Method for enhancing biomolecular condensate targeting function of drug by means of charge modification

Abstract

Disclosed in the present invention is a method for enhancing a biomolecular condensate targeting function of a drug by means of charge modification. The method comprises the following steps: (1) determining the electrostatic potential range of a condensate that is formed by or enriched by, in vitro or intracellularly, a target biomolecule associated with a disease; and (2) carrying out a charge modification on a drug with a known target, the drug with the known target being a drug having a prevention and / or treatment effect on the disease described in step (1), the modified drug with the known target and the condensate carrying opposite net charges, so that the targeting specificity of the drug with the known target after the charge modification can be enhanced. The present invention performs charge modification on a drug with a known target on the basis of the electric potential of a target biomolecular condensate, and adds a functional group or amino acid carrying net charges that are opposite to that of the target biomolecular condensate, so as to enhance the targeting specificity of the drug with the known target, thereby improving the treatment effect of the drug.

Description

A method for enhancing drug targeting of biomolecular aggregates through charge modification Technical Field

[0001] The present invention belongs to the technical field of drug screening, and in particular relates to a method for enhancing drug-targeted biomolecular aggregates through charge modification. Background Art

[0002] The liquid phase separation phenomenon of biomacromolecules such as proteins plays an important role in the molecular distribution, functional regulation, cell signaling, and disease development of tissue cells. Neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, as well as certain types of cancer such as lymphoma and multiple myeloma, are often associated with abnormal protein aggregation and the formation of insoluble amyloid deposits. Studies have shown that disorders in the phase separation process of proteins may promote their liquid-to-solid transition, thereby accelerating protein deposition. Therefore, the development of drugs targeting biomolecular aggregates formed by phase separation will hopefully intervene in the aggregation process of target proteins and delay the development of diseases, which is a new therapeutic strategy with great potential.

[0003] In the past, people simply believed that small molecule drugs would be evenly distributed after entering cells. However, in 2020, Richard A. Young's team linked the differences in the efficacy of anti-tumor drugs to the phase separation mechanism of biomacromolecules, and proposed that the selective distribution and concentration of small molecule drugs by biomolecular aggregates help the drugs to take effect. At the same time, cells can also develop drug resistance by changing the properties of aggregates. In another study report by Christian Betzel's team, the small molecule suramin effectively dissolves Tau protein:RNA aggregates into Tau monomers through electrostatic interactions, and can interfere with the formation and stability of Tau aggregates in cells. The inspiration given by these two studies is that we may be able to enhance the targeting of small molecule drugs to target aggregates through chemical modification, thereby enhancing the effect of drugs.

[0004] Enhancing the targeting of small molecule drugs to specific biomolecular aggregates will help enhance their effectiveness. To date, research in this field has been based on drug screening, searching compound libraries for molecules that improve targeting to target aggregates or alter their condensed phase properties. However, the crucial role of electrostatic interactions in the targeting properties of biomolecular aggregates has remained largely unexplored.

[0005] Summary of the Invention

[0006] In order to address the deficiencies in the prior art, the present invention aims to provide a method for enhancing drug targeting of biomolecular aggregates by charge modification.

[0007] The specific technical solutions are as follows:

[0008] The present invention provides a method for enhancing drug targeting of biomolecular aggregates by charge modification, comprising the following steps:

[0009] (1) Determine the electrostatic potential range of aggregates formed or enriched by target biomolecules related to diseases in vitro or in cells;

[0010] (2) performing charge modification on a known target drug, wherein the known target drug is a drug having a preventive and / or therapeutic effect on the disease described in step (1), and the charge-modified known target drug has an opposite net charge to the agglomerate, and the charge-modified known target drug can enhance the targeting of the known target drug.

[0011] Furthermore, the known target drug is a small molecule compound or a peptide, and the charge of the known target drug is modified through functional groups or amino acid residues;

[0012] The functional groups include positively charged and negatively charged functional groups;

[0013] The amino acid residues include positively charged and negatively charged amino acid residues.

[0014] Furthermore, small molecule compounds are charge-modified through functional groups; and polypeptides are charge-modified through amino acid residues.

[0015] Furthermore, the positively charged functional group is selected from one or more of alkylamine, quaternary ammonium salt, pyrrolidine, piperidine, guanidine salt and aryl diazonium salt;

[0016] The negatively charged functional group is selected from one or more of carboxylates, phosphates and sulfonates;

[0017] The positively charged amino acid residue is selected from arginine and / or lysine;

[0018] The negatively charged amino acid residue is selected from aspartic acid and / or glutamic acid.

