CN112794880B - Stable polypeptide protein targeted inhibitor and application thereof - Google Patents

Abstract

The invention provides a stable polypeptide protein targeted inhibitor, the amino acid sequence of which is shown as Ac-Q-S-Q-Q-F-K-N-M_q‑W‑R‑L‑L‑D‑Q‑N‑NH₂Wherein M is_qIs composed of

The invention also provides application of the stable polypeptide protein targeted inhibitor in preparation of an MDM4 protein inhibitor. The polypeptide of the invention can be covalently combined with lysine on MDM4 protein through nuclear magnetic and secondary mass spectrometry, and the like, thereby influencing the binding activity of MDM4 protein and p53 protein. Meanwhile, cell proliferation experiments also prove that the polypeptide can inhibit the proliferation of breast cancer cells.

Description

Stable polypeptide protein targeted inhibitor and application thereof

Technical Field

The invention belongs to the field of bioengineering, relates to a polypeptide, and particularly relates to a stable polypeptide protein targeted inhibitor and application thereof.

Background

Among the human tumor genes known so far, p53 plays a very important role as a cancer suppressor in intracellular stability and maintenance of microenvironment, and is also known as a "genomic guard". When the intracellular environment in vivo is damaged, such as DNA damage, etc., the transcription factor p53 can correspondingly regulate and control the related gene, so that the expression of the gene can promote the repair of cells. p53 plays a crucial role in preventing malignant transformation of cells, and nearly 50% of human tumors are currently found to be unable to function normally due to p53 protein mutation or inactivation of p53 gene. It can be further proved that p53 has a protective effect on the normal operation of life activities.

MDM2 and MDM4 are negative regulators of p53 protein and are also major factors in p53 ubiquitination and degradation. It directly inhibits p53, primarily by linking to the N-terminal transcriptionally activated binding region of p 53. MDM2 also has activity against E3 ubiquitin ligase, and can cause ubiquitination and degradation of p53 directly by the protease system. Scientists have found that overexpression of p53 inhibits its function and can be found in many tumors known to date.

MDM4 is one of the most important negative regulators of p 53. In human cancers, scientists have found that elevated levels of MDM4 result in the inactivation of oncostatin p 53. The structure of the protein is similar to that of MDM2 protein, but the function is different, MDM2 plays the role by degrading p53, and MDM4 plays the role of inhibiting p53 by binding with a transcription activation region. And the discovery that the content of MDM4 is abnormally excessive in breast cancer also makes MDM 4a potential anti-tumor therapeutic target.

In 2010, the subject group of the professor of Sawyer utilizes RCM loop-closing strategy to design a polypeptide inhibitor of ATSP-7041, and the polypeptide inhibitor is optimized on the basis of a previously reported polypeptide sequence, so that the polypeptide inhibitor can be combined with target MDM2 and MDMX at nanomolar level, and is also the currently reported polypeptide inhibitor of double-targeting MDM2 and MDMX with highest activity. In 2017, Li Jung teaches that a subject group developed a strategy of using chirally induced helical polypeptides to construct polypeptide inhibitors targeting p53-MDM 2/MDMX. Both polypeptides MeR and PhR with helical configuration showed better target binding and cell penetrating ability than polypeptides MeS and PhS. The close connection between the secondary structure and the biological activity of the polypeptide is proved. And research shows that the polypeptide has good tumor stem cell killing effect, and provides a new idea for a subsequent polypeptide inhibitor aiming at the tumor stem cells.

In 2016, Wang Lei professor designed polypeptide covalent inhibitors that effectively block the interaction between p53-MDM 4. This study replaced the leucine at position 22 in the reported sequence of SAH-p53-8 with a sulfonyl fluoride compound containing an orientation group. The p53 polypeptide helix is stabilized by the full carbon side chain, and simultaneously the sulfonyl fluoride compound is introduced into the side chain of the polypeptide, and when the polypeptide is combined with MDM4, the sulfonyl fluoride group on the side chain is close to lysine on the protein in space, thereby triggering covalent reaction and forming a protein-polypeptide covalent body. The study shows that the strategy can increase the inhibitory effect of SAH-p53-8 by 10 times, and systematic study on the modification site on the benzene ring finally determines the optimal chemical modification group.

