A method for selectively modifying protein lysine and its application

Abstract

The present invention provides a method for selectively modifying protein lysine, wherein the protein and phenyl isocyanide are incubated in a phosphate buffer to form modified lysine. The phenyl isocyanide of the present invention has electrophilic reaction characteristics, and can be used alone to specifically modify and add lysine residues in proteins, thereby selectively labeling lysine in protein molecules, especially lysine in protein active sites. In addition, the phenyl isocyanide of the present invention also has potential uses such as protein imaging in cells, regulating protein activity and cancer targeted medication.

Description

Technical Field

[0001] The present invention relates to the technical field of amino acid modification, and in particular to a method for selectively modifying protein lysine and application thereof. Background Art

[0002] Small molecules play a vital role in the study of protein function and in the development of drugs aimed at modulating protein activity. However, more than 85% of human proteins are currently classified as "druggable" because they lack identified catalytic active sites or binding pockets that cannot be functionally and selectively targeted by traditional small molecules. To address this challenge, covalent ligands have emerged as a promising strategy. These ligands contain a recognition unit and a reactive group that enables them to interact with shallow or dynamic binding pockets on proteins that are typically inaccessible to reversible small molecules. Therefore, there is an urgent need to develop innovative reactive electrophiles that can reach binding sites beyond traditional electrophiles.

[0003] The current toolkit of probes for selective binding to functional residues other than cysteine ​​remains rather limited, highlighting the great need for the development of amino electrophiles. Advances in this area are being made with the FDA-approved drug voxelotor (GBT440), which demonstrates the potential for lysine-targeted therapeutics. In addition, recent advances have led to the creation of a variety of electrophilic head groups. For example, isoheteroaryl sulfones, sulfates, 2-sulfone pyridines, thiotriazoles, 2-methylthiopyridinium oxazolinium ions, oxazole epoxides, and bicyclobutanecarboxamides, each designed to selectively target cysteine, lysine, tyrosine, and methionine. These innovative compounds have various advantages and have found applications in active proteomics (ABPP) for target discovery and the development of novel covalent inhibitors.

[0004] For the selective modification of lysine in proteins, the existing technology (NHS-Esters as Versatile Reactivity-Based Probes for Mapping Proteome-Wide Ligandable Hotspots) uses NHS-ester (N-hydroxysuccinimide ester) to modify amino acid residues such as lysine, serine, threonine and tyrosine in proteins. Since the reaction activity of NHS-ester is strong, the modification reaction specificity is not high. Therefore, a modification method with high specificity for lysine modification reaction is needed. Summary of the invention

[0005] In view of this, the present invention proposes a method with high specificity for lysine modification reaction and its application.

[0006] The technical solution of the present invention is achieved as follows: In the first aspect, the present invention provides a method for selectively modifying protein lysine, wherein the probe is incubated with a protein containing a lysine residue in a phosphate buffer, and click-reacted with TAMRA-N3, and then separated by SDS-PAGE to obtain the modified lysine; the probe is an isocyanide compound containing phenyl.

[0007] On the basis of the above technical solution, preferably, the probe is one of YJ-1, YJ-2, YJ-3, YJ-4, YJ-5 and YJ-6, and the structural formulas are as follows:

[0008]

[0009] On the basis of the above technical solutions, preferably, the probe and the aldehyde compound are used in combination to selectively modify protein lysine.

[0010] On the basis of the above technical solution, preferably, the concentration of the protein is 10 nM, the concentration of the probe is 1-50 μM, and the concentration of the aldehyde compound is 0.2-5 mM.

[0011] On the basis of the above technical solution, preferably, the aldehyde compound is one of formaldehyde, benzaldehyde and propionaldehyde.

[0012] On the basis of the above technical solution, preferably, the protein is one of bovine serum albumin, KAS G12D protein, β-Amylase protein, lactoferrin and catalase.

[0013] Based on the above technical solution, preferably, the incubation temperature is 20-25° C. and the incubation time is 1-2 h.