[0019] Furthermore, the target biological molecule is a target protein.

[0020] Furthermore, the method for determining the electrostatic potential range of the aggregates formed or enriched by the target protein in the cell includes the steps of: 1) constructing a first expression vector expressing a fusion protein of the target protein and the fluorescent protein mCherry; 2) constructing second expression vectors respectively expressing fluorescent proteins with different net charges; 3) co-transfecting the first expression vector and each second expression vector into the cell; 3) calculating the distribution coefficient of fluorescent proteins with different net charges based on the fluorescence intensity in the intracellular phase separation system and the fluorescence intensity distribution outside the aggregate, the distribution coefficient being the ratio of the fluorescence intensity inside and outside the aggregate, and determining whether the aggregate has a positive or negative electrostatic potential based on the distribution coefficient.

[0021] Preferably, when the fluorescence intensity of the target protein expressed by the second expression vector in the aggregates formed or enriched in the cell is lower than that outside the aggregates, the aggregates formed or enriched and the fluorescent protein expressed by the second expression vector have the same charge, and the stronger the fluorescence intensity expressed by the second expression vector outside the aggregates, the more positive or negative the electrostatic potential of the aggregates.

[0022] Furthermore, the method also includes screening charge-modified known target drugs, and the charge-modified known target drugs that have high targeting and the same function as the known target drugs are the target drugs.

[0023] Furthermore, the screening method is: a charge-modified known target drug with a fluorescent label or with fluorescence itself is applied to a biomolecular aggregate, the fluorescence intensity in the phase separation system and the fluorescence intensity distribution outside the aggregate are detected, and the distribution coefficient of the charge-modified known target drug is calculated. The distribution coefficient is the ratio of the fluorescence intensity inside and outside the aggregate. The distribution coefficient is proportional to the targeting. The higher the distribution coefficient value, the higher the targeting of the charge-modified known target drug.

[0024] The present invention also provides a charge-modified known target drug obtained by the method.

[0025] The beneficial effects of the present invention are:

[0026] This invention charges known target drugs based on the potential of target biomolecular aggregates. By adding functional groups or amino acids with a net charge opposite to that of the target biomolecular aggregates, the targeted nature of the known target drug is enhanced, thereby improving the drug's therapeutic efficacy. By directly chemically modifying known drugs, adding functional groups or amino acids with corresponding net charges opposite to those of the target biomolecular aggregates, and then screening for candidate drug molecules with the highest targeting, the invention significantly narrows the search area for optimizing drug targeting, thereby reducing the cost of drug research and development and increasing the success rate of new drug discovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1-a) Molecular structural formulas of molecules A, B, and C;

[0028] Figure 1-b) Fluorescence confocal imaging of an α-synuclein phase separation system in vitro. The white trace in the figure represents the fluorescence intensity distribution outside the aggregates.

[0029] Figure 1-c) Partition coefficients of molecules A, B, and C in the in vitro phase separation system, with the average values ​​in brackets;

[0030] Figure 2-A Fluorescence image of SH-SY5Y cells after co-transfection of the αSyn-mCherry expression vector and the mEGFP(-8) expression vector in Example 2;

[0031] FIG2-B is a fluorescence image of SH-SY5Y cells after co-transfection of the αSyn-mCherry expression vector and the mNeonGreen(-1) expression vector in Example 2;

[0032] FIG3 shows the structural formulas of functional groups listed in the present invention. DETAILED DESCRIPTION

[0033] To more clearly understand the present invention, the present invention is further described with reference to the following examples and accompanying drawings. The examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. In the examples, all raw materials and reagents are commercially available. Experimental methods without specific conditions are conventional methods and conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0034] Example 1

[0035] Applicants have discovered that the aggregates formed by α-synuclein both in vitro and in cells have high electrostatic potentials, thereby regulating the spatial distribution of biomacromolecules and small chemical molecules. Example 1 uses an in vitro phase separation experiment of α-synuclein as an example to illustrate how charge modification can enhance the targeting of drugs to aggregates formed by target proteins. The specific steps are as follows:

[0036] (1) Lyophilized α-synuclein was obtained by overexpressing and purifying α-synuclein in Escherichia coli as described in general literature. The lyophilized protein sample was dissolved in a buffer solution (25 mM HEPES, 150 mM NaCl, pH 7.4) and 20% (w / v) PEG8000 was added. At room temperature and when the final protein concentration was higher than 400 μM, the protein phase separated and α-synuclein aggregates were formed in the in vitro phase separation system of α-synuclein. The electrostatic potential range of the aggregates formed by α-synuclein in vitro was determined by measuring the zeta potential. The α-synuclein aggregates formed in vitro had a negative electrostatic potential.