Disclosure of Invention

Aiming at the technical problems in the prior art, the invention provides a stable polypeptide protein targeted inhibitor and application thereof, and aims to solve the technical problem that the effect of a medicament in the prior art on treating breast cancer is poor.

The invention provides a stable polypeptide protein targeted inhibitor, the amino acid sequence of which is shown as Ac-Q-S-Q-Q-F-K-N-M_q-W-R-L-L-D-Q-N-NH₂，M_qIs composed of

(shown in SEQ ID NO. 2).

Furthermore, the structural formula is shown as follows,

the invention provides application of the stable polypeptide protein targeted inhibitor in preparation of an MDM4 protein inhibitor.

The invention provides application of the stable polypeptide protein targeted inhibitor in preparation of a medicine for treating breast cancer.

The stabilized polypeptide protein targeted inhibitors of the present invention are capable of covalently binding to the p53 polypeptide through the specificity of reactive lysine residues on the binding interface of MDM 4.

Experiments such as hydrogen spectrum, carbon spectrum, heteronuclear single quantum relationship, secondary mass spectrum and the like prove that the polypeptide can utilize propargyl sulfonium salt center (s-alkyne) and is covalently bonded with the p53 polypeptide through the specificity of reactive lysine residue on a bonding interface of MDM 4. The polypeptide can inhibit the growth of breast cancer cells.

The novel propargyl sulfonium salt reagent with stable polypeptides can realize sulfydryl-alkyne coupling reaction without free radical action at room temperature in a weakly alkaline aqueous solution environment. Under the action of polypeptide ligand guidance, the propargyl sulfonium salt and the lysine can be covalently combined under the condition of sufficiently close space, so that selective covalent modification of the lysine under mild conditions is further realized, and the application range of the stabilized polypeptide is widened.

The invention selects the previously reported SAH-p53-8 sequence (Ac-Q-S-Q-Q-T-F- + -N-X-W-R-L-L- # -Q-N-NH)₂) Is the original sequence, with a methionine substitution at its X-position. In combination with the research strategy of the subject group taught by Wang Lei, the invention utilizes lysine and aspartic acid to perform side chain closure at the i position and the i +7 position, thereby further stabilizing the polypeptide.

Compared with the prior art, the invention has remarkable technical progress. The polypeptide of the invention can be covalently combined with lysine on MDM4 protein through nuclear magnetic and secondary mass spectrometry, and the like, thereby influencing the binding activity of MDM4 protein and p53 protein. Meanwhile, cell proliferation experiments also prove that the polypeptide can inhibit the proliferation of breast cancer cells.

Drawings

FIG. 1 is a mechanism for the reaction of lysine residue nucleophiles with an aminoalkyne.

FIG. 2 shows the reaction of wild-type MDM4-WT and cysteine-free MD3C mutant proteins with FAM-labeled sulfonium salt-alkyne probes.

FIG. 3 is a schematic representation of the cyclisation of Lys-Asp (KD) to an immobilised peptide.

Fig. 4 is a nuclear magnetic spectrum of the reaction of a sulfonium salt-alkyne with a lysine amino group, wherein a: 1H NMR:¹H NMR(400MHz DMSO-d6)δ7.85–7.32(m,1H),6.29(d,J＝7.5Hz,1H),4.39(s,1H),3.90–3.57(m,1H),2.83(s,7H),2.05(s,3H),1.34(m,17H)；B：¹³C{¹H}NMR：¹³C{¹H}NMR(400MHz DMSO-d6)δ174.7,158.6,158.5,158.2,155.3,118.8,115.8,77.6,69.1,54.6,52.0,44.9,42.9,31.9,31.5,28.3,27.1,23.1,18.1,8.8,7.7；C：¹H-¹H COSY；D：¹H-¹³C HSQC；E：¹H-¹³C HMBC；F：¹H-¹H TOCSY。

FIG. 5 shows the reaction scheme for the covalent coupling of three polypeptides to the MDM4 protein.

FIG. 6 is a graph of the protein of the MD3C mutant without cysteine reacted with Peptide-2 at 20-fold equivalent.

