CN116421712A - D-configuration oncolytic peptide-camptothecine conjugate and preparation method and application thereof - Google Patents

D-configuration oncolytic peptide-camptothecine conjugate and preparation method and application thereof Download PDF

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CN116421712A
CN116421712A CN202310317960.XA CN202310317960A CN116421712A CN 116421712 A CN116421712 A CN 116421712A CN 202310317960 A CN202310317960 A CN 202310317960A CN 116421712 A CN116421712 A CN 116421712A
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peptide
oncolytic
yjf
cancer
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齐昀坤
姚景方
王克威
杜姗姗
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Qingdao University
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Abstract

The invention provides a D-type oncolytic peptide-camptothecin conjugate, a preparation method and application thereof, belonging to the technical field of polypeptide preparation and biological medicine, comprising the following steps: synthesizing D-type polypeptide by using 9-fluorenylmethoxycarbonyl-based solid-phase polypeptide synthesis method, then covalently connecting the synthesized D-type polypeptide with a small molecule antitumor drug through a connecting group, and obtaining the D-type oncolytic peptide-camptothecine conjugate through peptide cutting, separation, purification and freeze drying. The D-type oncolytic peptide-camptothecine conjugate can obviously improve the solubility of camptothecine, and has the advantages of high enzymolysis stability, long half-life, stronger inhibition effect on tumor cell proliferation and the like. The invention realizes the space-time monitoring of the release condition of the camptothecine conjugate, thereby having good practical application value.

Description

D-configuration oncolytic peptide-camptothecine conjugate and preparation method and application thereof
Technical Field
The invention belongs to the technical field of polypeptide preparation and biological medicine, and particularly relates to a D-configuration oncolytic peptide-camptothecin conjugate, and a preparation method and application thereof.
Background
The disclosure of this background section is only intended to increase the understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art already known to those of ordinary skill in the art.
Tumors are one of the major factors that endanger human health in today's world. Current traditional methods of treatment for tumors are surgical treatment, radiation therapy and chemotherapy. Traditional anticancer drugs such as Paclitaxel (PTX), camptothecine (CPT) and Doxorubicin (DOX) exhibit remarkable tumor inhibition, but due to their disadvantages of low water solubility, strong systemic toxicity, multi-drug resistance, etc., development of novel effective anticancer drugs with small toxic and side effects is urgently required.
Camptothecin (CPT) is a small molecule anticancer drug, and is isolated from bark and branch of camptotheca acuminata (camptotheca acuminata) produced in China. Camptothecins have strong cytotoxicity and obvious anti-tumor activity on various tumor cell lines. Studies have shown that DNA topoisomerase I (TopoI) is the target of camptothecins, and camptothecins cause DNA fragmentation and cell death by blocking the last step of the reaction of TopoI with DNA, i.e., the recombination of single-or double-stranded DNA at the nick site. However, camptothecins have high cytotoxicity to normal cells, poor water solubility, and weak ability to penetrate cell membranes, which limits the use of camptothecins.
The oncolytic peptide is a bioactive peptide with tumor inhibition effect, and has a plurality of advantages in the aspect of treating tumors compared with other medicines, such as small molecular weight, simple structure, high activity, high selectivity, less side effect, multiple administration modes, difficult drug resistance and the like. The antitumor activity of peptides is related to polypeptide chain length, overall charge/hydrophobicity, helix structure, amino acid composition and sequence, and the like. Oncolytic peptide generally consists of tens to tens of amino acid residues, has positive charges and amphipathy, can inhibit proliferation of tumor cells at different stages of growth and differentiation of the tumor cells by combining with negatively charged tumor cell membranes, achieves specific killing effect on the tumor cells, and finally leads to death of the tumor cells.
LTX-315 (amino acid sequence: H-KWWKKW-Dip-K-NH) modified based on the antibacterial peptide bovine lactoferrin sequence 2 ) Is a cationic oncolytic peptide which shows obvious antitumor activity on tumor cell lines. LTX-315 breaks the mitochondrial and cell membranes causing the cellular contents to flow out and can activate the immune system itself to attack tumor cells. LTX-315 can achieve efficient killing of tumor cells through a dual mechanism.
Frog toxins (Aureins) are a family of polypeptides consisting of 13-25 amino acid residues, wherein a variety of peptides exhibit anti-gram positive and anti-tumor effects, originally isolated from the skin secretions of the australian frog. Experiments prove that the antitumor peptide Aurein1.2 (amino acid sequence H-GLFDIIKKIAESF-NH) with 13 amino acid residues 2 ) Has obvious anti-tumor activity on various conventional tumor cell lines and drug-resistant tumor cell lines, and has low toxicity on normal cells. Aurein1.2 is a cationic amphiphilic polypeptide, and can induce neutral or anionic membrane dissolution through a carpet mechanism, damage tumor cell membranes and mitochondrial membranes, enable cell contents to flow out, excite an immune system to generate a strong immune response, and cause cell death.
