Interfering with short peptide CC1 and its applications

Abstract

This invention belongs to the field of biomedical technology and discloses a short interfering peptide CC1 targeting the binding interface between CLCF1 and its secretory chaperone CRLF1. Its amino acid sequence is: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Gly-His-Ser-Asp-Phe-Leu-Gln-Lys-Met-Asp-Asp, with a molecular weight of 2891.24 g / mol. This short peptide can be obtained through classic Fmoc solid-phase synthesis. This invention is the first to independently design an analgesic peptide CC1 using molecular docking technology, targeting the specific binding interface between CLCF1 and its secretory chaperone CRLF1. Molecular experiments show that the short peptide CC1 can specifically bind to the CRLF1 protein. Furthermore, in a mouse pain model, the short peptide CC1 exhibits good analgesic activity, with both rapid onset and long duration of action. This invention will provide new molecular tools and drug development targets for the precision treatment of neuropathic pain.

Description

Technical Field

[0001] This invention belongs to the field of short peptide pharmacology technology, specifically relating to the interference of short peptide CC1 and its applications. Background Technology

[0002] Neuropathic pain is a type of chronic pain directly caused by damage or disease of the somatosensory nervous system, and is an important subtype of chronic pain. Its pathophysiological mechanisms are complex, involving multiple aspects such as peripheral and central sensitization, ion channel dysfunction, and neuroimmune interactions. Clinical features mainly include spontaneous pain (such as burning or electric shock-like pain), hyperalgesia (an excessively heightened response to painful stimuli), and anomalous pain (pain triggered by non-nociceptive stimuli). This disease is often accompanied by anxiety, depression, and sleep disorders, leading not only to a significant decline in patients' quality of life but also imposing a certain socioeconomic burden.

[0003] Currently, first-line drugs for treating neuropathic pain are mostly based on regulating signal transmission in the central nervous system, such as pregabalin, gabapentin (a calcium channel α2-δ ligand), tricyclic antidepressants (such as amitriptyline), and serotonin-norepinephrine reuptake inhibitors (such as duloxetine). However, these drugs generally have central nervous system side effects (such as dizziness, drowsiness, and cognitive impairment), and their efficacy response rate is limited, with only a portion of patients achieving satisfactory analgesia. In recent years, research has gradually shifted towards targeting peripheral mechanisms. For example, specifically blocking sodium ion channel subtypes (such as Nav1.7 and Nav1.8) that are highly expressed on peripheral nociceptive neurons, or antagonizing pain-promoting factor receptors (such as NGF and ATP receptors) located at peripheral nerve endings. This is believed to be able to intervene in the generation and transmission of pain signals at the source, while also potentially avoiding blood-brain barrier-related challenges and central nervous system side effects. Therefore, developing novel analgesics with high selectivity and action on peripheral targets has become an important strategy to break through the current bottleneck in the treatment of neuropathic pain, with clear unmet clinical needs and translational prospects.

[0004] The dorsal root ganglion (DRG) is a key "gating point" for peripheral sensory signals entering the central nervous system and a core hub for pain perception regulation. As a "transmitter" of pain signals, it gathers primary sensory neurons that transmit different sensory modalities such as mechanical pain, thermal pain, and itch. It also collaborates closely with non-neuronal cells such as satellite glial cells and Schwann cells to construct a highly structurally and functionally coupled pain-modulating microenvironment. Under physiological conditions, this microenvironment precisely mediates sensory signal transduction. After peripheral nerve injury, abnormal neuro-glial cell interactions in the DRG and gene expression reprogramming initiate and maintain pain sensitization, playing a central role in the development and progression of neuropathic pain. Furthermore, the DRG is also a core initiating element in the pain cascade response. A single-cell transcriptome study published in *Cell Research* in 2021 confirmed that in the classic neuropathic pain model, the mouse spared nerve injury (SNI) model, mouse DRG neurons undergo significant transcriptome remodeling. This remodeling, through neuronal subtype switching and dynamic regulation of gene expression, becomes a key molecular basis for the development and progression of neuropathic pain. Specifically, cardiotrophin-like cytokine factor 1 (CLCF1) was significantly upregulated in DRG injury-specific neuronal subsets following nerve injury. The preliminary mechanism may involve CLCF1 ascending into the spinal cord, specifically activating dorsal horn astrocytes, and mediating the formation of mechanosensitive pain by regulating the neuroinflammatory microenvironment. However, the specific mechanism remains unclear (Wang et al., 2021). As an important cytokine in the interleukin-6 family, CLCF1 needs to form a complex with its secretory partner cytokine receptor-like factor 1 (CRLF1), and then specifically bind to the receptor protein ciliary neurotrophic factor receptor α (CNTFRα) to form a complex. Only then can it exert its biological effects by activating downstream signaling pathways such as JAK / STAT3 (Crisponi et al., 2022; Elson et al., 2000).

