CN117903245A - Fluorescent probe for detecting deacylating and/or demethylating enzyme activity and preparation method and application thereof - Google Patents

Abstract

The present invention provides a fluorescent probe for detecting deacylase and/or demethylase activity and a preparation method and application thereof. The present invention first provides a compound, the compound comprising a structure shown in formula (I), wherein _R1 and _R2 are each independently selected from H, an acylation modification group or a methylation modification group, _R1 and _R2 are not H at the same time, and _R1 and _R2 are not acylation modification groups at the same time; _R3 is an N-terminal protecting group; _R4 is a carboxyl group or an amide group; S1 is an amino acid or a polypeptide; S2 is an amino acid or a polypeptide; n is 2, 3 or 4. The present invention discloses a general design strategy for a fluorescent probe for detecting deacylase and/or demethylase activity based on a long-distance intramolecular reaction of nitrobenzofurazan (NBD). Based on the above design strategy, a detection probe for enzyme activity and lysine demodification enzyme function identification can be developed, and used for the role of related enzymes in epigenetic control and regulation.

Description

Fluorescent probe for detecting deacylating and/or demethylating enzyme activity and preparation method and application thereof

Technical Field

The invention relates to a fluorescent probe for detecting deacylation and/or demethylation enzyme activity and a preparation method and application thereof, belonging to the technical field of biological detection.

Background technique

Post-translational modifications (PTMs) of the ε-amino group of lysine residues in proteins play diverse roles in regulating biological processes in living cells. To date, a variety of lysine PTMs have been discovered, many of which have been identified as playing key roles in regulating cellular processes. For example, lysine acetylation (Kac) is a key PTM involved in DNA replication, repair, and gene transcription. Lysine succinylation (Ksucc) regulates multiple metabolic pathways in mitochondria. A variety of diseases, including cancer and cardiovascular disease, have been associated with dysregulation of Ksucc. Lysine crotonylation (Kcro) is highly enriched in active promoter and enhancer regions. Recently, lysine lactylation has been identified as a new histone PTM associated with inflammation and cancer. Unlike lysine acetylation, which is usually associated with gene activation, lysine methylation can activate or repress genes depending on the site and state of methylation in the histone. Lysine methylation is closely related to transcriptional regulation, heterochromatin assembly, and cell cycle progression. Given that dynamic changes in histone acylation and methylation affect multiple cellular functions, analysis of their writers, erasers, and readers is essential and addresses a pressing need to understand epigenetic regulation and its impact on disease progression.

Histone deacetylases (HDACs) are epigenetic erasers that remove acetyl groups from lysine residues in histones and non-histone proteins. Emerging evidence suggests that HDACs are able to remove various lysine acylations through enzymatic catalysis. For example, the deacetylase Sirt3 was found to remove lysine crotonylation. The deacetylase Sirt5 has been identified as a demalonylating/desuccinylating enzyme that removes malonylating/succinylating lysine residues, thereby regulating cellular metabolism. Recently, HDACs1-3 were identified as demalonylating enzymes in HeLa cells. Meanwhile, dysfunction of HDAC activity is frequently found in cancer and neurological diseases, making HDACs attractive drug targets for the treatment of these pathologies. On the other hand, lysine methylation is removed through the catalytic action of lysine demethylases (KDMs). For example, Jumonji C (JmjC) lysine demethylase is an Fe(II)-dependent hydroxylase that catalyzes the oxidative demethylation of methyl lysine residues in proteins. Dysregulation of KDMs has been associated with developmental disorders and various cancers. Therefore, KDMs have become a popular target for drug development. Developing reliable analytical methods to detect the enzymatic activities of lysine deacylases and demethylases will be key to understanding how these epigenetic erasers affect cellular activities.

To date, a variety of methods have been developed to detect HDAC and KDM activity, such as mass spectrometry, high performance liquid chromatography, radioisotope methods, and antibody-based methods. However, these methods have various disadvantages, such as the need for expensive instruments, complex experimental procedures or multi-step reactions, time-consuming and lack of high-throughput capabilities. Fluorescence-based methods have become a popular strategy for detecting enzyme activity due to their simplicity, high sensitivity and high throughput. Therefore, researchers have shown great interest in developing fluorescent probes for measuring HDAC and KDM activity. Due to the importance of HDAC and KDM in diseases and the lack of fluorescent probes to detect them, there is an urgent need to develop a new generation of fluorescent probes to detect their activities and study their complex roles in biology.

Summary of the invention

In order to solve the above problems, the present invention proposes a fluorescent probe for detecting deacylating and/or demethylating enzyme activity and a preparation method and application thereof.