The traditional oncolytic peptide is L-configuration polypeptide, consists of L-type amino acid and glycine, can be extracted from the natural world, chemically synthesized or recombinantly expressed, has poor stability, is easy to degrade by protease, has short half-life period, and has potential immunogenicity and the like. The D-configuration polypeptide consists of D-type amino acid and glycine, and is not existed in nature and can only be obtained by chemical total synthesis. Because the D-configuration polypeptide or D-configuration protein cannot be obtained by the existing central rule, the D-configuration polypeptide is not easy to identify by an enzyme system in a human body, and has the advantages of low immunogenicity, good stability, difficult degradation by protease and the like.
The inventors found Aurein1.2 (H-GLFDIIKKIAESF-NH) 2 ) And LTX-315 (H-KWWKKW-Dip-K-NH) 2 ) Is composed of L-type amino acid and glycine, and has the advantages of high anti-tumor activity, wide anti-cancer spectrum, uneasy induction of tumor drug resistance, etc. Aurein1.2 and LTX-315 are readily degraded by proteases, have a short half-life, and are potentially immunogenic. This limits the widespread use of Aurein1.2 and LTX-315.
Disclosure of Invention
Aurein1.2 and LTX-315 have the problems of poor stability, short half-life period, immunogenicity, high normal cytotoxicity, poor water solubility, weak cell membrane penetrating ability and the like. Based on the defects of the prior art, the invention provides a D-configuration oncolytic peptide-camptothecin conjugate, and a preparation method and application thereof. Aiming at the defects that the L-type oncolytic peptide is easy to be degraded, has immunogenicity, poor camptothecine solubility, weak cell membrane penetrating capability and the like, the invention obtains the D-type oncolytic peptide consisting of D-type amino acid residues through a series of structural transformation, and further synthesizes the D-type oncolytic peptide-camptothecine conjugate which is covalently combined by the D-type oncolytic peptide and camptothecine. The D-type oncolytic peptide is not easy to be degraded by amino acid, has higher stability and low immunogenicity. Aiming at the problem of high price of D-isoleucine (D-Ile), the invention uses D-leucine (D-Leu) to replace D-Ile, thereby greatly reducing the preparation cost of oncolytic peptide. The D-type oncolytic peptide-camptothecine conjugate prepared by the experiment is connected with camptothecine through a degradable ester bond, so that the inhibition activity of oncolytic peptide on tumor cells is increased. In addition, the invention utilizes the principle of intramolecular fluorescence self-quenching to monitor the release of camptothecin from time and space levels by introducing a fluorescence quenching group dinitrophenyl (Dnp) into the D-type oncolytic peptide-camptothecin conjugate. The antitumor dose-response curve shows that the solubility of the modified D-type oncolytic peptide-camptothecine conjugate is increased, and the antitumor activity is obviously improved. Meanwhile, an anti-tumor aging curve shows that the D-type oncolytic peptide-camptothecine conjugate greatly increases the stability of oncolytic peptide and prolongs the action time. Therefore, the D-type oncolytic peptide-camptothecine conjugate has good practical application value.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the invention, there is provided a D-type oncolytic peptide-camptothecin conjugate comprising the amino acid residue sequence:
YJF-599H-Glfkllkklaksf-NH 2
YJF-600 CPT-AEEA-Glfkllkklaksf-NH 2
YJF-601(CPT) 2 -k-AEEA-Glfkllkklaksf-NH 2
YJF-602(CPT) 2 -k-k(CPT)-AEEA-Glfkllkklaksf-NH 2
YJF-603(CPT) 2 -k-k(Dnp)-AEEA-Glfkllkklaksf-NH 2
YJF-604CPT-AEEA-kkwwkkw-Dip-kk(Dnp)-NH 2
YJF-605(CPT) 2 -k-AEEA-kkwwkkw-NH 2
the D-type oncolytic peptide-camptothecine conjugate greatly improves the antitumor activity, the stability and the like of the oncolytic peptide, can not be degraded by protease and the like while destroying cell membranes, can further enter cells to release camptothecine, and can simultaneously destroy the cell membranes and mitochondrial membranes and block the recombination of single-stranded or double-stranded DNA at a notch position so as to lead the DNA to be broken, thereby playing the role of antitumor cooperatively. Meanwhile, the spatial-temporal level monitoring of the release condition of the camptothecine is realized by utilizing the intramolecular fluorescence self-quenching effect of the Dnp and the camptothecine.
In a second aspect of the present invention, there is provided a method for preparing the above-mentioned D-type oncolytic peptide-camptothecin conjugate, which comprises synthesizing a polypeptide by a solid-phase polypeptide synthesis method, and connecting the polypeptide with a linking group and a small molecule drug group by condensation reaction.
Specifically, the above oncolytic peptide was synthesized by solid-phase polypeptide synthesis (Fmoc-SPPS) based on 9-fluorenylmethoxycarbonyl.
