The present invention verifies the regulatory effect of protein conformation targeting regulatory molecules on the conformation and functional activity of target proteins by using live cell in situ cross-linking mass spectrometry, nuclear magnetic resonance technology, X-ray crystal diffraction, cryo-electron microscopy and a series of functional experiments (such as CCK-8 experiment, clone formation test, immunofluorescence, immunoprecipitation, post-translational modification site identification, etc.). The experimental operation in the present invention is usually carried out according to conventional experimental conditions, and can also be operated according to the experimental conditions in the embodiments, and the reagents can be used according to the instructions attached by the manufacturer.

[0017] The present invention has the following advantages:

[0018] (1) The present invention integrates the reported targeted drugs or ligands with the in situ cross-linking concept to develop a targeted method for regulating the conformation of target proteins in cells. The method has high specificity and reaction site selectivity, and realizes active regulation of the activity and function of target proteins in cells.

[0019] (2) The present invention modularizes protein conformation regulation molecules, which can be flexibly modified. According to the conformation of the target protein and the relationship between function and structure, different targeting groups and active groups are selected, and combined with calculation methods, the degree of regulation can be controlled.

[0020] (3) The protein conformation targeting regulatory molecule developed by the present invention has a good ability to distinguish between the target protein and proteins with similar conformations or sequences, because it simultaneously introduces a targeting group that specifically binds to the target protein and an active group and a rigid connecting arm developed for the reaction site of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 .The chemical structure of the targeted conformational regulatory molecule KP1 of the PIN1 protein prepared in Example 1.

[0022] Figure 2 .Schematic diagram of the synthesis route of KP1 in Example 1.

[0023] Figure 3 .The crystal structure of the KP1 and PIN1 complex.

[0024] Figure 4 .Line graph showing the inhibitory effect of KP1, a conformational targeted regulatory molecule of PIN1, on the proliferation of HeLa cells.

[0025] Figure 5 .Immunoblotting image of PIN1 degradation induced by KP1, a conformational targeted regulatory molecule of PIN1, in MCF-7 cells. DETAILED DESCRIPTION

[0026] Example 1

[0027] Synthetic PIN1 protein targeting conformational regulatory molecule KP1

[0028] (1) A targeted conformational regulatory molecule KP1 of PIN1 protein was developed, and its structural formula is as follows Figure 1 As shown. The PIN1 targeted inhibitor ZL-PIN13 is used as the targeting group. It already has an active group chloroacetamide that can covalently bind to Cys113. A vinylsulfonyl imide fluoride group is modified on the biphenyl group of the targeting group so that it can react with Lys132, thereby regulating the conformation of the catalytic enzyme activity pocket of the PPIase domain of PIN1.

[0029] (2) Synthesis route: Figure 2 shown.

[0030] Step a: Add mercaptoacetic acid (1.2 mmol) to a toluene solution (15 mL) of 2-furan-amine (1) (1 mmol) and 1-Boc-4-piperidone (1.1 mmol) and mix at room temperature. The reaction mixture was stirred at room temperature for 5 minutes and refluxed for 12 hours. The reaction mixture was cooled to room temperature, diluted with water (10 mL), and extracted with ethyl acetate (15 mL×2). The combined organic phases were washed with brine, dried over anhydrous Na2SO4 and evaporated. The residue was purified by flash chromatography to obtain compound 2. 1 H NMR (500MHz, CDCl3) δ7.55(s,3H),6.38(s,2H),6.28(s,3H),4.06(s,6H),1.53(s,6H).

[0031] Step b: At 0°C, compound 2 (1 mmol) was dissolved in anhydrous DMF (10 mL) and N-bromosuccinimide (NBS, 1.2 mmol) was added. After stirring at room temperature overnight, the reaction mixture was quenched with water (15 mL) and extracted with ethyl acetate (EtOAc, 20 mL×3). The combined organic phase was washed with brine, dried over anhydrous Na2SO4 and evaporated. The residue was purified by flash chromatography to obtain compound 3. 1 H NMR(500MHz, CDCl3)δ7.55(s,3H),6.38(s,2H),6.28(s,3H),5.30(s,3H),4.80(s,3 H),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 isopropanol (IPA) / H2O with a volume ratio of 3:1 (24 mL), and Pd(PPh3)2Cl2 (0.05 mmol) and Na2CO3 (5 mmol) were added. 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×3). The combined organic phases were washed with brine, dried over anhydrous Na2SO4 and evaporated. The residue was purified by flash chromatography to obtain compound 4. 1 H NMR(500MHz, CDCl3)δ6.48(s,2H),6.15(s,2H),5.34(s,2H),4.84(s,2H),3 .61(d,J=18.1Hz,7H),3.49(s,3H),2.43(s,1H),2.15(s,1H),1.42(s,18H).