[0014] In a second aspect, the present invention also provides an application of the probe in protein imaging, characterized in that the probe is one of YJ-1, YJ-2, YJ-3, YJ-4, YJ-5 and YJ-6.

[0015] In a third aspect, the present invention also provides an application of a probe in regulating protein activity, characterized in that the probe is one of YJ-1, YJ-2, YJ-3, YJ-4, YJ-5 and YJ-6.

[0016] In a fourth aspect, the present invention also provides an application of a probe in the preparation of cancer targeted drugs, characterized in that the probe is one of YJ-1, YJ-2, YJ-3, YJ-4, YJ-5 and YJ-6.

[0017] The lysine modification method of the present invention has the following beneficial effects compared with the prior art:

[0018] The phenyl isocyanide of the present invention has electrophilic reaction characteristics and can be used alone or in combination with formaldehyde to specifically modify and add lysine residues in proteins, thereby selectively labeling lysine in protein molecules, especially lysine in protein active sites, and has potential uses in protein imaging in cells, regulating protein activity, and targeting cancer pharmaceuticals. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The figure shows the modification effect of YJ-1 and YJ-1 combined with formaldehyde on protein lysine residues;

[0021] Figure 2 The SDS-Page gel electrophoresis diagram of lysine modified by phenylisocyanide combined with formaldehyde;

[0022] Figure 3 This is the SDS-Page gel electrophoresis diagram of protein lysine modification by phenylisocyanide;

[0023] Figure 4 The lysine sites detected from BSA using formaldehyde in combination with the probe;

[0024] Figure 5 Cell imaging of probe YJ-1 / YJ-3 in HepG2 cells;

[0025] Figure 6 The distribution map of lysine residue binding sites identified by the probe from the protein;

[0026] Figure 7 This is a diagram showing the effect of the probe fitting into the protein binding pocket with K183 and K12;

[0027] Figure 8 This is a diagram showing the effect of probe labeling GLUD1 K183;

[0028] Fig. 9 This is a graph showing the anti-proliferation effect of the probe on HepG2, H3255 and PC9 cancer cells;

[0029] Fig.10 It is the mass spectrometry analysis diagram of YJ-1 / YJ-3 binding protein;

[0030] Fig.11Pull-down WB validation target result diagram for YJ-1 / YJ-3 and corresponding antibodies. DETAILED DESCRIPTION

[0031] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0032] The invention provides a method for selectively modifying protein lysine, comprising the following steps: incubating a probe with a protein containing a lysine residue in a phosphate buffer, performing a click reaction with TAMRA-N3, and then separating the modified lysine through SDS-PAGE; the probe is an isocyanide compound containing phenyl.

[0033] Specifically, a total of 6 phenyl isocyanides, namely YJ-1, YJ-2, YJ-3, YJ-4, YJ-5 and YJ-6, are selected as probes in the embodiment, and the structural formulas of YJ-1 / 2 / 3 / 4 / 5 / 6 are shown in the following table:

[0034] Table 1 Structural formula of YJ-1 / 2 / 3 / 4 / 5 / 6 probe

[0035]

[0036] The preparation method of the above probe is: firstly, amine is treated with formic acid to generate formamide, and then dehydrated by oxidative phosphorous acid treatment. The stability of YJ-1 / 2 / 3 / 4 / 5 / 6 probe in aqueous solution was analyzed and evaluated by 1H NMR, and it was found that the spectrum remained unchanged after 7 days in DMSO-d6 / D2O (2 / 1, v / v) solution, indicating that YJ-1 / 2 / 3 / 4 / 5 / 6 probe has good stability in aqueous solution and is also an electrophilic reagent for modifying amino acid residues in proteins.