In a third aspect of the invention, there is provided the use of an oncolytic peptide-camptothecin conjugate as described above in the preparation of an anti-cancer or anti-tumor drug.
Preferably, the cancer or tumor may be selected from breast cancer, nasopharyngeal cancer, thymus cancer, bladder cancer, bronchogenic cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, gangliocytoblastoma, gastric cancer, gangliocytoneuroma, papillary thyroid cancer, squamous cell carcinoma of the head and neck, testicular cancer.
In a fourth aspect of the invention there is provided the use of a drug release profile monitor for a D-type oncolytic peptide-camptothecin conjugate as described above. The oncolytic peptide-camptothecine conjugate used by the Dnp and camptothecine can emit specific blue fluorescence after camptothecine is released, so that space-time level monitoring of camptothecine release conditions is realized, and the oncolytic peptide-camptothecine conjugate can be applied to cell, tissue and living animal imaging, and further explores antitumor activity and action mechanism of the D-oncolytic peptide-camptothecine conjugate at molecular, cell, tissue and animal levels.
The beneficial technical effects of the technical scheme are as follows:
1. the above technical scheme can greatly improve the anti-tumor activity of the oncolytic peptide. The D-type oncolytic peptide-camptothecine conjugate synthesized by the invention improves the inhibition activity of oncolytic peptide on tumor cells. Wherein the IC of each peptide 50 (24h) The following are provided: in Jurkat cells, 14.4.+ -. 2.7. Mu.M (YJF-599), 10.9.+ -. 2.3. Mu.M (YJF-600), 17.9.+ -. 1.2. Mu.M (YJF-601), 10.4.+ -. 2.1. Mu.M (YJF-605), 19.3.+ -. 2.2. Mu.M (YJF-599), 4.5.+ -. 1.8. Mu.M (YJF-600), 7.9.+ -. 2.4. Mu.M (YJF-601), 18.3.+ -. 3.9. Mu.M (YJF-602), 7.9.+ -. 3.3. Mu.M (YJF-605), 44.5.+ -. 5.5. Mu.M (YJF-599) in PC-3 cells, 21.7+ -1.2 μM (YJF-600), 26.1+ -1.1 μM (YJF-601), 38.6+ -5.5 μM (YJF-602), 48.9+ -1.1 μM (YJF-605), and finally, in Hela-S3 cells, 76.1+ -5.6 μM (YJF-599), 11.8+ -2.8 μM (YJF-600), 14.7+ -3.2 μM (YJF-601), 10.8+ -2.2 μM (YJF-605), which are significantly improved over the protooncolytic peptides LTX-315 and Aurein 1.2.
2. The technical scheme can greatly improve the stability of the oncolytic peptide. Aurein1.2, LTX-315 and the like developed by the former are oncolytic peptides composed of L-type amino acids, are easy to degrade by protease in the body, have insufficient stability and short half-life, and have high immunogenicity and are easy to be repelled by the body. The project uses more stable D-type amino acid to synthesize novel D-type oncolytic peptide. The D-type oncolytic peptide has high enzymolysis stability, is more stable than the traditional L-type oncolytic peptide, can obviously prolong the anti-tumor action time and improve the anti-tumor activity, reduces the immunogenicity and has larger application value.
3. The technical scheme can greatly reduce the synthesis cost of the oncolytic peptide. The Aurein1.2 peptide sequence developed before contains three isoleucine residues, the D-type isoleucine (D-Ile) is expensive, and the synthesis cost of the D-configuration oncolytic peptide is high. The project replaces D-Ile with D-leucine (D-Leu), so that the synthesis cost of the polypeptide is greatly reduced, and the antitumor activity of the polypeptide is not influenced.
4. According to the technical scheme, the release and distribution space-time monitoring of the small-molecule antitumor drug is realized through intramolecular self-quenching. The Dnp is used together with the camptothecine to enable the camptothecine to emit blue fluorescence after being released, so that the time and space level monitoring of the camptothecine release condition is realized. Can be applied to imaging cells, tissues and living animals, and further explores the antitumor activity and action mechanism of the oncolytic peptide-camptothecin conjugate at the molecular, cell, tissue and animal levels.
5. The technical proposal can greatly reduce the toxicity of the camptothecine to normal cells and enhance the solubility of the camptothecine. By covalently linking camptothecin to a polypeptide, the water solubility of the camptothecin is improved by more than 6 times (on a molar basis).