The first aspect of the present invention provides a compound, wherein the compound comprises a structure shown in formula (I), wherein

R ₁ and R ₂ are each independently selected from H, an acylation modification group or a methylation modification group, R ₁ and R ₂ are not H at the same time, and R ₁ and R ₂ are not acylation modification groups at the same time;

_R3 is the N-terminal protecting group of the amino acid;

_R4 is a carboxyl group or an amide group;

S1 is an amino acid or a polypeptide;

S2 is an amino acid or a polypeptide;

n is 2, 3 or 4.

According to a specific embodiment of the present invention, the length of S2 is 1-7 amino acids; most preferably, it is 1 amino acid;

Preferably, the length of S1 is 1-10 amino acids; most preferably, it is 4 amino acids.

It is known that the activity of most lysine demodification enzymes depends on the recognition of long and diverse lysine adjacent polypeptide sequences. In the present invention, determining the long and diverse amino acid spacers between the target lysine and the O-NBD group can also induce effective intramolecular reactions. Changing the length and sequence of different polypeptide substrates can make them suitable for performing a variety of lysine demodification enzyme activity tests.

Preferably, the acylation modification includes one or a combination of two or more of acetylation (Kac), propionylation (Kpr), butyrylation (Kbu), crotonylation (Kcro), malonylation (Kmal), succinylation (Ksucc), glutarylation (Kglu), 2-hydroxyisobutyrylation (Khib), β-hydroxybutyrylation (Kbhb), myristoylation (Kmys), lactylation (Klac), benzoylation (Kbz), lipoylation (Klip) and isonicotinoylation (Kinic); preferably, the acylation modification is acetylation (Kac) or succinylation (Ksucc);

The methylation modification is one or a combination of two or more of monomethylation (Kme1), dimethylation (Kme2) and trimethylation (Kme3); preferably, the methylation modification is dimethylation (Kme2).

The compound includes a structure shown in formula (I-1), formula (I-2) or formula (I-3), wherein

Formula (I-1): S1 is QTAR; S2 is STG; one of R ₁ and R ₂ is H, the other is Ac, R ₃ is Ac, and R ₄ is CONH ₂ ; the compound represented by formula (I-1) of the present invention is also called Pac-NBD;

Formula (I-2): S1 is QTAR; S2 is STG; one of R ₁ and R ₂ is H, and the other is COCH ₂ CH ₂ COOH, R ₃ is Ac, and R ₄ is CONH ₂ ; the compound represented by formula (I-2) of the present invention is also called Psucc-NBD;

Formula (I-3): S1 is QTAR; S2 is STGGK(ac)AP; R ₁ is methyl, R ₂ is methyl, R ₃ is Ac, and R ₄ is CONH ₂ . The compound represented by formula (I-3) of the present invention is also called Pme-NBD;

Formula (I-1), formula (I-2) and formula (I-3) are defined on the basis of formula (I).

It can be known that the present invention can use any suitable amino acid N-terminal protecting group, including but not limited to acetylation protection, Fmoc protection, Boc protection, Biotin protection, Z-Glycine (CAS 1138-80-3) protection, etc.; preferably, the protecting group is an acetylation protecting group. The present invention uses acetylation to protect the N-terminus of the amino acid, which has the advantages of small size, simple synthesis, and no other biological effects.

The second aspect of the present invention provides a compound precursor, wherein the compound precursor comprises a structure shown in formula (II), wherein

S1, S2, _R1 , _R2 , _R3 and _R4 are the same as described above.

The third aspect of the present invention provides the use of the compound as a fluorescent probe in detecting deacylase activity and/or demethylase activity.

The fourth aspect of the present invention provides a method for preparing the compound, the method comprising:

Designing and synthesizing the compound precursor; and

An O-NBD group is coupled to the precursor.

Preferably, the synthesis method of the compound precursor is solid phase synthesis.

Preferably, the method for coupling O-NBD with the compound precursor is a click chemistry reaction;

Preferably, the method for coupling O-NBD with a compound precursor comprises a copper-catalyzed azide-alkyne Husigen cycloaddition reaction and/or a copper-free click chemistry reaction.

According to a specific embodiment of the present invention, the method further comprises:

Designing a synthetic compound precursor, and mixing the precursor with O-NBD azide; preferably, the molar ratio of the compound precursor to O-NBD azide is 1:0.4-2.5; further preferably, the molar ratio of the compound precursor to O-NBD azide is 1:2;

The mixture is then added to an aqueous solution containing copper sulfate, tris(3-hydroxypropyltriazinemethyl)amine and sodium ascorbate; preferably, the molar ratio of copper sulfate, tris(3-hydroxypropyltriazinemethyl)amine and sodium ascorbate is 1:1-5:1-20, more preferably 1:5:2; further preferably, the molar ratio of polypeptide to copper sulfate is 1:0.2-5, more preferably 1:5. The reaction solution is stirred at room temperature for 4-12 hours. The final product is purified by HPLC and confirmed by mass spectrometry.