[0037] Experiment 1: Effect of probe modification (labeling) on ​​lysine-containing proteins

[0038] 1.1 Modification effect of different concentrations of probe on bovine serum albumin lysine

[0039] The protein selected was bovine serum albumin (BSA), with a concentration of 10 nM, and the concentrations of YJ-1 were set to 1 μM, 5 μM, 10 μM, 20 μM and 50 μM, with a control concentration of 0. BSA and YJ-1 were incubated in phosphate buffer at 25°C for 2 h, then clicked with TAMRA-N3, separated by SDS-PAGE, and imaged by in-gel fluorescence scanning. The results are shown in Figure 2-A.

[0040] Figure 2 As shown in A, YJ-1 can successfully label BSA in a concentration-dependent manner (upper gel). YJ-1 at a concentration of 10 μM was used for time-dependent labeling (see Figure 2 -D), experiments showed that only 15 minutes were sufficient to produce a distinct marker band (lower gel).

[0041] 1.2 Probe modification effects on pure KAS G12D protein and pure β-Amylase protein

[0042] The modification effect of YJ-1 / 2 / 3 / 4 / 5 probes on pure KAS G12D protein and pure β-Amylase protein was detected. The concentration of pure KAS G12D protein and pure β-Amylase protein was 10 nM, and the concentration of YJ-1, YJ-2, YJ-3, YJ-4 and YJ-5 was 20 μM. The incubation was carried out at 20° C. The rest of the methods were the same as in Example 1. The results are shown in FIG. Figure 2 -B and 2-C.

[0043] Figure 2 As shown in Figure 2B and Figure 2C, YJ-1, YJ-2, YJ-3, YJ-4 and YJ-5 probes can modify pure KAS G12D protein and pure β-Amylase protein.

[0044] 1.3 Probe modification effects on lactoferrin and catalase

[0045] We then tested the effects of YJ-1 / 2 / 3 / 4 / 5 on lactoferrin ( Figure 2 -E) and catalase ( Figure 2 Although YJ-1 / 2 / 3 / 4 / 5 showed different labeling abilities for each protein, they all demonstrated robustness for protein labeling.

[0046] Experiment 2: Probe combined with aldehyde compounds to modify lysine

[0047] 2.1 Use of probes in combination with different concentrations of formaldehyde

[0048] The probe was YJ-1 at a concentration of 20 μM; the modified protein was bovine serum albumin at a concentration of 10 nM, the formaldehyde concentrations were 0.2 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, and the rest was the same as in Example 1. The results are shown in Figure 3 -A.

[0049] 2.2 Modification effects of different incubation times

[0050] The probe was YJ-1 at a concentration of 20 μM; the modified protein was bovine serum albumin at a concentration of 10 nM, the formaldehyde concentration was 5 mM, the incubation time was 1 h, 2 h, 3 h, 5 h, 8 h, and the rest was the same as in Example 1. The results are shown in Figure 3 -B.

[0051] 2.3 Effect of combining probe with different aldehydes

[0052] The probe was YJ-1 at a concentration of 20 μM; the modified protein was bovine serum albumin at a concentration of 10 nM; the aldehyde reagents were C7H6O (benzaldehyde), HCHO (formaldehyde) and C3H6O (propionaldehyde) at a concentration of 5 mM; the incubation time was 1 h; the rest of the contents were the same as in Example 1. The results are shown in Figure 3 C.

[0053] 2.4 Comparison of the modification effects of probes with and without formaldehyde

[0054] The probes selected were YJ-1, YJ-2, YJ-3, YJ-4 and YJ-5, with a concentration of 20 μM; the modified protein selected was bovine serum albumin, with a concentration of 10 nM; the aldehyde reagent selected was HCHO (formaldehyde), with a concentration of 5 mM; the incubation time was selected as 1 h; the rest of the contents were the same as in Example 1, and the results are shown in Figure 3 D.

[0055] Figure 3 As shown in D, benzaldehyde, formaldehyde and propionaldehyde can be used in combination, among which formaldehyde is the most effective reagent for protein labeling in multi-component reactions. Formaldehyde has the best effect; when formaldehyde is used in combination, a concentration of 1mM can produce obvious protein modification effects. The intensity of the labeled protein using formaldehyde and phenylisocyanide is better than that of isocyanide alone, and the fluorescence intensity is significantly increased.