In conclusion, the synthetic cost of the oncolytic peptide Aurein1.2 and LTX-315 developed by the former is high, the enzymolysis stability is poor, the half-life period is short, the immunogenicity is high, and the antitumor activity is relatively low. In order to avoid the defects, the novel D-type oncolytic peptide-camptothecin conjugate is designed and synthesized, and has the advantages of high enzymolysis stability, long half-life period, lower immunogenicity, stronger activity and the like. Meanwhile, the Dnp and the camptothecine are used together to enable the camptothecine to emit blue fluorescence after being released, so that the time and space level monitoring of the camptothecine release condition is realized, and the camptothecine has a good application prospect.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is a diagram showing a comparison of the characteristics of L-type and D-type polypeptides;
FIG. 2 is a schematic diagram of a method for synthesizing a solid-phase polypeptide according to the present invention;
FIG. 3 is a chemical structural formula, a primary amino acid sequence, an analytical reverse phase high performance liquid chromatogram and a mass spectrum of YJF-599 of the invention;
FIG. 4 shows the chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatography and mass spectrum of YJF-600 of the invention;
FIG. 5 shows the chemical structural formula, the primary amino acid sequence, the analytical reverse phase high performance liquid chromatography and the mass spectrum of YJF-601 of the invention;
FIG. 6 is a chemical structural formula, a primary amino acid sequence, an analytical reverse phase high performance liquid chromatogram and a mass spectrum of YJF-602 of the invention;
FIG. 7 shows the chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatography and mass spectrum of YJF-603 of the invention;
FIG. 8 shows the chemical structural formula, primary amino acid sequence, analytical reverse phase high performance liquid chromatography and mass spectrum of YJF-604 of the present invention;
FIG. 9 is a chemical structural formula, a primary amino acid sequence, an analytical reverse phase high performance liquid chromatogram and a mass spectrum of YJF-605 of the invention;
FIG. 10 is a schematic diagram of intramolecular self-quenching monitoring of camptothecin release;
FIG. 11 is a graph showing the evaluation of cell level of D-type oncolytic peptide-camptothecin conjugates and IC for combination use in accordance with the present invention 50 ；
FIG. 12 is a graph of the cellular level evaluation of the inhibition of tumor cell proliferation by the D-type oncolytic peptide-camptothecin conjugates of the present invention;
FIG. 13 is a graph showing the inhibition of tumor cell proliferation by a combination of the D-type oncolytic peptide-camptothecin conjugates of the present invention at the cellular level;
FIG. 14 is a graph showing the aging profile of the D-type oncolytic peptide-camptothecin conjugates of the present invention inhibiting tumor cell proliferation.
FIG. 15 is a graph showing the monitoring of the stability of the L-type and D-type oncolytic peptides of the present invention in serum.
FIG. 16 shows the use of the principle of intramolecular self-quenching to monitor the release of camptothecin in accordance with the present invention.
Detailed Description
The following detailed description of specific embodiments of the invention refers to the accompanying drawings and detailed description. In particular embodiments, each of the original reagents and starting materials are commercially available. It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As described above, aurein1.2 and LTX-315 are composed of L-type amino acids, and have the disadvantages of low enzymolysis stability, short half-life, high immunogenicity, high cost and the like, thus limiting the wide application thereof.
In view of this, in one exemplary embodiment of the present invention, there is provided a D-type oncolytic peptide-camptothecin drug conjugate comprising the amino acid residue sequence:
YJF-599H-Glfkllkklaksf-NH 2
YJF-600 CPT-AEEA-Glfkllkklaksf-NH 2
YJF-601(CPT) 2 -k-AEEA-Glfkllkklaksf-NH 2
YJF-602(CPT) 2 -k-k(CPT)-AEEA-Glfkllkklaksf-NH 2
YJF-603(CPT) 2 -k-k(Dnp)-AEEA-Glfkllkklaksf-NH 2
YJF-604CPT-AEEA-kkwwkkw-Dip-kk(Dnp)-NH 2
YJF-605(CPT) 2 -k-AEEA-kkwwkkw-NH 2
the oncolytic peptide-drug conjugate can greatly improve the enzymolysis stability of oncolytic peptide, reduce the synthesis cost and improve the antitumor activity; meanwhile, through the joint use of Dnp and camptothecine, the camptothecine emits blue fluorescence after being released, so that the time and space level monitoring of the camptothecine release condition is realized.
In yet another embodiment of the present invention, there is provided a method for synthesizing the above-described D-type oncolytic peptide-camptothecin conjugate, the method comprising: synthesizing polypeptide by adopting a solid-phase polypeptide synthesis method; and connecting the polypeptide with a connecting group and a small molecule drug group through condensation reaction.
Specifically, the above polypeptide was synthesized by solid-phase polypeptide synthesis (Fmoc-SPPS) based on 9-fluorenylmethoxycarbonyl.
According to the invention, rink Amide Am resin (substitution degree is 0.38 mmol/g) is selected to synthesize polypeptide containing an Amide terminal, if not specified; the amino acids used in the synthesis process are Fmoc-D-type amino acids except glycine (Gly, G).
More specifically, the method for synthesizing the D-type oncolytic peptide-camptothecin conjugate comprises the following steps:
step 1, synthesizing D-type polypeptide by a solid-phase polypeptide synthesis method based on 9-fluorenylmethoxycarbonyl;
step 2, connecting a connecting group with the synthesized polypeptide through condensation reaction, and connecting a small molecule drug group with the synthesized polypeptide;
step 3, adding a peptide-cutting reagent into the condensation product, and cutting the condensation product from the resin;
and 4, separating, purifying and freeze-drying the product after peptide cutting to obtain the D-type oncolytic peptide-camptothecin conjugate.
