Cell apoptosis inducer based on death receptor signaling pathway activation and preparation method thereof

Abstract

The invention relates to the field of biological medicine, and discloses a cell apoptosis inducer based on death receptor signaling pathway activation and a preparation method thereof. The inducer takes polypeptide molecules as an active component, activates a Caspase cascade reaction to induce apoptosis through specific binding with a death receptor, and has low toxicity to non-recipient cells. The core technology comprises the following steps: 1, polypeptide optimization design: through 1-5 amino acid substitution, chemical modification or fusion protein strategy, stability and targeting are improved; 2, preparing the polypeptide by a solid-phase synthesis or gene recombination technology, and combining purification identification processes such as HPLC (High Performance Liquid Chromatography) and mass spectrum; and 3, the preparation adopts a nano delivery system or sustained release microspheres, so that the in-vivo delivery efficiency and half-life period are enhanced. The problems of poor polypeptide stability, low delivery efficiency and insufficient safety in the prior art are solved, the polypeptide has the advantages that the tumor affinity is improved by 10 times, the half-life period is prolonged to 72 hours, the off-target toxicity is remarkably reduced, and the polypeptide is suitable for the fields of tumor treatment and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and specifically relates to an apoptosis inducer based on activation of the death receptor signaling pathway and its preparation method. Background Technology

[0002] Apoptosis is a programmed cell death process that plays a crucial role in maintaining normal development, tissue homeostasis, and disease resistance. The death receptor signaling pathway is one of the important ways to induce apoptosis.

[0003] Apoptosis is a crucial mechanism of programmed cell death, playing a key role in maintaining tissue homeostasis and inhibiting tumor growth. Death receptors (DRs) are a class of transmembrane proteins on the cell membrane surface, mainly including Fas (CD95), DR4 (TRAIL-R1), and DR5 (TRAIL-R2). By binding to ligands (such as FasL or TRAIL), they activate downstream caspase-8 / 10, initiating a caspase cascade reaction and ultimately leading to apoptosis. However, natural ligands suffer from insufficient specificity, short in vivo half-life, and a tendency to induce systemic toxicity. Furthermore, tumor cells often evade apoptosis signals by downregulating death receptor expression or through mutations, limiting the effectiveness of traditional therapies. Therefore, there is an urgent need to develop a stable, specific, and easily prepared apoptosis-inducing agent based on the activation of the death receptor signaling pathway.

[0004] The shortcomings of existing technologies are mainly reflected in the following aspects: (1) peptide activators are easily degraded by proteases and have poor in vivo stability; (2) the delivery efficiency is low and it is difficult to effectively reach target cells; (3) there is potential toxicity to non-recipient cells and the safety needs to be improved. Therefore, there is an urgent need to develop a cell apoptosis inducer with optimized structure, stable activity and strong targeting. Summary of the Invention

[0005] This invention proposes an apoptosis inducer based on activation of the death receptor signaling pathway and its preparation method, which solves the problems in the prior art.

[0006] The technical solution of the present invention is as follows: an apoptosis inducer based on the activation of the death receptor signaling pathway, the active ingredient of which is a polypeptide molecule that can specifically bind to the death receptor and activate the downstream Caspase cascade reaction to induce apoptosis, and has no significant toxicity to non-receptor cells or cells with low expression of death receptor.

[0007] In a preferred embodiment, the polypeptide molecule is: Optimized variants of the wild-type sequence, containing 1-5 amino acid substitutions, insertions, or deletions, while maintaining binding activity with the death receptor; Chemically modified peptides, including PEGylation, acetylation, palmitoylation, or glycosylation, are used to improve stability or prolong half-life. Fusion proteins coupled with carrier proteins enhance in vivo delivery rates.

[0008] In a preferred embodiment, the death receptor is Fas (CD95) or DR4 (TRAIL-R1) with a binding affinity KD value ≤ 10 nM.

[0009] A method for preparing an apoptosis inducer includes the following steps: S1. Polypeptide synthesis: Polypeptides are synthesized according to a predetermined sequence using solid-phase synthesis or gene recombination technology. S2. Purification and identification: Separation and purification are performed by HPLC, ion exchange chromatography or gel electrophoresis, and the structure and purity are confirmed by mass spectrometry and circular dichroism spectroscopy. S3. Formulation preparation: Mix the peptide with pharmaceutical excipients to prepare injections, nanodelivery systems, oral microspheres, or sustained-release formulations.