[0005] Since the secretion and functional activation of CLCF1 depend on its specific binding with its secretory partner CRLF1, designing specific interfering peptides targeting the binding interface between the two to block its abnormal extracellular secretion has certain research value and application prospects for the treatment of neuropathic pain. Summary of the Invention

[0006] The purpose of this invention is to provide an interfering short peptide CC1 and its applications, offering new molecular tools and drug development targets for the precise treatment of neuropathic pain.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides an interfering short peptide CC1, the amino acid sequence of which is shown below: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Gly-His-Ser-Asp-Phe-Leu-Gln-Lys-Met-Asp-Asp (SEQ ID No. 1) has a molecular weight of 2891.24 g / mol.

[0008] Secondly, the present invention provides the application of the above-mentioned interfering short peptide CC1 as an active ingredient in the preparation of drugs for treating neuropathic pain.

[0009] Furthermore, the neuropathic pain is peripheral neuropathic pain.

[0010] Furthermore, the peripheral neuropathic pain is SNI-induced neuropathic pain.

[0011] Furthermore, the neuropathic pain is spontaneous pain induced by the human CLCF1-CRLF1 complex protein.

[0012] Thirdly, the present invention provides a medicament for treating neuropathic pain, comprising the aforementioned interfering short peptide CC1.

[0013] Furthermore, the drug also includes pharmaceutical excipients.

[0014] Furthermore, the drug is an injectable or oral formulation.

[0015] The present invention has the following beneficial effects: Based on the binding interface between CLCF1 and its secretory partner CRLF1, this invention designs a specific interfering short peptide CC1 that targets their interaction, which can be obtained through classic Fmoc-solid-phase synthesis. The interfering short peptide CC1 can specifically block the abnormal secretion of CLCF1, thereby inhibiting the activation of downstream signaling pathways triggered by CLCF1, and ultimately blocking the pro-pain pathobiological effects mediated by this pathway. Compared with existing drugs, the interfering short peptide CC1 has the following beneficial effects: (1) Rapid onset and long duration of action: DRG local targeted delivery of the short interfering peptide CC1 can rapidly, efficiently and time-limitedly alleviate SNI-induced mechanical hyperalgesia. Animal experiments show that DRG local administration of CC1 can rapidly increase the mechanical pain threshold in mice. The pain threshold increases significantly 0.5 h after administration, and a certain analgesic effect can still be observed 24 h after a single administration. (2) It has a long-lasting analgesic effect: Von Frey mechanical pain test results showed that intrathecal administration of CC1 short peptide could significantly increase the mechanical withdrawal threshold of mice from the 3rd day after surgery and exert an analgesic effect continuously during the 14-day administration period. (3) Good safety: Interfering short peptide CC1 achieves the effect of treating neuropathic pain through peripheral local administration, avoiding the risks of a series of adverse reactions such as tolerance, dependence, gastrointestinal disorders, respiratory depression and pain hypersensitivity of existing analgesics, and has better clinical application prospects. Attached Figure Description

[0016] Figure 1 To quantitatively detect DRG tissues in model group and control group mice using qPCR technology clcf1 mRNA expression level ( Figure 1 A) The content of secretory CLCF1 protein in DRG supernatant was detected by enzyme-linked immunosorbent assay (ELISA). Figure 1 B) Result.

[0017] Figure 2 The experimental procedure to verify the effect of knocking down the CLCF1 gene expression in the DRG on SNI-induced mechanical hyperalgesia ( Figure 2 A) and animal behavioral test results ( Figure 2 B).

[0018] Figure 3 The experimental procedure to verify the induction effect of local administration of CLCF1 / CRLF1 complex protein on mechanical hyperalgesia in DRG ( Figure 3 A) and animal behavioral test results ( Figure 3 B).