Preferably, the preparation method of the O-NBD azide comprises:

4-Fluoro-7-nitrobenzo-2-oxa-1,3-diazole, 2-azidoethanol and N,N-diisopropylethylamine were dissolved in dichloromethane, stirred at room temperature overnight, and separated by column chromatography.

A fifth aspect of the present invention provides a method for detecting histone deacylase and/or demethylase activity, comprising the following steps:

Using the compound as a fluorescent probe to detect the activity of deacylase and/or demethylase to evaluate the enzyme activity;

Preferably, the detection comprises incubating the compound with the deacylase and/or demethylase to be detected in a HEPES buffer, wherein the HEPES buffer comprises 20 mM HEPES buffer at pH=8.0; preferably, the buffer further comprises the coenzyme NAD ⁺ ; preferably, the buffer further comprises ammonium ferrous sulfate (II), 2-oxoglutaric acid and ascorbic acid;

Preferably, the incubation temperature is 37° C. and the incubation time is 60-180 minutes.

According to a specific embodiment of the present invention, wherein the method for evaluating enzyme activity comprises colorimetry, high performance liquid chromatography, mass spectrometry, ultraviolet visible spectroscopy analysis and fluorescence spectroscopy analysis;

Preferably, the method for evaluating enzyme activity is achieved by changes in the fluorescence spectrum of a fluorescent probe.

The fluorescent probe for detecting the activity of lysine deacetylase, desuccinylase or demethylase provided by the present invention has high selectivity and sensitivity. The compound prepared by the present invention provides a useful tool for promoting drug discovery in epigenetics as a fluorescent probe.

The sixth aspect of the present invention provides a kit for detecting the activity of a deacylating and/or demethylating enzyme, wherein the kit comprises the compound; and a HEPES buffer at pH=8.0; preferably, the concentration of HEPES is 20 mM;

Preferably, the kit further comprises coenzyme NAD ⁺ ; the kit comprising coenzyme NAD ⁺ is particularly suitable for detecting the activity of the third major class of deacylases (Sirt1-7);

Preferably, the kit further comprises ammonium ferrous sulfate (II), 2-oxoglutaric acid and ascorbic acid; the kit comprising ammonium ferrous sulfate (II), 2-oxoglutaric acid and ascorbic acid is particularly suitable for detecting the activity of JmjC family demethylases.

On the other hand, the present invention proposes a general design strategy for fluorescent probes for detecting deacylase and demethylase activity based on long-range intramolecular reactions of nitrobenzofuroxan (NBD). The fluorescent probe in this design strategy comprises a polypeptide sequence of post-translation modification of the ε-amino group of a lysine residue and an O-NBD fluorophore portion coupled by a click chemistry reaction. Based on the above design strategy, detection probes for enzyme activity and lysine demodification enzyme function identification can be developed and used for the role of related enzymes in epigenetic control and regulation. Beneficial effects of the present invention:

A major advantage of the NBD structure used in the present invention is that the O-NBD group does not fluoresce, while the N-NBD group exhibits strong fluorescence at longer wavelengths. Under mild aqueous conditions, the long amino acid interval between the O-NBD group and the unmodified lysine residue can effectively undergo long-distance intramolecular reactions to produce N-NBD and emit fluorescence. Therefore, this is a detection method that "turns on" fluorescence. It is different from the conventionally used fluorescence quenching detection method. Another advantage is that the small size of the NBD group helps to minimize interference when the probe binds to the target protease. After the enzymatic reaction removes the acyl group in the lysine residue, the released free amine reacts intramolecularly with the O-NBD part, resulting in fluorescence turning on.

The present invention can provide a powerful tool to effectively analyze the activity of lysine deacylase and/or demethylase, and is conducive to accelerating the development of epigenetic drugs in the future.

In addition, the NBD-based platform of the present invention has a wide range of applications, for example, it can be easily extended to the study of the enzymatic activity of epigenetic erasers, which can remove other novel lysine PTMs, such as lysine lactylation, lysine isonicotinoylation, etc. The present invention will help to further clarify their biological effects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows a general design method of fluorescent probes based on histone deacylase and demethylase according to the present invention.

FIG2 is a diagram showing the mechanism of the fluorescent probe Pac-NBD of the present invention for detecting enzymatic deacetylation reaction through long-distance intramolecular reaction, HPLC, mass spectrum and color change diagram.