[0010] In a preferred embodiment, the solid-phase synthesis step includes: S11. Use royal jelly resin or Rink amide resin as a carrier; S12, when coupling and protecting amino acids, use the HBTU / DIEA or PyBOP activation system; S13, cleavage and deprotection conditions: treatment with 95% TFA solution for 2 hours to ensure complete release of the peptide.

[0011] In a preferred embodiment, the purification step includes: S21. Reversed-phase HPLC was used with 20% acetonitrile / 80% phosphate buffer as the mobile phase. The purity of the target peak was collected to be ≥95%. The pH of the 20% acetonitrile / 80% phosphate buffer was 7.0. The molecular weight of S22 was confirmed to be 543.21 Da by ESI-MS or MALDI-TOF mass spectrometry.

[0012] In a preferred embodiment, the formulation preparation steps include: S31. Pharmaceutical excipients include stabilizers, buffers, and surfactants, wherein the stabilizer is mannitol, the buffer is phosphate, and the surfactant is Tween 80. S32, the nanodelivery system is prepared by thin film hydration or self-assembly technology, with particle size controlled in the range of 100-200 nm.

[0013] After adopting the above technical solution, the beneficial effects of the present invention are: Through precise sequence optimization and modification, this invention increases the affinity of tumor cells by more than 10 times compared to normal cells, greatly reducing off-target effects and thus reducing side effects.

[0014] By employing PEGylation and a nanodelivery system, the invention's half-life was extended to 72 hours, reducing the frequency of dosing and improving patient adherence. This characteristic allows the drug to maintain an effective concentration in the body for a longer period, enhancing therapeutic efficacy.

[0015] By utilizing the EPR effect and active targeting strategies, nanosystems can achieve preferential accumulation in tumor tissue, thereby reducing damage to healthy tissue. Detailed Implementation

[0016] The embodiments described below are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0017] An apoptosis inducer based on activation of the death receptor signaling pathway, capable of specifically binding to death receptors and activating the apoptosis signaling pathway, including: (1) Wild-type sequence optimized variants: Through bioinformatics simulation and experimental verification, the natural ligand sequence is precisely modified (such as 1-5 amino acid replacements, insertions or deletions) to improve the affinity (KD value ≤10 nM) and stability with the receptor.

[0018] Among them, hydrophilic Ser is replaced with hydrophobic Leu to enhance the interaction with the transmembrane region of the receptor; Among these measures, rigid residues (such as Pro) are introduced to stabilize the polypeptide conformation and resist protease degradation. Terminal modifications (such as acetylation or palmitoylation) promote cell membrane penetration and improve intracellular delivery efficiency.

[0019] (2) Chemical modification strategies: PEGylation: Extending the in vivo half-life and reducing immunogenicity through polyethylene glycol (PEG) coupling; Glycosylation: enhances water solubility and stability, and reduces non-specific adsorption; Lipid modification: By binding lipid groups such as palmitic acid, the affinity of tumor cells to the membrane is enhanced.

[0020] (3) Fusion protein design: It is coupled with carrier proteins (such as albumin, Fc fragments) to enhance drug efficacy by utilizing the targeting or cycling stability of the carrier proteins. After fusion with albumin, it can achieve active targeting through the gp60 receptor highly expressed in tumor tissue. Specificity and toxicity are controlled through rational design and high-throughput screening to ensure that the KD value of the peptide molecule for the target receptor (such as Fas, DR4) is ≤5 nM, while having no significant activating effect on non-receptor cells or normal cells with low expression of death receptors.

[0021] A method for preparing an apoptosis inducer, comprising: (1) Polypeptide synthesis Solid-phase synthesis: The Fmoc strategy was adopted, using Rink amide resin or Wang resin as a carrier, and amino acids were coupled through HBTU / DIEA or PyBOP activation system to ensure condensation efficiency ≥99%; Gene recombination: Construct expression vectors (such as the pET system), express them in E. coli or yeast, and then purify them by affinity chromatography.

[0022] (2) Purification and identification: Separated by reversed-phase high-performance liquid chromatography (RP-HPLC), with a purity ≥98%; Mass spectrometry analysis (ESI-MS / MALDI-TOF) confirmed the molecular weight; Circular dichroism (CD) spectroscopy is used to analyze the secondary structure, ensuring a conformation similar to that of the natural ligand.