[0033] Step d: Ethylenesulfonyl fluoride (0.108mmol), Pd(OAc)2(10mol), Cu(OAc)2(0.072mmol), LiOAc(0.043mmol) and 2.0mL anhydrous tetrahydrofuran were loaded into a sealed tube under nitrogen protection. Subsequently, compound 4 (0.036mmol) was dissolved in anhydrous tetrahydrofuran (2.0mL) and added dropwise to the reaction mixture in the sealed tube using a syringe over about 30 minutes. The reaction mixture was stirred at room temperature for 4 hours. Upon completion, 10mL of methylamine solution was immediately added, stirred for 10 minutes, and then filtered through a diatomaceous earth plug. The filtrate was concentrated in vacuo and purified by neutral alumina column chromatography using 30% ethyl acetate in hexane as eluent to obtain compound 5. 1H NMR(500MHz, CDCl3)δ8.40(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: Compound 5 (1 mmol) was added to a solution of HCl / EtOAc (2 mol / L, 20 mL) at room temperature. After stirring overnight, the reaction was concentrated in vacuo and basified to pH 8-9 with a 1 M NaOH aqueous solution. The mixture was extracted with EtOAc (25 mL×3), washed with brine, dried over anhydrous Na2SO4, filtered and evaporated to give a crude product. Under nitrogen protection, the crude product was dissolved in anhydrous dichloromethane (25 mL) solution, and potassium carbonate (1.1 mmol) and chloroacetyl chloride (1.1 mmol) were added. 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(500MHz, CDCl3)δ8.39(s,2H),8.04–7.73(m,10H),7.70(s,2H),7.16(s,1H),6.97(s,3H),6.12(s,2H) ,5.49(s,2H),4.88(s,2H),4.21(s,4H),3.66(s,4H),3.59(s,2H),3.49(s,3H),2.42(s,1H),2.16(s,1H).

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

[0036]

[0037] Example 2

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

[0039] (1) The developed PIN1 protein conformation-targeted regulatory molecule KP1 is used to regulate the conformation of the catalytic enzyme activity pocket of the PPIase domain of PIN1, hinder the recognition and binding of the PPIase domain to the substrate, and inhibit the biological activity of PIN1 protein in promoting cancer cell proliferation.

[0040] (2) Regulating the binding of molecules to target proteins: Hela cells were seeded in a 96-well plate at a density of 5,000 cells / well. After the cells adhered to the plate and grew well, KP1 was added to 100 μL of cell culture medium at final concentrations of 0, 10 nM, 50 nM, 100 nM, 200 nM, 500 nM, 750 nM, and 1 μM, respectively. The mixture was mixed and incubated at 37°C in a cell culture incubator containing 5% CO2 and 95% air by volume for 24 h.

[0041] (3) CCK8 assay for cell proliferation activity: 10 μL of CCK8 (Biyuntian, China) solution was added to the cells and incubated for 1 h in a cell culture incubator at 37° C. with 5% CO 2 and 95% air.

[0042] (4) The absorbance at 450 nm was measured using an ELISA reader, with 3 replicates at each concentration point. Figure 4 shown.

[0043] (5) Figure 4 The results show that KP1, a conformation-targeted regulatory molecule of PIN1, can effectively inhibit the proliferation of HeLa cells, with an IC50 of 450nM.

[0044] Example 3

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

[0046] (1) Regulatory molecule KP1 binds to PIN1: KP1 (prepared with dimethyl sulfoxide as the stock solution) was added to MCF-7 cells in good growth condition at final concentrations of 0, 10 nM, 100 nM, and 1 μM, respectively, and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 h.

[0047] (2) Protein extraction: discard the cell culture medium, wash twice with 4°C pre-cooled PBS, add 1 ml of RIPA (strong) lysis buffer containing 1% protease inhibitor, let stand on ice for 30 min, and collect broken cells and proteins.

[0048] (3) Use the BCA protein concentration assay kit to measure the protein concentration and adjust the protein concentration to 2 mg / mL using PBS.

[0049] (4) Take a protein solution containing 50 μg of whole protein at 2 mg / mL in each experimental group, add 6x Loading Buffer, and heat at 95 degrees for 5 minutes. The heated protein is subjected to SDS-PAGE gel electrophoresis.

[0050] (5) The bands on the gel were transferred to a PVDF membrane using the wet transfer method. After transfer, the membrane was blocked with 5% skim milk powder (dissolved in TBST) at room temperature for 1 h. The membrane was then immersed in a TBST (Tris buffered saline solution, containing 0.1% Tween20) solution containing 2 μg / mL PIN1 antibody at 4°C overnight. The membrane was washed three times with TBST, and a TBST solution containing 2 μg / mL goat anti-rabbit secondary antibody was added and incubated at room temperature for 30 min. The membrane was washed three times with TBST.