[0056] In the presence of formaldehyde, 31 lysine sites were detected from BSA, while only 8 lysine sites were detected in the absence of formaldehyde. Similarly, 398 lysine sites were detected from HepG2 cell lysate in the presence of formaldehyde, while only 165 lysine sites were detected in the absence of formaldehyde ( Figure 4 ), indicating that formaldehyde can enhance the modification effect of the probe.

[0057] Application of the Triple Probe in Protein Imaging

[0058] To track the subcellular localization of the probes in living cells, cell imaging experiments were performed. YJ-1 and YJ-3 probes (at a concentration of 20 μM) were incubated with HepG2 cells for 2 h, fixed and permeabilized, and then click-reacted with TAMRA-N3. The cells were imaged under previously optimized conditions. The imaging results are shown in Figure 5 .

[0059] As shown in the figure, the binding proteins of YJ-1 and YJ-3 are distributed throughout the cell, including the nucleus. It is worth noting that under the same conditions, YJ-1 shows higher fluorescence intensity than YJ-3 probe, which is consistent with the trend observed in the pure protein labeling analysis. The combination of these labeling and imaging experiments shows that the probe of the present invention is a reactive warhead suitable for protein labeling in living cells.

[0060] Application of four-probe assay in protein activity regulation

[0061] To characterize the binding sites of the probes in cell lysates or living cells, binding site analysis was performed. We first evaluated the amino acid selectivity in cell lysates using the pChem search engine, and the results showed that probe YJ-1 mainly labeled lysine residues, which once again demonstrated that the YJ series probes (YJ-1 / 2 / 3 / 4 / 5 / 6) have excellent amino acid selectivity and specificity.

[0062] Binding site analysis was performed in living cells. After incubation of HepG2 cancer cells with YJ-1 probe (200 μM) for 5 h, the cells were lysed and click-reacted with UV-cleavable biotin azide (PC-Biotin). The peptides labeled with the probe were affinity purified and tested by LC-MS / MS. A total of 221 binding sites were identified from 130 proteins, 36% of which were not found in drugbank (Table 2 and Figure 6 ).

[0063] Table 2 Lysine residue subsets identified by YJ-1 in living HepG2 cells

[0064]

[0065]

[0066] As shown in Table 2, YJ-1 identified a series of functional sites of key proteins, including K12 of nucleoside diphosphate kinase A (NME1) and K183 of glutamate dehydrogenase 1 (GLUD1). NME1 (K12) plays an important role in nucleoside triphosphate synthesis and is involved in cell proliferation, differentiation and development. The active site of GLUD1 (K183) has been reported to play a key role in glutamate splicing through the production of α-ketoglutarate. Targeting these sites may regulate the function of proteins. Other binding sites include K110 of malate dehydrogenase (MDH1) and K270 of aldo-keto reductase family 1 member C3 (AKR1C3), which were found to be located in the binding or active pocket. MDH1 plays an important role in the malate-aspartate cycle and the tricarboxylic acid cycle and is essential for the mitochondrial NADH supply for oxidative phosphorylation.

[0067] Docking experiments showed that YJ-1 could fit well into the protein binding pocket with K183 of GLUD1 and K12 of NME1 ( Figure 7 ). Inhibitory activity experiments showed that YJ-1 had a moderate inhibitory effect on recombinant GLUD1 protein ( Figure 8 A), and YJ-1 can successfully label the recombinant GLUD1 protein in a concentration-dependent manner ( Figure 8 B). Overexpression of GLUD1 protein in HepG2 cells resulted in a stronger antiproliferative effect of the probe ( Figure 8 C), indicating that GLUD1 is an important target for the probe to exert its anti-proliferative effect. These findings suggest that probe YJ-1 has the ability to modulate the function of these important proteins.