The specific experimental method of the step 1 is as follows:
weighing Rink-Amide AM resin, pre-activating and activating the resin, removing Fmoc protecting groups, performing amino acid condensation according to an amino acid sequence, cleaning after all amino acid condensation is completed, and cleaning again after the last protecting group is removed.
In yet another embodiment of the present invention, the above-mentioned pre-activation and activation experimental methods are respectively: the washing was performed alternately with DMF and DCM, and the washing was performed with DMF at room temperature for 1-2 h. The resin was soaked with a DMF/DCM mixture (1:1, volume ratio, v: v) and reacted at 28℃for 0.5h in a thermostatted shaker.
The experimental method for removing Fmoc protecting groups comprises the following steps: the protecting group was removed at 28℃using a 20% piperidine in DMF (v: v) for 5 minutes and 10 minutes, respectively.
The experimental method of the condensation reaction comprises the following steps: each amino acid was condensed twice at 28 ℃ for 30 min and 40 min, respectively, and the ratio of the reactants was Fmoc-D-amino acid: HCTU: DIPEA = 3-fold equivalent: 2.8 times equivalent: 6 times the equivalent.
The cleaning experiment method comprises the following steps: the residual solvent was then pumped with water after alternating washes with DMF and DCM.
The specific experimental method in the step 2 is as follows:
condensing the D-type oncolytic peptide synthesized in the step 1 with an AEEA connecting group, and then condensing with a micromolecular drug group, wherein the proportion of reactants of YJF-600 is camptothecine: HATU: HOAT: DIEA = 2-fold equivalent: 1.8 times equivalent: 2 times equivalent: 4 times equivalent (molar ratio), the reactant ratio of YJF-601 is camptothecine: HATU: HOAT: DIEA = 3-fold equivalent: 2.8 times equivalent: 3 times equivalent: 6 times equivalent (molar ratio), the reactant ratio of YJF-602 is camptothecin: HATU: HOAT: DIEA = 4-fold equivalent: 3.6 times equivalent: 4-fold equivalent: 8 times equivalent (molar ratio), the reactant ratio of YJF-603 is camptothecine: HATU: HOAT: DIEA = 3-fold equivalent: 2.8 times equivalent: 3 times equivalent: 6 times equivalent (molar ratio).
The specific experimental method in the step 3 is as follows:
to the prepared condensation product was added a peptide-cleaving reagent in the ratio TFA: phenol: water: tips=88:5:5:2 (v: v: v). Placing the mixture in a shaking table at 28 ℃ to react for 1.5-2 h in a dark place, washing resin for 2 times by using 0.5mL of TFA, concentrating the reaction product to 3mL by using high-purity nitrogen, adding pre-cooled anhydrous diethyl ether into the concentrated solution, and precipitating target polypeptide. Centrifuging to obtain crude peptide, adding anhydrous diethyl ether, and centrifuging repeatedly for three times. The product was dried in a fume hood.
The specific experimental method in the step 4 is as follows:
the crude peptide product obtained in step 3 was dissolved using a mixed solution of acetonitrile and water containing 1% TFA, and the obtained crude peptide was then analyzed and identified using analytical reverse-phase high performance liquid chromatography and ESI-MS. The dissolved crude peptide was placed at-80℃for further use. Freeze-drying to obtain flocculent crude peptide. The crude peptide was dissolved by a mixed solution of acetonitrile and water containing 1% TFA, and was separated and purified by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC), and the collected pure peptide solution was allowed to stand at-80℃overnight, followed by freeze-drying to give the target pure peptide.
In yet another embodiment of the present invention, there is provided the use of the above-described oncolytic peptide-camptothecin conjugates in the preparation of an anticancer or antitumor drug.
In yet another embodiment of the invention, the invention can treat solid tumors and hematological tumors. Wherein the solid tumor can be breast cancer, nasopharyngeal carcinoma, thymus cancer, bladder cancer, bronchogenic carcinoma, non-small cell lung cancer, prostatic cancer, ovarian cancer, neuroblastoma, gangliocytoma, gastric cancer, gangliocytoma, papillary thyroid cancer, head and neck squamous cell carcinoma, testicular cancer, and liver cancer. The hematological neoplasm may be acute myelogenous leukemia, acute lymphoblastic leukemia, granulocytic leukemia, and malignant lymphoma.
In yet another embodiment of the present invention, there is provided the use of the above-described D-type oncolytic peptide-camptothecin conjugates as a drug release profile monitor. The oncolytic peptide-drug conjugate used by the Dnp and the camptothecine can emit blue fluorescence after camptothecine is released, so that the space-time level monitoring of camptothecine release condition is realized, and the oncolytic peptide-drug conjugate can be applied to cell, tissue and living animal imaging, and further explore the antitumor activity and action mechanism of the oncolytic peptide-drug conjugate at the molecular, cell, tissue and animal level.
