A kind of stable polypeptide inhibitor targeting hdac and use thereof

A technology of polypeptides and inhibitors, applied in the field of bioengineering, can solve problems such as large toxic side effects and poor effects, and achieve the effects of inhibiting proliferation, solving toxic and side effects, and improving toxic and side effects.

Active Publication Date: 2022-07-05
PEKING UNIV SHENZHEN GRADUATE SCHOOL
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • A kind of stable polypeptide inhibitor targeting hdac and use thereof
  • A kind of stable polypeptide inhibitor targeting hdac and use thereof
  • A kind of stable polypeptide inhibitor targeting hdac and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] Example 1: Design of Polypeptide Inhibitors

[0042] The present invention adopts the way of polypeptide-small molecule covalent coupling to design HDAC polypeptide inhibitor, such as figure 1 As shown, we selected a polypeptide sequence (TVALREIRRYQK(Ac)) near the H3K56 (the 56th amino acid lysine side chain amino group of histone H3 was modified by acetylation), a selective substrate of HDAC1, because K56 of this polypeptide is in a segment of The C-terminus of the helix is ​​suitable for us to use the "N-terminal aspartate 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 substrate polypeptides to HDAC, we mutated the amino acid of K56 into a hydroxamic acid structure, as in figure 1 A shown.

[0043] It has been reported that this structure can chelate with the zinc ion in the catalytic region of HDAC enzyme to inhibit the activity of HDAC enzyme. The bas...

Embodiment 2

[0046] Example 2: Synthetic Preparation of Polypeptide Inhibitors

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

[0048] Specific steps:

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

[0050] (2) Solid-phase synthesis of peptides: Weigh 100 mg of 2-Chlorotity-N-Fmoc-hydroxylamine resin into a 10 ml peptide tube, add dichloromethane (DCM), and swell with nitrogen for 10 min. 50% (v / v) morpholine in N,N-dimethylformamide (DMF) solution was added, and nitrogen was bubbled for 30min×2 to remove the Fmoc protecting group. After alternately washing the resin 6 times 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, DMF) solution, N,N-dii...

Embodiment 3

[0055] Example 3: Identification of Polypeptide Secondary Structure and Research on Inhibitory Effect on HDAC Enzyme Activity

[0056] For each peptide, circular dichroism was used to determine the conformation in water, demonstrating that the peptides stabilized in water by the method of terminal side chain-tail linking chiral diacid-modified polypeptides (e.g. Figure 5 A) and in 20% TFE solution (as Figure 5 B) has a stable α-helical conformation, in which TFE (trifluoroethanol) has the effect of promoting the helix. To identify the inhibitory effect of HDAC enzyme activity, use a commercially purchased HDAC enzyme activity detection kit for operation, and each operation is repeated at least three times. The enzyme activity results are as follows: Image 6 B (design of polypeptide inhibitory library and screening of the enzymatic activity inhibitory effect of HeLa nuclear extract) and Image 6 C( Image 6 C: The inhibitory effect of different polypeptide inhibitors on t...

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Abstract

The present invention provides a stable polypeptide inhibitor targeting HDAC, the amino acid sequence structure of which is as follows, and the present invention also provides the above-mentioned stable polypeptide inhibitor targeting HDAC in preparation for inhibiting the enzymatic activity of the HDAC family use in medicines. The present invention also provides the use of the above-mentioned stable polypeptide inhibitor targeting HDAC in preparing a medicine for inhibiting tumor stem cells with high expression of HDAC1. The present invention also provides the use of the above-mentioned stable polypeptide inhibitor targeting HDAC in the preparation of a medicament for treating human ovarian teratoma or testicular embryonic carcinoma. Cell proliferation, apoptosis and cell cycle arrest experiments prove that the polypeptide of the present invention can effectively inhibit the proliferation of tumor stem cells, and the polypeptide has no obvious tissue damage or toxicity to mice at high doses.

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 use thereof. Background technique [0002] Cancer is a major public health problem worldwide, and in China, it is the leading cause of death and a serious disease burden. According to the 2014 National Center Cancer Registry (NCCRC) cancer statistics in China, there are currently 3,804,000 cases of cancer diagnosed in China each year, and the cancer incidence rate is about 278.07 / 100,000 (male incidence rate 301.67 / 100,000, female incidence rate 253.29 / 100,000). Tumor types and causes of tumor pathogenesis vary by gender and age, and the complexity of tumor disease poses challenges for humans to overcome this disease. The occurrence, development, invasion, metastasis and treatment of tumors have always been hot spots and difficult problems in medical research. The tumor stem cell theory believes that ...

Claims

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Application Information

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