FIG3 is a graph showing the ultraviolet absorption spectrum, fluorescence spectrum, selectivity and time variation of the enzymatic reaction between the fluorescent probe Pac-NBD of the present invention and deacetylase.

FIG4 is a diagram showing the mechanism of the fluorescent probe Psucc-NBD of the present invention for detecting enzymatic desuccinylation reaction through long-distance intramolecular reaction, HPLC, mass spectrum and color change diagram.

FIG5 shows the ultraviolet absorption spectrum, fluorescence spectrum, selectivity and time variation of the enzymatic reaction between the fluorescent probe Psucc-NBD of the present invention and desuccinylase.

FIG6 is a mechanism diagram, HPLC, mass spectrum, and ultraviolet absorption and fluorescence spectra of the fluorescent probe Pme-NBD of the present invention for detecting enzymatic demethylation reactions through long-distance intramolecular reactions.

FIG. 7 is a HPLC spectrum of the fluorescent probe precursors Pac, Psucc and Pme of the present invention.

FIG8 is an ESI-MS spectrum of the fluorescent probe precursors Pac, Psucc and Pme of the present invention.

Detailed ways

Since these epigenetic erasers have recognition specificity for substrate polypeptide sequences, it is very difficult to design a general strategy for detecting their activity. Here, the present invention reports a general strategy for constructing a fluorescent probe that can detect the enzymatic activity of epigenetic "erasers" through long-range intramolecular reactions based on NBD. Fluorescent probes can be easily prepared by installing the O-NBD group on the C-terminal residue of a specific peptide substrate through click chemistry. In addition, the fluorescent probe provided by the present invention is a single-step fluorescent probe. Based on this strategy, the detection of lysine deacetylase, desuccinylase or demethylase activity has been successfully achieved through the development of fluorescent probes, with excellent sensitivity and selectivity. The fluorescent probe of the present invention will provide a powerful tool to promote drug discovery in future epigenetics.

The general strategy for preparing a fluorescent probe for detecting deacylase and demethylase activity by long-range intramolecular reaction based on NBD is shown in FIG1 . The strategy of the present invention is briefly described as follows:

① Installing an O-NBD group on the C-terminal residue of the precursor of the fluorescent probe: The NBD-based fluorescent probe for detecting deacylase and demethylase activity is to install an O-NBD group on the C-terminal residue of the precursor polypeptide through a copper-catalyzed click chemistry reaction (Click chemistry); by changing the length and sequence of the polypeptide substrate, the corresponding fluorescent probe can be designed to detect different lysine demodification enzyme activities;

② Enzyme-catalyzed deacylation or demethylation reaction: When the probe interacts with the corresponding deacylase or demethylase, an enzyme-catalyzed deacylation or demethylation reaction occurs, thereby releasing the primary or secondary amine group of the side chain of the lysine residue;

③ Formation of N-NBD: O-NBD will undergo spontaneous long-range intramolecular exchange with primary or secondary amine groups to form N-NBD, resulting in fluorescence on.

The luminescent substance whose fluorescence is “turned on” is nitrobenzofuroxan (O-NBD).

In order to make the technical means, creative features, objectives and effects of the present invention easy to understand, the present invention is further described below in conjunction with specific implementation methods. However, the protection scope of the present invention is not limited to the following embodiments.

Example 1

This embodiment provides a method for synthesizing O-NBD azide. The preparation steps are as follows:

Fluoro-7-nitro-2,1,3-benzoxadiazole (40 mg, 0.218 mmol) was dissolved in DCM. Then 2-azidoethanol (20 μL, 0.262 mmol) and 76 μL DIEA were added. The mixture was stirred at room temperature overnight. After the solvent was removed under reduced pressure, the crude product was chromatographed on a silica gel column (DCM: PE = 1: 1). The product was further purified by recrystallization in DCM/hexane to give O-NBD azide as a yellow solid (31.0 mg, 56.9% yield) after vacuum drying.

¹ H NMR (300 MHz, CDCl ₃ ) δ8.55 (d, J=8.3 Hz, 1H), 6.75 (d, J=8.3 Hz, 1H), 4.58 (t, J=4.9 Hz, 2H), 3.86 (t, J=4.9 Hz, 2H); ¹³ C NMR (100 MHz, CDCl ₃ ) δ153.9, 145.1, 144.0, 133.7, 130.4, 105.3, 69.8, 49.6.

Example 2

This example provides a method for preparing fluorescent probe precursors Pac, Psucc and Pme. The preparation steps are as follows:

The fluorescent probe precursors QTARK(ac)STG-Pra(Pac), QTARK(succ)STG-Pra(Psucc), and QTARK(me2)STGGK(ac)AP-Pra(Pme) were synthesized using the standard Fmoc protocol of the solid phase peptide synthesis (SPPS) method.