[0023] (3) Formulation preparation: Injectable formulation: The formula contains mannitol, phosphate buffer, and Tween 80 to ensure long-term storage stability (activity remains ≥90% after 12 months of storage at -20℃). Nanoparticle delivery systems: Liposomes: Utilized by thin-film hydration method, with a particle size of 100-200 nm, and surface modified with PEG to reduce non-specific adsorption; Polymer nanoparticles: encapsulated with PLGA (polylactic acid-glycolic acid copolymer), which enhances tumor accumulation through the EPR effect; Exosome delivery: Using engineered exosomes to carry peptides to achieve natural targeting and immune evasion.

[0024] Through precise sequence optimization and modification, this invention exhibits a more than 10-fold increased affinity for tumor cells compared to normal cells, significantly reducing off-target effects and thus minimizing side effects. By employing PEGylation and a nanodelivery system, the invention's half-life is extended to 72 hours, reducing dosing frequency and improving patient adherence. This characteristic allows the drug to maintain effective concentrations in the body for a longer period, enhancing therapeutic efficacy. Utilizing the EPR effect and active targeting strategy, the nanosystem can preferentially accumulate in tumor tissue, thereby reducing damage to healthy tissues.

[0025] This invention proposes an apoptosis inducer based on the activation of the death receptor signaling pathway and its preparation method. This inducer specifically activates the death receptor by mimicking the key structural domain of the ligand, efficiently inducing apoptosis in target cells with extremely low cytotoxicity to normal cells. Its optimized polypeptide sequence exhibits high stability, can be mass-produced using solid-phase synthesis, and is available in various formulations, suitable for the treatment of multiple diseases such as tumors and viral infections, demonstrating significant clinical application potential.

[0026] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An apoptosis inducer based on activation of the death receptor signaling pathway, wherein the active ingredient is a polypeptide molecule, characterized in that, It can specifically bind to death receptors and activate downstream Caspase cascades to induce apoptosis, and has no significant toxicity to non-receptor cells or cells with low expression of death receptors.

2. The apoptosis inducer based on activation of the death receptor signaling pathway according to claim 1, characterized in that, The polypeptide molecule is: Optimized variants of the wild-type sequence, containing 1-5 amino acid substitutions, insertions, or deletions, while maintaining binding activity with the death receptor; Chemically modified peptides, including PEGylation, acetylation, palmitoylation, or glycosylation, are used to improve stability or prolong half-life. Fusion proteins coupled with carrier proteins enhance in vivo delivery rates.

3. The apoptosis inducer based on activation of the death receptor signaling pathway according to claim 1, characterized in that, The death receptor is either Fas (CD95) or DR4 (TRAIL-R1), with a binding affinity KD value ≤ 10 nM.

4. A method for preparing an apoptosis inducer according to any one of claims 1-3, comprising the following steps: S1. Polypeptide synthesis: Polypeptides are synthesized according to a predetermined sequence using solid-phase synthesis or gene recombination technology. S2. Purification and identification: Separation and purification are performed by HPLC, ion exchange chromatography or gel electrophoresis, and the structure and purity are confirmed by mass spectrometry and circular dichroism spectroscopy. S3. Formulation preparation: Mix the peptide with pharmaceutical excipients to prepare injections, nanodelivery systems, oral microspheres, or sustained-release formulations.

5. The method for preparing an apoptosis inducer according to claim 4, characterized in that, The solid-phase synthesis step includes: S11. Use royal jelly resin or Rink amide resin as a carrier; S12, when coupling and protecting amino acids, use the HBTU / DIEA or PyBOP activation system; S13, cleavage and deprotection conditions: treatment with 95% TFA solution for 2 hours to ensure complete release of the peptide.

6. The method for preparing an apoptosis inducer according to claim 4, characterized in that, The purification steps include: S21. Reversed-phase HPLC was used with 20% acetonitrile / 80% phosphate buffer as the mobile phase. The purity of the target peak was collected to be ≥95%. The pH of the 20% acetonitrile / 80% phosphate buffer was 7.

0. The molecular weight of S22 was confirmed to be 543.21 Da by ESI-MS or MALDI-TOF mass spectrometry.

7. The method for preparing an apoptosis inducer according to claim 4, characterized in that, The formulation preparation steps include: S31. Pharmaceutical excipients include stabilizers, buffers, and surfactants, wherein the stabilizer is mannitol, the buffer is phosphate, and the surfactant is Tween 80. S32, the nanodelivery system is prepared by thin film hydration or self-assembly technology, with particle size controlled in the range of 100-200 nm.