Stable polypeptide inhibitor targeting HDAC and application of stable polypeptide inhibitor

A technology of polypeptides and inhibitors, applied in the field of bioengineering, can solve the problems of large toxic and side effects and poor effects, and achieve the effects of inhibiting proliferation, solving toxic and side effects, and improving the ability of cells to penetrate membranes.

Active Publication Date: 2019-06-21
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a stable HDAC-targeting polypeptide inhibitor and its use. The HDAC-targeting stable polypeptide inhibitor and its use should solve the existing HDAC polypeptide inhibitors in the technology have poor efficacy in treating cancer, and the technical problem of relatively large toxic and side effects

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  • Stable polypeptide inhibitor targeting HDAC and application of stable polypeptide inhibitor
  • Stable polypeptide inhibitor targeting HDAC and application of stable polypeptide inhibitor
  • Stable polypeptide inhibitor targeting HDAC and application of stable polypeptide inhibitor

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Experimental program
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Effect test

Embodiment 1

[0041] Embodiment 1: the design of polypeptide inhibitor

[0042] The present invention adopts the way of polypeptide-small molecule covalent coupling to design HDAC polypeptide inhibitors, such as figure 1 As shown, we selected a polypeptide sequence (TVALREIRRYQK(Ac)) near the HDAC1 selective substrate H3K56 (the 56th amino acid lysine side chain amino of histone H3 was modified by acetylation), because the K56 of this polypeptide is in a The C-terminus of the helix is ​​suitable for us to use the "N-terminal aspartic acid strategy" method to stabilize the polypeptide without affecting the recognition of the target by the C-terminal K56. In order to further improve the binding of the substrate polypeptide to HDAC, we mutated the amino acid of K56 into a hydroxamic acid structure, such as figure 1 As shown in A.

[0043] This structure was reported to be able to chelate zinc ions in the catalytic region of HDAC enzymes to inhibit HDAC enzyme activity. The basic structure o...

Embodiment 2

[0046] Embodiment 2: the synthetic preparation of polypeptide inhibitor

[0047] We use the internationally accepted solid-phase peptide synthesis technique (SPPS) to synthesize peptides. Its synthetic route is as follows figure 2 shown.

[0048] Specific steps:

[0049] (1) Synthesis of unnatural amino acid (Fmox-Hx-OH) with hydroxamic acid: the synthetic route is completely carried out according to the method reported in the literature.

[0050] (2) Solid-phase synthesis of polypeptides: Weigh 100 mg of 2-Chlorotity-N-Fmoc-hydroxylamine resin into a 10 ml peptide tube, add dichloromethane (DCM), and swell with nitrogen gas for 10 min. Add 50% (v / v) morpholine in N,N-dimethylformamide (DMF) solution, blow nitrogen gas for 30min×2, and remove the Fmoc protecting group. After washing the resin 6 times alternately with DMF and DCM, the prepared (1) Fmoc-Hx-OH (5eq, 0.4M, DMF) solution, 6-chlorobenzotriazole-1,1,3,3-tetramethyl Urea hexafluorophosphate (HCTU) (5eq, 0.38M, D...

Embodiment 3

[0055] Example 3: Identification of the secondary structure of the polypeptide and its inhibitory effect on HDAC enzyme activity

[0056] For each peptide, conformation in water was determined by circular dichroism chromatography, demonstrating that peptides stabilized by terminal side chain-to-tail linkage of chiral diacid-modified peptides in water (e.g. Figure 5 A) and in 20% TFE solution (such as Figure 5 B) has a stable α-helical conformation, in which TFE (trifluoroethanol) has the effect of promoting the helix. The identification of HDAC enzyme activity inhibitory effect is carried out with commercially purchased HDAC enzyme activity detection kits, and each operation is repeated at least three times. The enzyme activity results are as follows: Figure 6 B (the design of peptide inhibition library and the screening of the enzyme activity inhibitory effect of HeLa nucleus extract) and Figure 6 C( Figure 6 C: the inhibitory effect of different polypeptide inhibitor...

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Abstract

The invention provides a stable polypeptide inhibitor for targeting HDAC. The amino acid sequence structure of the stable polypeptide inhibitor is shown in specification. The invention also provides application of the stable polypeptide inhibitor targeting HDAC in preparation of a drug for inhibiting the enzymatic activity of HDAC family. The invention also provides application of the stable polypeptide inhibitor targeting HDAC in preparation of a drug for inhibiting tumor stem cells with high expression of HDAC1. The invention further provides application of the stable polypeptide inhibitor targeting HDAC in preparation of a drug for treating human ovarian teratoma or testicular embryonic carcinoma. Experiments of cell proliferation, apoptosis and cell cycle arrest prove that the polypeptide can effectively inhibit proliferation of the tumor stem cells, and the polypeptide has no obvious tissue damage or toxicity to mice at a high dose.

Description

technical field [0001] The invention belongs to the field of bioengineering and relates to a polypeptide, in particular to a stable polypeptide inhibitor targeting HDAC and its application. Background technique [0002] Cancer is a major public health problem worldwide, and in China it is the leading cause of death and causes a significant disease burden. According to the 2014 National Center for Cancer Registry (NCCRC) cancer statistics in China, there are currently 3,804,000 cases of cancer diagnosed in China each year, and the incidence of cancer is about 278.07 / 100,000 (male incidence rate is 301.67 / 100,000, female incidence rate is 253.29 / 100,000). The types of tumors and the causes of tumors are different in different genders and ages. The complexity of tumor diseases has brought challenges to human beings to overcome this disease. The occurrence, development, invasion, metastasis and treatment of tumors have always been hot spots and difficult problems in medical r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K7/06A61K38/08A61P35/00
Inventor 李子刚尹丰王冬园
Owner PEKING UNIV SHENZHEN GRADUATE SCHOOL
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