In the fluorescent probe precursor, the sequence numbers of the polypeptides other than Pra (propargylglycine) are:

SEQ ID NO.1: QTARK(ac)STG;

SEQ ID NO.2: QTARK(succ)STG;

SEQ ID NO.3: QTARK(me2)STGGK(ac)AP.

The coupling in each step was carried out on Rink-amide MBHA resin (loading 0.868 mmol/g) using Fmoc amino acid (4.0 equiv.), HATU (4.0 equiv.) and DIEA (8.0 equiv.). After washing the reaction mixture with DMF, DCM and DMF, the Fmoc protecting group was removed using 20% piperidine/DMF. After coupling, a solution of pyridine: acetic anhydride: dichloromethane (1:1:2) was added with the resin for 30 minutes to obtain the N-terminally acetylated peptide. After the peptide synthesis was completed, the resin was treated with TFA/TIS/H ₂ O (95:2.5:2.5) for 2 hours to release the peptide. The peptide was harvested by precipitation with ice-cold diethyl ether. The crude product was collected after resuspension in ice-cold diethyl ether and centrifugation. The peptide was purified using reverse phase HPLC on a C18 column and verified by HPLC and ESI-MS. The results are shown in Figures 7 and 8. In Figure 7: A is the HPLC spectrum of Pac; B is the HPLC spectrum of Psucc; C is the HPLC spectrum of Pme. In Figure 8: A is the ESI-MS spectrum of Pac, LCMS m/z = 1026.9 [M+H] ⁺ , B is the ESI-MS spectrum of Psucc, LCMS m/z = 1084.7 [M+H] ⁺ , C is the ESI-MS spectrum of Pme, LCMS m/z = 1408.1 [M+H] ⁺ . In the synthesis of these peptides, the Fmoc-amino acids used were: Fmoc-Gln(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-Lys(Ac)-OH, Fmoc-Lys(Dde)-OH, Fmoc-Lys(Me)2-OH·HCl and Fmoc-Pra-OH.

Example 3

This embodiment provides a method for preparing fluorescent probes Pac-NBD, Psucc-NBD and Pme-NBD. The preparation steps are as follows:

The fluorescent probe precursor (Pac, Psucc or Pme) obtained in Example 2 (0.2 mM) was mixed with O-NBD azide (0.4 mM) solution. The mixture was then added to an aqueous solution of CuSO ₄ (1 mM), THPTA (5 mM) and sodium ascorbate (2 mM). The reaction solution was stirred at room temperature for 4 hours. The final products Pac-NBD, Psucc-NBD and Pme-NBD were purified by HPLC and confirmed by mass spectrometry. LCMS m/z of fluorescent probe Pac-NBD = 1276.51 [M+H] ⁺ ; LCMS m/z of Psucc-NBD = 1334.66 [M+H] ⁺ ; LCMS m/z of Pme-NBD = 829.87 [M+2H] ²⁺ .

Example 4

This example provides a method for detecting enzymatic deacetylation reactions using the fluorescent probe Pac-NBD via long-range intramolecular reactions. The steps are as follows:

Pac-NBD was incubated with sirtuins and coenzyme NAD ⁺ in HEPES buffer (pH=8.0, 20 mM HEPES) at 37° C. for 95 minutes.

Figure 2 A shows the mechanism of Pac-NBD probe detecting enzymatic deacetylation reaction through long-range intramolecular reaction. The peptide sequence used is histone H3 peptide (amino acids 5-12), with an acetylated lysine residue at position 9.

Next, we studied whether the Pac-NBD fluorescent probe could be recognized and deacetylated by Sirt1 by HPLC, mass spectrometry, and colorimetry, and the results are shown in Figure 2B, Figure 2C, and Figure 2D, respectively. The results showed that the color of the reaction solution turned yellow after 95 minutes of reaction and showed strong green fluorescence under ultraviolet light, which produced the expected deacetylation/exchange product.

These data indicate that: ① the NBD-based H3K9(ac) peptide probe can be recognized by the known Kac "eraser"Sirt1; ② a long-range intramolecular reaction occurs between the O-NBD and the _NH2 group, despite the presence of three different amino acids in between.

Example 5

This example provides a method for measuring the absorption spectrum and fluorescence spectrum of the enzymatic reaction between Pac-NBD and sirtuin.

The measurement steps are: Set the reaction to a total reaction volume of 200 μL. Collect the absorption spectrum from 300 to 600 nm. Collect the fluorescence spectrum of the enzymatic reaction using Pac-NBD between 520 and 700 nm using an excitation wavelength of 480 nm.