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1
In this example all polypeptides of interest were prepared using solid-phase polypeptide synthesis technology (Fmoc-SPPS) based on 9-fluorenylmethoxycarbonyl. According to the invention, rink Amide Am resin (substitution degree is 0.38 mmol/g) is selected to synthesize polypeptide containing an Amide terminal, if not specified; the amino acids used in the synthesis process except glycine (Gly, G) are achiral amino acids, and the rest amino acids are Fmoc-D-amino acids.
The scale of the synthesized polypeptide is generally 0.15mmol, and the basic flow of polypeptide synthesis is shown in FIG. 1.
Solid-phase polypeptide synthesis experiments: 400mg of Rink Amide AM resin (1-fold equivalent) was weighed out, alternately washed with DMF and DCM, and pre-activated by soaking in DMF for 1-2 h at room temperature. After the alternate washing again, the resin was soaked with a DMF/DCM mixture (1:1, volume ratio, v: v) and shaken in a constant temperature shaker at 28℃for 0.5h to activate the resin. Fmoc protecting groups were then removed using 20% piperidine in DMF (v: v) for 5 minutes first and 10 minutes second. Each amino acid was condensed twice at 28 ℃ for 30 min and 40 min, respectively, and the ratio of the reactants was Fmoc-D-amino acid: HCTU: DIPEA = 3-fold equivalent: 2.8 times equivalent: 6 times the equivalent. After the final amino acid has been condensed, its Fmoc protecting group is removed and alternately washed with DMF and DCM.
Synthesis of D-oncolytic peptide-camptothecin conjugates: the synthetic oncolytic peptide and the connecting group are subjected to condensation reaction through an amide bond, and then the connecting group and the micromolecular drug group are subjected to condensation through the amide bond. The proportion of the reactants of YJF-600 is camptothecine: HATU: HOAT: DIEA = 2-fold equivalent: 1.8 times equivalent: 2 times equivalent: 4 times equivalent (molar ratio), the reactant ratio of YJF-601 is camptothecine: HATU: HOAT: DIEA = 3-fold equivalent: 2.8 times equivalent: 3 times equivalent: 6 times equivalent (molar ratio), the reactant ratio of YJF-602 is camptothecin: HATU: HOAT: DIEA = 4-fold equivalent: 3.6 times equivalent: 4-fold equivalent: 8 times equivalent (molar ratio), the reactant ratio of YJF-603 is camptothecine: HATU: HOAT: DIEA = 3-fold equivalent: 2.8 times equivalent: 3 times equivalent: 6 times equivalent (molar ratio).
Peptide cutting and separation and purification: to the prepared condensation product was added a peptide-cleaving reagent in the ratio TFA: phenol: water: tips=88:5:5:2 (v: v: v). Placing the mixture in a shaking table for reaction for 1.5-2 h at 28 ℃, washing the resin for 2 times by using 0.5mL of TFA, concentrating the reaction product to 3mL by using high-purity nitrogen, adding pre-cooled anhydrous diethyl ether into the concentrated solution, and precipitating the target polypeptide. The crude peptide was obtained by centrifugation and repeated three times with the addition of anhydrous diethyl ether. The product was dried in a fume hood. The crude peptide product obtained in step 3 was then dissolved using a mixed solution of acetonitrile and water containing 1% TFA, and the resulting crude peptide was then analyzed and identified using analytical reverse-phase high performance liquid chromatography and ESI-MS. The dissolved crude peptide was placed at-80℃for further use. Freeze-drying to obtain flocculent crude peptide. Dissolving the prepared crude peptide with a mixed solution of acetonitrile and water containing 1%o TFA, separating and purifying by semi-preparative reversed phase high performance liquid chromatography (RP-HPLC), standing the collected pure peptide solution at-80 ℃ overnight, and freeze-drying to obtain the target pure peptide.
The chemical structural formula, the primary amino acid sequence, the analytical reverse phase high performance liquid chromatography and the ESI-MS mass spectrum of the 7D-type oncolytic peptide-camptothecine conjugates prepared by the invention are shown in figures 3-9.
CCK-8 assay for tumor cell proliferation inhibition assay:
a20, jurkat, hela S3 and PC-3 cells in the logarithmic growth phase were selected, and cells were collected and inoculated in 96-well plates after cell counting. After seeding the cells, the outer walls were sterilized and the cells were placed in an incubator for 24 hours, 50. Mu.L of YJF-599, YJF-600, YJF-601, YJF-602, YJF-605, CPT, LTX-315 and Aurein1.2 were added at final concentrations (100. Mu.M, 50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M) and 3 multiplex wells were placed in each group. The cells were continuously cultured in the incubator for 24 hours. After 24h, CCK-8 (final concentration 5. Mu. Mol.L) was isolated from light -1 ) Added into each well, gently mixed, placed into an incubator for incubation for 4 hours, and the culture is terminated. And measuring the absorbance value under the condition of 450nm wavelength, repeating the measurement for three times, recording the result, and carrying out subsequent calculation according to a formula.