[0051] (6) Add luminescent liquid to the membrane, and allow it to react for 2 minutes in the dark before exposure. Figure 5 shown.

[0052] (7) Figure 5 The results of using KP1, a conformation-targeted regulatory molecule of PIN1, to induce PIN1 degradation in MCF-7 cells. As shown in the results, using the internal reference protein β-Actin as the indicator protein loading amount, after applying KP1, PIN1 protein in MCF-7 was degraded, and the degradation of PIN1 was concentration-dependent.

Claims

1. A targeted regulatory molecule for the conformation of a target protein in a cell, characterized in that: The target protein conformation targeted regulatory molecule includes: a) a targeting group that selectively binds to the target protein as a skeleton, b) two active functional groups are modified on the skeleton, and c) a rigid connecting arm is introduced between the targeting group and the active functional group.

2. The target protein conformation targeting regulatory molecule according to claim 1, characterized in that: The targeting group includes one or more of polypeptides, small molecules, nucleic acids, antibodies, nanoparticles, etc.; the binding affinity between the targeting group and the target protein is 1E-6 to 1E-13 moles.

3. The target protein conformation targeting regulatory molecule according to claim 1, characterized in that: Reactive functional groups include functional groups that can undergo covalent reactions when the distance from an amino acid is less than about 5E-10 meters, or photoreactive functional groups that acquire covalent reaction activity after photoactivation; The active functional group can only undergo covalent reaction with the amino acids near the binding pocket of the target protein after the targeting group binds to the target protein or is activated by light. The size of the targeted regulatory molecule is 1E-10 to 1E-8 meters.

4. The target protein conformation targeting regulatory molecule according to claim 1 or 3, characterized in that: The active functional group can be selected from acrylamide, α-cyanoacrylamide, alkynyl, aldehyde, 2-formylphenylboronic acid, nitrile group connected to aromatic heterocycle, fumarate, allenamide, propiolenitrile, alkenyl substituted heteroaromatic hydrocarbon, alkynyl substituted heteroaromatic hydrocarbon, propargylamide, arylsulfonyl, halogenated hydrocarbon, α-halomethylamide / ester / ketone, epoxide, aziridine, nitroalkane, nitroheteroaromatic hydrocarbon, α-cyanenone, heteroaryl nitrile, cyanamide, isothiocyanate, vinylsulfonamide, vinyl sulfone, sulfonyl fluoride, sulfonimidoyl fluoride, aryl fluorosulfate, N-acyl-N-alkylsulfonamide, N-methylisoxazolium, oxaziridine, and one or two of the aforementioned derivatives; The photoreactive functional group is selected from one or two of a benzophenone group, a phenylazide group, a diaziridine group or a 2-aryl-5-carboxytetrazole group, and the light excitation wavelength of the photoreactive functional group is 200-800 nm.

5. The target protein conformation targeting regulatory molecule according to claim 3, characterized in that: The amino acids near the binding pocket of the target protein include: one or more of glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.

6. The target protein conformation targeting regulatory molecule according to claim 1, characterized in that: The rigid connecting arm includes a double bond, a triple bond, a glycosidic bond, a cyclic group, etc., and one or more of the derivatives of the aforementioned groups.

7. A method for targeted regulation of the conformation of a target protein in a living cell using the targeted regulation molecule according to any one of claims 1 to 6, characterized in that: The targeted regulatory molecule is added to the cell sample culture medium and incubated with the cell sample at 37°C, 5% (v / v) CO2 for 0.5-120 hours; if the active functional group contains a photoreactive group, illumination is implemented after incubation to predict and analyze the selectivity and reaction site of the targeted regulatory molecule binding to the target protein.

8. The method according to claim 7, characterized in that The final concentration of the targeted regulatory molecule in the cell culture medium is 1nM-100mM, and the cell culture medium is one of the commonly used culture mediums such as RPMI-1640, DMEM, MEM, F12 culture medium, etc.; the illumination wavelength is 200-800nm, and the illumination time is 10s-60min.

9. The method according to claim 7, characterized in that: The predictive analysis uses one or more of the following methods: X-ray crystallography, cryo-electron microscopy, molecular docking, molecular dynamics simulation, virtual drug screening, conformational search and optimization.

10. The method according to claim 7, characterized in that The cell samples include: one or more of tumor cells (sensitive strains, drug-resistant strains), immune cells, stem cells, healthy cells, bacteria, fungi, and microorganisms.

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