The measurement results are as follows: At the beginning, a clear absorption peak was observed for Pac-NBD at 380nm, corresponding to the absorption peak of the O-NBD structure. After the enzymatic reaction with Sirt1 and NAD ⁺ , a new absorption peak was detected at 480nm (Figure 3A), indicating the formation of N-NBD. Subsequently, a more detailed fluorescence study was performed.

The results showed that Pac-NBD alone and Pac-NBD showed negligible fluorescence (λ _ex = 480 nm) in the presence of denatured Sirt1 and in the absence of Sirt1 or NAD ⁺ . However, after the addition of Sirt1 and NAD ⁺ , a strong emission peak was observed at the same excitation wavelength ( FIG. 3B ).

The results of studying the response of Pac-NBD probe to other HDACs showed that Pac-NBD probe showed good activity against Sirt1, Sirt2, Sirt3 and HDAC3 ( Figure 3C ).

By using the Pac-NBD probe to monitor the activity of Sirt1 (D in Figure 3), the results showed that the fluorescence intensity of the reaction solution was weak at first, but increased rapidly during the incubation process and stabilized after one hour. By fitting the fluorescence data to an exponential equation, the first-order rate constant k of Sirt1 was determined to be 0.033min ^-1 . It is worth mentioning that the Pac-NBD probe is quite stable in the buffer solution, and the background signal of the probe is negligible.

All experiments together confirmed the feasibility of the NBD-based fluorescent probe of the present invention for long-distance intramolecular reactions. The versatility of the strategy provided by the present invention was verified, that is, by using different post-translationally modified polypeptide substrates, synthesizing stable and sensitive probes for single-step detection of other lysine PTM "erasers" is a potential universal strategy.

Example 6

This example provides a method for detecting enzymatic desuccinylation reactions using Pcucc-NBD via long-range intramolecular reactions.

Figure 4 A shows the mechanism of the Psucc-NBD probe detecting enzymatic desuccinylation reaction through long-range intramolecular reaction. The peptide sequence used is histone H3 peptide (amino acids 5-12), with a succinylated lysine residue at position 9.

Next, we verified whether the Psucc-NBD peptide probe could be recognized and erased by the desuccinylase Sirt5. Pcucc-NBD was incubated with sirtuins and coenzyme NAD ⁺ in HEPES buffer (pH=8.0, 20 mM HEPES) at 37°C for 115 minutes.

The results of HPLC, mass spectrometry and colorimetric studies are shown in Figure 4B, Figure 4C and Figure 4D, respectively, and the results showed that the color of the reaction solution turned yellow after 2 hours of reaction and showed strong green fluorescence under ultraviolet light, which produced the expected desuccinylation/exchange product.

Example 7

This example provides a method for measuring the absorption spectrum and fluorescence spectrum of the enzymatic reaction between Psucc-NBD and desuccinylase.

The measurement steps are: Set the reaction to a total reaction volume of 200 μL. Collect the absorption spectrum from 300 to 600 nm. Collect the fluorescence spectrum of the enzymatic reaction using Psucc-NBD between 520 and 700 nm using an excitation wavelength of 480 nm.

The measurement results are as follows: At the beginning, an obvious absorption peak was observed for Psucc-NBD at 380 nm (Figure 5A), corresponding to the absorption peak of the O-NBD unit. After the enzymatic reaction with Sirt5 and NAD ⁺ , a new absorption peak was detected at 480 nm, indicating the formation of N-NBD.

Subsequently, more detailed fluorescence studies were performed. The results showed that Psucc-NBD alone and Psucc-NBD showed negligible fluorescence (λ _ex = 480 nm) in the presence of denatured Sirt5 and in the absence of Sirt5 or NAD ⁺ . However, after the addition of Sirt5 and NAD ⁺ , a strong emission peak was observed at the same excitation wavelength (Figure 5B).

The results of studying the response of the Psucc-NBD probe to other HDACs showed that the Psucc-NBD probe showed good activity against Sirt5 (Figure 5C). By using the Psucc-NBD probe to monitor the activity of Sirt5, the fluorescence intensity was weak at first, and increased rapidly during the incubation process, and stabilized after one hour (Figure 5D). By fitting the fluorescence data to an exponential equation, the first-order rate constant k of Sirt5 was determined to be 0.025min ^-1 . It is worth mentioning that the Psucc-NBD probe is quite stable in the buffer solution, and the background signal of the probe is negligible.

These data suggest that the probe Psucc-NBD can provide a useful tool to screen potential desuccinylase inhibitors. The Psucc-NBD probe will provide a powerful tool for epigenetic studies and the discovery of Sirt5-targeted drugs for the treatment of breast cancer.