[0019] Figure 4 This is the mass spectrum of CC1, the target compound synthesized in Example 2.

[0020] Figure 5 The results of surface plasmon resonance (SPR) detection of the interaction between CC1 short peptide and CRLF1 protein are shown in Example 3.

[0021] Figure 6 Example 4: Procedure for determining the analgesic effect of a single dose ( Figure 6 A) and the results of the analgesic effect test ( Figure 6 B).

[0022] Figure 7 Example 4: Determination of the efficacy of long-acting analgesics ( Figure 7 A) and the results of the analgesic effect test ( Figure 7 B). Detailed Implementation

[0023] The present invention will be further described below with reference to the embodiments and accompanying drawings, but the description of the embodiments does not limit the scope of protection of the present invention in any way.

[0024] Unless otherwise defined, 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 pertains. The terminology used herein in the description of this invention is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0025] Unless otherwise specified, all substances or instruments used in the following examples can be obtained from conventional commercial sources.

[0026] Example 1: Verification of the regulatory role of CLCF1 in neuropathic pain in DRG 1. Validating the regulatory role of CLCF1 in DRG in neuropathic pain using the SNI model. First, establish the SNI model. The specific construction method is as follows: After anesthetizing the experimental mice, they were fixed in a prone position on the operating table. The surgical area was shaved and routinely disinfected. The skin and superficial fascia were incised sequentially along the surface projection of the sciatic nerve. The biceps brachii muscle was exposed using blunt dissection techniques, thereby exposing the main trunk of the sciatic nerve. The three main branches of the sciatic nerve were carefully dissected and identified along its course: the common peroneal nerve, the sural nerve, and the tibial nerve. The common peroneal nerve and the tibial nerve were tightly ligated with 5-0 silk sutures, and then severed at the distal end of the ligation, removing a nerve stump of approximately 4 mm, while ensuring that the sural nerve remained intact. Subsequently, the muscle and skin layers were sutured sequentially. Iodine was evenly applied to the suture wound, and penicillin was injected into the muscle of the left hind limb to prevent infection.

[0027] The control group mice underwent only sham surgery, with the surgical procedure being the same as that in the model group. Only the main trunk of the sciatic nerve was exposed, and no nerve ligation, transection, or resection was performed. All other postoperative disinfection and anti-infection treatments were completely uniform.

[0028] Postoperative validation results showed that on the first day of SNI modeling, the mechanical pain threshold of mice was significantly reduced, and the Von Frey test value dropped to below 0.4, indicating that the neuropathic pain model was initially successfully constructed.

[0029] To further investigate the molecular expression changes in early neurological injury caused by spontaneous neural tube defects (SNI), DRG tissues were collected from mice in both the model group and the control group on day 3 of SNI. The expression of DRG tissues in both groups was quantitatively detected using qPCR. clcf1 The expression level of mRNA was measured, and the content of secretory CLCF1 protein in the DRG supernatant was detected by enzyme-linked immunosorbent assay (ELISA). All results were compared with the gene and protein expression levels of the control group mice. The relative expression folds were calculated, and statistical significance analysis was performed.

[0030] like Figure 1 As shown, compared with the blank control group, the DRG of mice 3 days after SNI modeling was significantly higher. clcf1 The expression levels of mRNA and secreted CLCF1 were significantly increased, among which clcf1 The relative expression level of mRNA was 4.88 times that of the control group, and the relative expression level of secreted CLCF1 protein was 5.17 times that of the control group. These results suggest that peripheral nerve injury induced by SNI can rapidly induce transcriptional activation and protein secretion of CLCF1 in the DRG, providing direct experimental evidence for the early regulation of neuropathic pain by CLCF1.

[0031] 2. Effect of knockdown of CLCF1 expression in DRG on SNI-induced mechanohyperalgesia To verify the regulatory role of endogenous CLCF1 in neuropathic pain, this experiment injected small interfering RNA (siCLCF1) targeting CLCF1 into the DRG of mice before SNI modeling to specifically knock down CLCF1 expression in the DRG; a scrambled siRNA treatment group was set up as a negative control. Changes in mechanical pain threshold in each group of mice were dynamically monitored on days 1, 3, and 5 after SNI surgery. Figure 2 A).