[0037] (2) Organic small molecules with inherent fluorescence were selected and modified by chemical modification to obtain molecules A(-2), B(-1), and C(+1) with different net charges under physiological conditions of pH 7.4. The structural formulas of molecules A, B, and C are shown in Figure 1-a).

[0038] (3) Adding molecules A(-2), B(-1), and C(+1) to the α-synuclein aggregates with negative electrostatic potential prepared in step (1), detecting the fluorescence intensity of molecules A, B, and C in the in vitro phase separation system of α-synuclein, as well as the fluorescence intensity distribution outside the aggregates, and calculating the distribution coefficients of molecules A, B, and C. The distribution coefficient is the ratio of the fluorescence intensity inside and outside the aggregates. The distribution coefficient is proportional to the molecular targeting, that is, the higher the value, the better the targeting.

[0039] The fluorescence intensities of molecules A, B, and C in the in vitro phase separation system of α-synuclein, as well as the fluorescence intensity distribution outside the aggregates, are shown in Figure 1-b). The calculated distribution coefficients of molecules A, B, and C in the in vitro phase separation system are shown in Figure 1-c).

[0040] As shown in Figure 1-c), the distribution coefficients of the three are significantly different and increase exponentially with the increase of positive charge. This demonstrates the feasibility of the method and principle of the present invention to enhance the targeting of drugs to target aggregates by reverse charge modification.

[0041] Example 2

[0042] In this example, the applicant expressed α-synuclein in a human neuroblastoma cell line (SH-SY5Y) and simultaneously expressed fluorescent proteins with different net charges to verify that the aggregates formed by α-synuclein within the cells all had a high negative electrostatic potential. The specific steps are as follows:

[0043] (1) SH-SY5Y cell culture and differentiation. SH-SY5Y cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin at 37°C in a 5% carbon dioxide incubator. After the cell confluence reached 80-90%, the culture medium was removed using a pipette and washed once with PBS. Trypsin-EDTA (0.05%) was added to the culture and incubated in a 37°C cell incubator for 2 minutes. Two volumes of culture medium were added to stop digestion, and the cells were gently blown to suspend the cells into a uniform cell suspension. The cell pellet was resuspended in DMEM medium (10% fetal bovine serum, 1% penicillin / streptomycin) and plated in a cell suspension added to a 4-well plate (ibidi GmbH). After 24 hours, the culture medium was switched to DMEM / F12 (5% FBS) containing 10 μM all-trans retinoic acid (EC23) to promote differentiation and induce neuronal phenotype. After 3 days, the culture medium was switched to Neurobasal medium containing 50 ng / mL brain-derived neurotrophic factor (BDNF). Thereafter, the culture medium was changed every 2-3 days.

[0044] (2) Transfection of SH-SY5Y cells. In this embodiment, the first expression vector is a pcDNA3.1+ expression vector with the fluorescent protein mCherry attached to the C-terminus of α-synuclein; the second expression vector is a pcDNA3.1+ expression vector with the fluorescent protein mEGFP-stop and the second expression vector is a pcDNA3.1+ expression vector with the fluorescent protein mNeonGreen-stop. Under physiological conditions of pH 7.4, the fluorescent proteins mEGFP and mNeonGreen carry different net charges, (-8) and (-1), respectively. The respective DNA constructs were cloned into the pcDNA3.1(+) vector containing the CMV promoter to obtain the corresponding plasmids. Lipo6000 was used. TM The solution was added to the DNA, mixed thoroughly, and then added to cultured SH-SY5Y cells. Data were collected after 48 hours of culture.