As shown in fig. 12, the novel D-type oncolytic peptide-camptothecin conjugates can kill a20, jurkat, hela S3, and PC-3 tumor cells in a concentration-dependent manner. IC of novel D-type oncolytic peptide-camptothecine conjugate 50 The value is greatly improved compared with Aurein1.2 and LTX-315, and the anti-tumor activity is obviously improved.
D-oncolytic peptide-camptothecin combinations inhibit tumor cell proliferation:
a20, jurkat, hela S3 and PC-3 cells in the logarithmic growth phase were selected, and cells were collected and inoculated in 96-well plates after cell counting. After inoculating the cells, the outer wall was sterilized and the cells were placed in an incubator for 24 hours, three drug groups were set, the concentration ratio of the first group YJF-599 to CPT was 1:1, a second set of fixed YJF-599 final concentrations of 12.5. Mu.M, CPT solution (50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M) was added, a third set of fixed CPT final concentrations of 12.5. Mu.M, YJF-599 solution (50. Mu.M, 25. Mu.M, 12.5. Mu.M, 6.25. Mu.M, 3.125. Mu.M) was added, and three multiplex wells were placed in each set for continuous culture of cells in an incubator for 24h. After 24h, CCK-8 (final concentration 5. Mu. Mol.L) -1 ) Added into each well, gently mixed, placed into an incubator for incubation for 4 hours, and the culture is terminated. And measuring the absorbance value under the condition of 450nm wavelength, repeating the measurement for three times, recording the result, and carrying out subsequent calculation according to a formula.
As shown in FIG. 13, the inhibition effect of CPT (i.e., YJF-600) on tumor cells by covalently-linked YJF-599 is stronger than that of the non-covalently-linked combination of the two.
Aging assay of D-oncolytic peptide-camptothecin conjugates to inhibit tumor cell proliferation:
a20 cells in the logarithmic growth phase were selected, and cells were collected and inoculated in 96-well plates (100. Mu.l per well) after cell counting. mu.L of YJF-599, YJF-600, YJF-601, YJF-602, YJF-605, CPT, LTX-315 and Aurein1.2 were added at a final concentration of (10. Mu.M), and 3 multiplex wells were set per group. The cells were further cultured in the incubator for 4h, 12h, 24h,36h,48h,72h, respectively. CCK-8 (final concentration 5. Mu. Mol.L) was isolated from light after the specified time had been reached -1 ) Added into each well, gently mixed, placed into an incubator for incubation for 4 hours, and the culture is terminated. Absorbance values were measured at a wavelength of 450nm and the measurements were repeated three times.
As a result, as shown in FIG. 14, the inhibition rate of the 12 hD-type oncolytic peptide-camptothecin conjugate at 10. Mu.M on A20 cells was 65%, while the inhibition rate of LTX-315 and Aurein1.2 was about 25% -30%, which suggests that the antitumor activity of the D-type polypeptide was higher. The type D oncolytic peptide-drug conjugates peaked at 36-48 hours. LTX-315 and Aurein1.2 reached peak values at 12h and 36h (inhibition rates of 30% and 35%, respectively), followed by a start of activity decrease with only 20% inhibition at 72h. The D-type oncolytic peptide-camptothecine conjugate can exist for a long time and play an anti-tumor role. The above results demonstrate that the D-type oncolytic peptide-camptothecin conjugates have better antitumor activity and stability.
Serum stability experiments:
dissolving serum in PBS to obtain 5% serum solution, and weighing appropriate amounts of Aurein1.2, LTX-315, YJF-600-1 (H-AEEA-Glfkllklaksf-NH) 2 ) And YJF-605-1 (H-AEEA-kkwkkwkkkW-NH) 2 ) After the polypeptide solid is dissolved by adding water, the prepared serum solution is added into the polypeptide solution to prepare a mixed solution with the final concentration of 200 mu M, and the mixed solution is stirred for 3 times by shaking up and down. 80. Mu.l of the reaction mixture was immediately taken, 20. Mu.l of acetonitrile containing 0.1% TFA was added, and the mixture was shaken three times, allowed to stand on ice for 2 minutes, and then 60. Mu.l of acetonitrile and 400. Mu.l of deionized water each containing 0.1% TFA were added, and the mixture was shaken three times, immediately set at-80℃for storage. The reaction tube was sealed and placed in a constant temperature water bath at 37℃for 72h, and samples were taken as above at 0.5h,1h,2h,4h,8h,16h,24h,36h,48h,72h, respectively.
The results are shown in FIG. 15, where the remaining percentage of Aurein1.2 and LTX-315 polypeptides gradually decreased with increasing time and were completely hydrolyzed at 48 h. Whereas the YJF-600-1 and YJF-605-1 conjugates were not easily hydrolyzed, the remaining percentages were 80% and 70% respectively at 72h. The above results demonstrate that the D-type oncolytic peptide is more stable than the L-type oncolytic peptide.