FIG. 7 is a competition experiment for the binding of Peptide-2 and IAA to MDM4 protein.

FIG. 8 shows the covalent coupling reaction of Peptide-2 to MDM4 protein mutant.

FIG. 9Peptide-2 reacts with wild-type MDM4 protein on a time gradient.

FIG. 10 is an analysis of the conjugation site of Peptide-2 with MDM4 protein at K93 by MS2 analysis.

FIG. 11 shows the proliferation inhibitory activity of Peptide-2 against human breast cancer cell line MCF-7 and normal cell HEK 293T.

FIG. 12 shows the regulatory effect of Peptide-2 on DNA damage in MCF-7 cells.

Fig. 13 is a schematic diagram of the present invention.

FIG. 14 is a mass spectrum of 6 polypeptides of the present invention after HPLC purification.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Example 1

For better study, the design of the control polypeptide is necessary. As shown in Table I, we designed a series of control polypeptides. The invention selects SAH-p53-8 sequence (Ac-Q-S-Q-Q-T-F- + -N-X-W-R-L-L- # -Q-N-NH2) as original sequence, and replaces the X site with methionine. And combined with the research strategy of the subject group taught by Wang, chem.Commun.2016, 52,5140, they replaced leucine at position 22 in the sequence of SAH-p53-8 with sulfonyl fluoride compound containing aromatic group, so that the inhibitory effect of SAH-p53-8 is increased by 10 times. The invention utilizes lysine and aspartic acid to carry out side chain ring closure at i position and i +7 position, thereby further stabilizing the polypeptide. The synthesized polypeptide (1equiv.) was reacted with bromopropyne reagent (5equiv.) under acidic conditions for 12 hours to construct the polypeptide in table one. The invention also designs cyclic peptide polypeptide 3, which uses bromopropionic acid as Linker to carry out intramolecular ring closure as a compared polypeptide sequence. (see FIGS. 1,3 and 13)

Table one: the polypeptide molecule sequence of the invention, polypeptide No. 1,2 and 3 is applied to FIG. 5, the best response effect of polypeptide No.2 is screened out, and the best response fluorescent polypeptide is selected from polypeptide No. 4,5 and 6 for reaction, as shown in FIG. 2.

Example 2 preparation and isolation and purification of the polypeptide:

solid phase synthesis of polypeptide according to amino acid sequence, the core steps for preparing the stable polypeptide are as follows (taking Peptide-2 as an example):

the specific operation steps are as follows:

(1) polypeptide solid phase synthesis: 100mg of Rink amide MBHA resin was weighed into a 10ml peptide connecting tube, Dichloromethane (DCM) was added, and nitrogen was bubbled for 30min for swelling. Adding a 50% (v/v) morpholine solution in N, N-Dimethylformamide (DMF), bubbling nitrogen for 30min, and removing the Fmoc protective group. After the resin was alternately washed 6 times with DMF and DCM, the prepared solution of Fmoc-Asn-OH (5eq,0.4M, DMF), solution of 6-chlorobenzotriazole-1, 1,3, 3-tetramethyluronium Hexafluorophosphate (HCTU) (5eq, 0.38M, DMF), and N, N-Diisopropylethylamine (DIPEA) (10eq) were mixed uniformly and added to the resin with nitrogen for 1 h. The reaction solution was withdrawn, and the resin was washed as described above and then subjected to the next step.

(2) The second to fourteenth amino acids are the same as the method, namely after the Fmoc protective group on the resin is removed by morpholine, the second amino acid is prepared, namely Fmoc-Gln-OH or Fmoc-Asp (alloc) -OH or Fmoc-Leu-OH or hex FMOC-Arg-OH or forpin-OH or Fmoc-Met-OH or Fmoc-Asn-OH or Fmoc-Lys (alloc) -OH or Fmoc-Phe-OH or Fmoc-Gln-OH or bridge Fmoc-Gln-OH or Fmoc-Ser-OH or Fmoc-Gln-OH is uniformly mixed, and the HCTU and DIPEA solution is added into the resin for 1h after being uniformly mixed; the reaction solution was withdrawn, and the resin was washed as described above and then subjected to the next step. Wherein the third, sixth, seventh and ten amino acids recommend a coupling time of 2-3 h.