[0068] Application of five experimental probes in the preparation of cancer targeted drugs

[0069] To evaluate the effect of the probes (YJ-1 / 2 / 3 / 4 / 5 / 6) on cancer cells, the antiproliferative effects of YJ-1 / 2 / 3 / 4 / 5 / 6 were first studied on various cancer cell lines including HepG2, H3255 and PC9. The results showed that YJ-1 / 2 / 3 / 4 / 5 / 6 had certain antiproliferative effects on HepG2, H3255 and PC9 cancer cells ( Fig. 9 -A / B / C); among them, YJ-1 and YJ-2 showed the strongest antiproliferative activity against HepG2 cell line, with IC50 values ​​of 0.83μM and 0.47μM, respectively, which indicates that the proteins interacting with these probes are essential for the survival of cancer cells and can be used as targeted drugs for cancer genes HepG2, H3255 and PC9.

[0070] HepG2 cells were incubated for 5 hours with probes (YJ-1 / YJ-3) or DMSO and then lysed. The resulting proteome was treated with biotin-N3 click chemistry. The samples were enriched with avidin beads and digested with trypsin, and then subjected to LC-MS / MS testing. Proteins were screened based on Log2>1 and -Log10 P value>1.3, such as Fig.10 As shown, 311 and 59 proteins were identified from YJ-1 and YJ-3, respectively.

[0071] In addition, a series of important proteins related to various cancers and other diseases were identified, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldehyde-keto reductase family 1 member AK1C3, and heat shock homologous protein 71kDa (HSP7C). In the YJ-1 / 3 labeling analysis, the main band of about 55kDa corresponds to ALDH1 protein, and the bands of about 40kDa and about 35kDa correspond to AK1C3 and G3P proteins. G3P, NOP2, GLUD1, ALDH1A1 and other proteins were further verified by pull-down / Western blotting using corresponding antibodies and YJ-1 / 3 ( Fig.11 ), knockdown of ALDH1A1 protein in HepG2 cells resulted in reduced labeling of YJ-1, and these results demonstrated the high reliability of these proteins. The YJ-3 probe can successfully label recombinant ALDH1 protein in a concentration-dependent manner, and modification site analysis of YJ-3 with ALDH1A1 showed covalent binding to lysine residues. Notably, up to 75% of the identified proteins were not found in drugbank, suggesting that this reaction head may provide a new approach to target these difficult-to-drug proteins.

[0072] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for selectively modifying protein lysine, characterized in that: The probe is incubated with a protein containing a lysine residue in a phosphate buffer, and click-reacted with TAMRA-N3, and then separated by SDS-PAGE to obtain the modified lysine; the probe is an isocyanide compound containing a phenyl group; The probe is one of YJ-1, YJ-2, YJ-3, YJ-4 and YJ-5, and the structural formulas are: ， ， ， ， 。 2. A method for selectively modifying protein lysine according to claim 1, characterized in that: The probe is combined with an aldehyde compound to modify protein lysine.

3. A method for selectively modifying protein lysine according to claim 2, characterized in that: The concentration of the protein is 10 nM, the concentration of the probe is 1-50 μM, and the concentration of the aldehyde compound is 0.2-5 mM.

4. A method for selectively modifying protein lysine according to claim 2, characterized in that: The aldehyde compound is one of formaldehyde, benzaldehyde and propionaldehyde.

5. The method for selectively modifying protein lysine according to claim 1, characterized in that: The protein is one of bovine serum albumin, lactoferrin and catalase.

6. The method for selectively modifying protein lysine according to claim 1, characterized in that: The incubation temperature is 20~25℃ and the incubation time is 1~2h.

7. Use of the probe in the preparation of cancer targeted drugs, characterized in that: The probes are YJ-1 and YJ-3 in claim 1, wherein YJ-1 targets cancers associated with G3P, NOP2 and GLUD1 proteins, and YJ-3 targets cancers associated with ALDH1A1 protein.

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