Real-time monitoring of camptothecin release experiments using intramolecular self-quenching principle:
dissolving serum in PBS to prepare 5% serum solution, weighing a proper amount of YJF-604 and camptothecine solid, adding water to dissolve, adding the prepared serum solution into the solution to prepare a mixed solution with the final concentration of 200 mu M, and shaking up and down for 3 times. 80. Mu.l of the reaction mixture was immediately taken, 20. Mu.l of acetonitrile containing 0.1% TFA was added, and the mixture was shaken three times, allowed to stand on ice for 2 minutes, and then 60. Mu.l of acetonitrile and 400. Mu.l of deionized water each containing 0.1% TFA were added, and the mixture was shaken three times, immediately set at-80℃for storage. The reaction tube was sealed and placed in a constant temperature water bath at 37℃for 72h, and samples were taken at 0.5h,1h,2h,4h,8h,16h,24h,36h,48h,72h, respectively. The release of camptothecins was monitored in real time by RP-HPLC and fluorescence spectrophotometry, respectively.
As a result, as shown in FIG. 16, the RP-HPLC and the spectrophotometer test showed that the hydrolysis peak of YJF-604 was significantly enhanced with the increase in fluorescence intensity of YJF-604 and reached almost the same fluorescence intensity as that of camptothecin monomer at 72 hours. The above results demonstrate that the incorporation of DNP in D-configuration oncolytic peptide-camptothecin conjugates allows for monitoring of camptothecin release at a spatiotemporal level.
Finally, it should be noted that the foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the invention, and although the present invention has been described in detail with reference to the foregoing embodiment, it will be apparent to those skilled in the art that modifications may be made to the technical solution described in the foregoing embodiment, or equivalents may be substituted for portions thereof. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention. While the foregoing describes the embodiments of the present invention, it should be understood that the present invention is not limited to the embodiments, and that various modifications and changes can be made by those skilled in the art without any inventive effort.

Claims (5)

1. A class of D-type oncolytic peptide-drug conjugates, wherein the oncolytic peptide-drug conjugates comprise the amino acid residue sequence:
YJF-599H-Glfkllkklaksf-NH 2
YJF-600CPT-AEEA-Glfkllkklaksf-NH 2
YJF-601(CPT) 2 -k-AEEA-Glfkllkklaksf-NH 2
YJF-602(CPT) 2 -k-k(CPT)-AEEA-Glfkllkklaksf-NH 2
YJF-603(CPT) 2 -k-k(Dnp)-AEEA-Glfkllkklaksf-NH 2
YJF-604CPT-AEEA-kkwwkkw-Dip-kk(Dnp)-NH 2
YJF-605(CPT) 2 -k-AEEA-kkwwkkw-NH 2
2. the method for preparing the D-type oncolytic peptide-camptothecine conjugate according to claim 1, which is characterized in that the preparation method comprises the steps of adopting a solid-phase polypeptide synthesis method to synthesize polypeptide, and carrying out condensation reaction connection on the polypeptide, a connecting group and a small molecule drug group;
preferably, the polypeptide synthesis method is a solid-phase polypeptide synthesis method (Fmoc-SPPS) based on 9-fluorenylmethoxycarbonyl;
preferably, the synthetic method of the oncolytic peptide-drug conjugate is as follows:
the solid-phase polypeptide synthesis method based on 9-fluorenylmethoxycarbonyl is used for synthesizing polypeptide, then the synthesized polypeptide is connected with a connecting group and a small molecule drug group through condensation reaction, a peptide cutting reagent is added into the condensation product, the condensation product is cut off from resin, and the product after peptide cutting is identified, separated and purified to prepare the D-type oncolytic peptide-drug conjugate.
3. Use of the D-type oncolytic peptide-camptothecin conjugate according to claim 1 for the preparation of an anticancer or antitumor drug.
Preferably, the tumor is selected from the group consisting of breast cancer, nasopharyngeal cancer, thymus cancer, bladder cancer, bronchogenic cancer, non-small cell lung cancer, prostate cancer, ovarian cancer, neuroblastoma, gangliocytoma, gastric cancer, gangliocytoma, papillary thyroid cancer, head and neck squamous carcinoma, testicular cancer, liver cancer, acute myelogenous leukemia, acute lymphoblastic leukemia, granulocytic leukemia, and malignant lymphoma.
4. A pharmaceutical composition comprising the D-type oncolytic peptide-camptothecin conjugate of claim 1.
5. Use of the D-type oncolytic peptide-camptothecin conjugate according to claim 1 as a drug release profile monitor.
CN202310317960.XA 2023-03-29 2023-03-29 D-configuration oncolytic peptide-camptothecine conjugate and preparation method and application thereof Pending CN116421712A (en)

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