(3) Palladium catalytic deprotection: dimethylbarbituric acid (4eq) and resin were added to a solution of tetratriphenylphosphine in palladium in DCM (1eq) under nitrogen, and stirred for 2h in the dark. And repeated again. After the reaction, the mixture was washed with a solution of sodium diethyldithiocarbamate (0.5%, DMF) 5 times, and then washed with DMF and DCM 6 times alternately.

(4) Intramolecular amide bond ring closure: benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP, 3.2eq), HOBt (3.2eq), N-methylmorpholine (NMM, 3.2eq) solution was added and nitrogen sparged for 4h, which was repeated once.

(5) Next, the Fmoc protecting group was removed with morpholine, the N-terminus of the polypeptide was acetylated, mixed with acetic anhydride and DIPEA (1: 2 in DCM), and then charged into the resin under nitrogen bubbling for 1 h.

(5) Polypeptide purification: with trifluoroacetic acid(TFA), Triisopropylsilane (TIPS) and H₂O (v: v: v ═ 9.5:0.25:0.25) shear cuts the polypeptide from the resin, and the shear is removed. Purifying and separating by high performance liquid chromatography. (see fig. 14)

Example 3 experiment of reaction of sulfonium salt-alkyne with lysine

The invention designs a model reaction, selects propargyl dimethyl sulfonium salt and Boc-Lys-OH as substrates to react in water for 12 hours, and then obtains a target product by HPLC purification. Careful Nuclear Magnetic Resonance (NMR) experiments were performed in the present invention, including 1H NMR, 13C {1H } NMR, Heteronuclear Single Quantum Correlation (HSQC), Heteronuclear Multiple Bond Correlation (HMBC), and Total correlation Spectroscopy (TOCSY), and it was confirmed that propargyl sulfonium salt could react with the amino group on lysine (see FIG. 4). FIG. 4A: 1H NMR:¹h NMR (400MHz DMSO-d6) δ 7.85-7.32 (m,1H),6.29(d, J ═ 7.5Hz,1H),4.39(s,1H), 3.90-3.57 (m,1H),2.83(s,7H),2.05(s,3H),1.34(m, 17H); FIG. 4B:¹³C{¹H}NMR：¹³C{¹h } NMR (400MHz DMSO-d6) delta 174.7,158.6,158.5,158.2,155.3,118.8,115.8,77.6,69.1,54.6,52.0,44.9,42.9,31.9,31.5,28.3,27.1,23.1,18.1,8.8, 7.7; FIG. 4C:¹H-¹h COSY; FIG. 4D:¹H-¹³c, HSQC; FIG. 4E:¹H-¹³c HMBC; FIG. 4F:¹H-¹H TOCSY。

EXAMPLE 4 covalent conjugation of the polypeptide to the MDM4 protein

To assess the ability of the polypeptide to target the MDM4 protein, the most intuitive SDS-PAGE separation experiment was used for validation. After protein concentration, SDS-PAGE separation was performed, and covalent coupling efficiency of the polypeptide and protein was observed by staining with Coomassie Brilliant blue. FIG. 5 shows that three polypeptides and proteins are covalently reacted, and the reaction effect of polypeptide No.2 is the best; FIG. 6 shows that, the covalent reaction of the No.2 polypeptide and the protein is selected, and the reaction efficiency of the polypeptide and the protein reaches about 40% under the condition that 20 times of equivalent of the polypeptide is added; FIG. 7 shows that blocking of cysteine with IAA reagent does not affect the reactivity of the polypeptide with the protein, demonstrating that the polypeptide does not react with cysteine of the protein; FIG. 8 shows that the effect of the polypeptide No.2 on the response of the MDM4 protein to wild-type WT and the mutant protein 3C without cysteine was essentially identical, and that it did not react with the mutant protein 3C-K without lysine, indicating that the lysine on the polypeptide and protein reacted rather than cysteine; FIG. 9 shows the time gradient of the reaction of the polypeptide with MDM4 protein.