Example 8

This example provides an experimental method for detecting enzymatic demethylation reactions of Pme-NBD through long-range intramolecular reactions.

Figure 6A shows the mechanism by which the Pme-NBD probe detects enzymatic desuccinylation reactions through long-range intramolecular reactions.

Next, we verified whether the Pme-NBD peptide probe could be recognized and erased by the desuccinylase Sirt5.

The Pme-NBD polypeptide probe was mixed with the JMJD2E enzyme in HEPES buffer (pH=8.0, 20 mM HEPES, 100 μM ammonium ferrous sulfate (II), 500 μM 2-oxoglutaric acid, 2 mM ascorbic acid).

The enzymatic reaction of Pme-NBD was measured between 520 and 850 nm using an excitation wavelength of 495 nm. HPLC and mass spectrometry results showed that Pme-NBD generated the expected monomethylation/exchange products (B, C, and D in Figure 6). Although the spacer contained seven amino acids, a long-range intramolecular reaction occurred between the non-fluorescent O-NBD group and the monomethylated amine group.

The enzymatic activity of the demethylase was further analyzed by detailed UV absorption and fluorescence spectroscopy using Pme-NBD. Initially, Pme-NBD had a distinct absorption peak at 380 nm, corresponding to the O-NBD group. After 2 h of enzymatic reaction with JMJD2E, a new absorption peak was detected at 495 nm instead of 480 nm, indicating the formation of N(Me)-NBD instead of N(H)-NBD probe product (Figure 6E).

In addition, a more detailed fluorescence study was performed (F in Figure 6). The results showed that the fluorescence of the Pme-NBD probe alone was negligible, without the enzyme JMJD2E or cofactor Fe ²⁺ (λ _ex = 495nm). After adding JMJD2E and cofactor Fe ²⁺ , a strong emission peak was observed at the same excitation wavelength. The fluorescence increase was determined to be up to 23 times. The present invention also used this probe in a JMJD2E inhibition test, and IOX1 is a powerful and specific JMJD2E inhibitor. When Pme-NBD was incubated with JMJD2E in the presence of IOX1, the fluorescence enhancement of the probe was significantly suppressed to background levels (F in Figure 6). Therefore, the probe can detect the inhibition of enzyme activity by IOX1.

The kinetics of the enzymatic reaction with the Pme-NBD probe were further examined. As shown in Figure 6G, during the incubation of the probe with JMJD2E at 37°C, the fluorescence intensity of the probe was initially weak and increased dramatically during the incubation process. By fitting the fluorescence data to an exponential equation, the first-order rate constant k for JMJD2E was determined to be 0.018 min ^-1 . Similar to the deacylase probes Pac-NBD and Psucc-NBD, the Pme-NBD probe was also stable in buffer solutions, and the background signal of the probe was negligible (Figure 6G).

These results indicate that the Pme-NBD probe is a stable and sensitive NBD-based fluorescent probe for detecting demethylase activity. It provides a powerful tool for the field of epigenetic research and for the development of demethylase inhibitors that may have the ability to act as anticancer agents.

In view of this, the present invention provides a fluorescent probe with high selectivity and sensitivity for detecting the activity of lysine deacetylase, desuccinylase or demethylase. The probe prepared by the present invention provides a useful tool for promoting drug discovery in epigenetics. It is of great significance to develop a probe capable of detecting enzyme activity and revealing more lysine demodification enzymes, and clarifying their role in epigenetic control and regulation.

The long-range intramolecular reaction of NBD-based polypeptide fluorescent probes strongly demonstrates that using click chemistry to connect NBD fluorophores to the C-termini of different polypeptide sequences recognized by enzymes is a universal and convenient strategy for developing new fluorescent probes. Although the present invention only lists the cases of HDAC and JmjC family enzymes that recognize Kac, Ksucc and Kme2 residues, it is not difficult to understand that the technical scheme of the present invention can be extended to recognize other PTMs, such as lysine propionylation, crotonylation, benzoylation, lactylation, methacrylylation and other newly discovered PTMs. This design strategy provided by the present invention is very useful for developing enzymes that can detect enzyme activity and reveal the identities of more lysine demodification enzymes, and is expected to clarify their roles in epigenetic control and regulation.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims (10)