[0045] (3) In SH-SY5Y cells, αSyn-mCherry was co-transfected with fluorescent proteins mEGFP(-8) and mNeonGreen(-1) with different net charges, and the formation of a large number of αSyn droplets was observed. In this example, αSyn-mCherry droplets were co-localized with the fluorescent protein mNeonGreen. However, a large number of cavities were observed in the co-transfected mEGFP cells, and the fluorescence intensity of mEGFP inside the αSyn-mCherry droplets was significantly lower than that outside the droplets, indicating that the αSyn-mCherry droplets had a strong repulsive effect on mEGFP, that is, the presence of a negative electrostatic potential within the αSyn-mCherry condensate (Figure 2). Based on the fluorescence intensity of mEGFP(-8) and mNeonGreen(-1) with different negative charges in the intracellular phase separation system of α-synuclein, as well as the fluorescence intensity distribution outside the condensate, the distribution coefficient (r) of the fluorescent protein was calculated. The distribution coefficient is the ratio of the fluorescence intensity inside and outside the condensate. rmNeonGreen / r mEGFP The larger the ratio, the more negative the electrostatic potential of the condensate.

[0046] Therefore, based on the above observations, in the SH-SY5Y cell model, α-synuclein is enriched in cells to form aggregates with negative electrostatic potential.

[0047] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for enhancing the targeting of drugs to biomolecular condensates by charge modification, characterized in that, It includes the following steps: (1) Determine the electrostatic potential range of condensates formed or enriched by target biomolecules related to diseases in vitro or in cells; (2) Perform charge modification on known target drugs, where the known target drugs are drugs with preventive and / or therapeutic effects on the diseases described in step (1). The charge-modified known target drugs have opposite net charges to the condensates, and the charge-modified known target drugs can enhance the targeting of the known target drugs.

2. The method according to claim 1, characterized in that, The known target drugs are small molecule compounds or polypeptides, and the charge modification of the known target drugs is performed through functional groups or amino acid residues; The functional groups include positively charged and negatively charged functional groups; The amino acid residues include positively charged and negatively charged amino acid residues.

3. The method according to claim 2, characterized in that, The charge modification of small molecule compounds is performed through functional groups; the charge modification of polypeptides is performed through amino acid residues.

4. The method according to claim 2, characterized in that, The positively charged functional groups are selected from one or more of alkylamines, quaternary ammonium salts, pyrrolidines, piperidines, guanidine salts, and aryl diazonium salts; The negatively charged functional groups are selected from one or more of carboxylates, phosphates, and sulfonates; The positively charged amino acid residues are selected from arginine and / or lysine; The negatively charged amino acid residues are selected from aspartic acid and / or glutamic acid.

5. The method according to claim 1, characterized in that, The target biomolecule is a target protein.

6. The method according to claim 5, characterized in that, The method for determining the electrostatic potential range of condensates formed or enriched by the target protein in cells includes the steps: 1) Construct a first expression vector expressing a fusion protein of the target protein and the fluorescent protein mCherry; 2) Construct second expression vectors expressing fluorescent proteins with different net charges respectively; 3) Co-transfect the first expression vector into cells with each of the second expression vectors respectively; 3) Calculate the partition coefficients of the fluorescent proteins with different net charges according to the fluorescence intensity in the intracellular phase separation system and the fluorescence intensity distribution outside the condensates. The partition coefficient is the ratio of the fluorescence intensity inside and outside the condensates. Determine whether the condensates have positive or negative electrostatic potential according to the partition coefficient. When the fluorescence intensity of the fluorescent protein expressed by the second expression vector in the condensates formed or enriched by the target protein in cells is lower than that outside the condensates, the formed or enriched condensates have the same charge as the fluorescent protein expressed by the second expression vector.

7. The method according to claim 1, characterized in that, The method also includes screening the charge-modified known target drugs. The charge-modified known target drugs with high targeting and the same function as the known target drugs are the target drugs.

8. The method according to claim 7, characterized in that, The screening method is: Act on the biomolecule condensates with the charge-modified known target drugs with fluorescent labels or self-fluorescent properties, detect the fluorescence intensity in the phase separation system and the fluorescence intensity distribution outside the condensates, calculate the partition coefficient of the charge-modified known target drugs. The partition coefficient is the ratio of the fluorescence intensity inside and outside the condensates. The partition coefficient is directly proportional to the targeting. The higher the value of the partition coefficient, the higher the targeting of the charge-modified known target drugs.

9. A charge-modified known target drug obtained by the method according to any one of claims 6 - 8.

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