Example 5 Secondary Mass Spectrometry identification of polypeptides covalently coupled to MDM4 protein

To assess the site of targeting of the polypeptide to the MDM4 protein, the protein was trypsinized using standard in-gel trypsin digestion protocols. After electrophoresis, the band of the protein of interest was excised from 15% SDS-PAGE gel, and the band was cut into fragments. The gel was then mixed with 50% Acetonitrile (ACN)/50mM NH₄HCO₃Incubate to decolorize. The digested protein fragment was mixed with 55mM iodoacetamide (at 25mM NH)₄HCO₃Medium) to carry out alkylation. The gel particles were then loaded with 200ng trypsin (Promega Sequence Grade Modified) 50mM NH₄HCO₃Incubated for 4 hours. Self-made C for peptide recovery liquid₁₈Desalting by a suction head. The treated samples were used for MS/MS analysis, and mass spectrometric analysis of the cleaved fragments confirmed ligand-directed and lysine-specific complex formation of the polypeptide and MDM4 protein, with the symbol "ˇ" in the peptide sequence indicating that the previous lysine residue had been ligated with the export [ QSQQTFKNM ˇ WRLLDQN (α) + QSFSVK ˇ DPSPLYDMLRK (β)]Crosslinker fragmentation spectroscopic reaction of the crosslinked peptide, some of the identified fragments are reported in the figure. The peak shown at 1350.67 corresponds to the mass of peptide covalently bound to MDM4, demonstrating that the polypeptide covalently reacts with lysine # 93 on the MDM4 protein. (see fig. 10).

EXAMPLE 6 Effect of the Polypeptides on cell proliferative Capacity

In order to evaluate the killing ability of the polypeptide on cancer cells, a breast cancer cell line MCF-7 cell was selected as a representative. Meanwhile, the normal cell HEK293T was selected for control.

Cell viability was determined by MTT assay (3- (4, 5-dimethylthiozol-2-yl) -2,5-diphenylt-etrazolium bromide)). Cells were plated at 4X 10 in 96-well plates³Inoculation, treatment with polypeptide in medium (serum free) for 24h, and addition of MTT reagent to medium for 4h incubation. Then adding DMSO to dissolve the precipitate, and measuring at 490nm with enzyme-labeling instrumentAbsorbance. Wherein the untreated cell viability was 100%.

The results show that the polypeptide only has certain cell proliferation inhibition and concentration dependence on MCF-7 cells. Has no toxic and side effects on HEK293T cells. These results indicate the specificity of the polypeptide. (see fig. 11)

EXAMPLE 7 Regulation of cellular DNA Damage by Polypeptides

In order to evaluate the regulatory effect of the polypeptide on cellular DNA damage, a breast cancer cell line MCF-7 cell was selected as a representative.

MCF-7 cells are inoculated in a six-hole cell culture plate, a DOX reagent is added to treat the cells for 4 hours, then polypeptide Peptide-2 is added to incubate for 12 hours, the lysed cells are detected by Western blot, and are detected by incubation of antibodies resisting gamma H2AX and GAPDH, and FIG. 12 shows that the addition of the DOX reagent causes the increase of gamma H2AX of the cells, which indicates that the DOX reagent increases DNA damage in the cells; after equal amount of DOX reagent is added for treatment, DNA damage in cells added with polypeptide Peptide-2 is reduced, which shows that polypeptide Peptide-2 can slow down DNA damage in cells.

Sequence listing

<110> Shenzhen institute of university of Beijing

Shenzhen Bay Laboratory Pingshan Biomedical R & D Conversion Center

<120> stable polypeptide protein targeting inhibitor and application thereof

<130> JSP12014679

<150> 2020112852351

<151> 2020-11-17

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Gln Ser Gln Gln Phe Lys Asn Met Trp Arg Leu Leu Asp Gln Asn

1 5 10 15

Claims (2)

1. A stable polypeptide protein targeted inhibitor, which is characterized in that: the amino acid sequence is shown as Ac-Q-S-Q-Q-F-K-N-M_q-W-R-L-L-D-Q-N-NH₂，M_{q is}

The structural formula of the compound is shown as follows,

2. use of the stable polypeptide protein targeted inhibitor of claim 1 in the preparation of a medicament for the treatment of breast cancer.

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