1. A compound comprising a structure shown in formula (I), wherein R ₁ and R ₂ are each independently selected from H, an acylation modification group or a methylation modification group, R ₁ and R ₂ are not H at the same time, and R ₁ and R ₂ are not acylation modification groups at the same time; _R3 is the N-terminal protecting group of the amino acid; _R4 is a carboxyl group or an amide group; S1 is an amino acid or a polypeptide; S2 is an amino acid or a polypeptide; n is 2, 3 or 4. 2. The compound according to claim 1, wherein the length of S2 is 1-7 amino acids; Preferably, the length of S1 is 1-10 amino acids. 3. The compound according to claim 1 or 2, wherein the acylation modification comprises one or a combination of two or more of acetylation (Kac), propionylation (Kpr), butyrylation (Kbu), crotonylation (Kcro), malonylation (Kmal), succinylation (Ksucc), glutarylation (Kglu), 2-hydroxyisobutyrylation (Khib), β-hydroxybutyrylation (Kbhb), myristoylation (Kmys), lactylation (Klac), benzoylation (Kbz), lipoylation (Klip) and isonicotinoylation (Kinic); preferably, the acylation modification is acetylation (Kac) or succinylation (Ksucc); The methylation modification is one or a combination of two or more of monomethylation (Kme1), dimethylation (Kme2) and trimethylation (Kme3); preferably, the methylation modification is dimethylation (Kme2). 4. The compound according to any one of claims 1 to 3, wherein the compound comprises a structure represented by formula (I-1), formula (I-2) or formula (I-3), wherein Formula (I-1): S1 is QTAR; S2 is STG; one of R ₁ and R ₂ is H, the other is Ac, R ₃ is Ac, and R ₄ is CONH ₂ ; Formula (I-2): S1 is QTAR; S2 is STG; one of R ₁ and R ₂ is H, and the other is COCH ₂ CH ₂ COOH; R ₃ is Ac, and R ₄ is CONH ₂ ; Formula (I-3): S1 is QTAR; S2 is STGGK(ac)AP; _R1 is methyl, _R2 is methyl, _R3 is Ac, and _R4 is _CONH2 . 5. A compound precursor, wherein the compound precursor comprises a structure represented by formula (II), wherein S1, S2, R ₁ , R ₂ , R ₃ and R ₄ are the same as any one of claims 1-4. 6. Use of the compound according to any one of claims 1 to 4 as a fluorescent probe in detecting deacylase activity and/or demethylase activity. 7. A method for preparing the compound according to any one of claims 1 to 4, comprising: Designing and synthesizing the compound precursor of claim 5; and coupling an O-NBD group to the precursor; Preferably, the method for coupling O-NBD with the precursor is a click chemistry reaction; Further preferably, the method for coupling O-NBD with the precursor comprises a copper-catalyzed azide-alkyne Husigen cycloaddition reaction and/or a copper-free click chemistry reaction. 8. The method according to claim 7, wherein the method further comprises: Designing a synthetic compound precursor, and mixing the precursor and O-NBD azide together; preferably, the molar ratio of the precursor to the O-NBD azide is 1:0.4-2.5; further preferably, the molar ratio of the precursor to the O-NBD azide is 1:2; Then, the mixture is added to an aqueous solution containing copper sulfate, tris(3-hydroxypropyltriazinemethyl)amine and sodium ascorbate; preferably, the molar ratio of copper sulfate, tris(3-hydroxypropyltriazinemethyl)amine and sodium ascorbate is 1:1-5:1-20, more preferably 1:5:2; further preferably, the molar ratio of the polypeptide to copper sulfate is 1:0.2-5, more preferably 1:5; Preferably, the preparation method of the O-NBD azide comprises: dissolving 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole, 2-azidoethanol and N,N-diisopropylethylamine in dichloromethane, stirring at room temperature overnight, and separating by column chromatography. 9. A method for detecting histone deacylase and/or demethylase activity, comprising the following steps: Using the compound according to any one of claims 1 to 4 to detect the activity of deacylase and/or demethylase to evaluate the enzyme activity; Preferably, the detection comprises incubating the compound with the deacylase and/or demethylase to be detected in a HEPES buffer, wherein the HEPES buffer comprises 20 mM HEPES buffer at pH=8.0; preferably, the buffer further comprises the coenzyme NAD ⁺ ; preferably, the buffer further comprises ammonium ferrous sulfate (II), 2-oxoglutaric acid and ascorbic acid; Preferably, the method for evaluating enzyme activity comprises colorimetry, high performance liquid chromatography, mass spectrometry, ultraviolet-visible spectroscopy analysis and/or fluorescence spectroscopy analysis. 10. A kit for detecting deacylating and/or demethylating enzyme activity, wherein the kit comprises the compound according to any one of claims 1 to 4; and pH=8.0 HEPES buffer; Preferably, the kit further comprises coenzyme NAD ⁺ ; preferably, the kit further comprises ammonium ferrous sulfate (II), 2-oxoglutaric acid and ascorbic acid.