[0032] Animal behavioral tests showed that, compared with the Scramble control group, the SiCLCF1 intervention group mice showed a significant increase in mechanical pain threshold on day 1 after SNI surgery, and maintained a high pain threshold level on days 3 and 5 post-surgery. Statistical analysis showed that the differences in pain threshold between the two groups at all time points were highly statistically significant (P<0.01). Figure 2 B).

[0033] The above results confirm that CLCF1 in the DRG is a key regulatory molecule mediating the development of neuropathic pain, and that targeting and inhibiting CLCF1 expression can significantly improve pain abnormalities after nerve injury.

[0034] 3. The induction of mechanical hyperalgesia by local DRG supplementation with CLCF1 / CRLF1 complex protein. To further validate the analgesic effect of CLCF1 signaling, this study administered different doses of the CLCF1 / CRLF1 complex protein (25 ng, 50 ng, 2 μL) to the mouse DRG via cannula implantation, with an equal volume of PBS as a blank control. Changes in the mechanical pain threshold of mice were measured at 2 h, 4 h, 6 h, 8 h, and 24 h post-administration. Figure 3 A).

[0035] Behavioral results showed that, compared with the Vehicle control group, the CLCF1 / CRLF1 25 ng group induced a significant decrease in the mechanical pain threshold in mice 2 h after administration, which remained at a low level for 4–8 h and gradually recovered after 24 h; the pain sensitivity effect was more significant in the CLCF1 / CRLF1 50 ng group, with a significant decrease in the pain threshold 2 h after administration, reaching a peak at 6 h, and not fully recovering to baseline levels until 24 h; statistical analysis showed that the differences between the two CLCF1 / CRLF1 treatment groups and the PBS group were highly significant (P<0.01), and there was a certain dose-dependent relationship. Figure 3 B).

[0036] The above results confirm that local administration of the CLCF1 / CRLF1 complex protein to the DRG can dose-dependently induce mechanical hyperalgesia in experimental mice, and this effect has a certain time-dependent effect, suggesting that the activation of the CLCF1 signaling axis in the DRG is a key link in mediating pain abnormalities.

[0037] Example 2: Design and synthesis of interfering short peptide CC1 The interfering short peptide CC1 was designed based on the pathological mechanism of CLCF1 secretion and downstream cascade activation. It is the core functional region where CLCF1 and CRLF1 interact, competitively binding to CRLF1 and blocking the formation of the CLCF1-CRLF1 complex, thereby inhibiting CLCF1 secretion into the extracellular space. The interfering short peptide CC1 adopts a modular structure of "membrane-transfer peptide-linker-core target": the N-terminus is a classic TAT membrane-transfer peptide (YGRKKRRQRRR), responsible for mediating the transmembrane entry of the short peptide into cells secreting CLCF1; the middle is a flexible linker (GG) to ensure the spatial freedom of the functional domain; the C-terminus of the short peptide mimics the core key functional sequence HSDFLQKMDD of CLCF1; the specific sequence is shown below: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Gly-His-Ser-Asp-Phe-Leu-Gln-Lys-Met-Asp-Asp (SEQ ID No. 1).

[0038] The synthesis of the short peptide CC1 is interfered with by the following steps: (1) The amino acid sequence shown in SEQ ID No. 1 was assembled stepwise on the resin using the Fmoc solid-phase synthesis method. The specific steps are as follows: (1-1) Place the Wang resin, the solid support in the solid-phase synthesis of Fmoc, in the reaction column and soak it in dichloromethane for at least 30 min to make it swell. (1-2) Add a 20% (v / v) solution of hexahydropyridine / N,N-dimethylformamide to the swollen Wang resin and shake at room temperature for 20 min to remove the Fmoc protecting group at the end of the resin. After the reaction, wash the resin three times with N,N-dimethylformamide for 5 min each time. Take a small amount of resin and use the ninhydrin colorimetric method to check whether the amino deprotection is complete. (1-3) Under an inert gas atmosphere, the condensing agent combination (N,N-diisopropylethylamine, 1-hydroxybenzotriazole, O-benzotriazole-tetramethylurea hexafluorophosphate) was added to the resin, and then followed SEQ ID NO. The amino acid sequence shown in No. 1, from C-terminus to N-terminus (Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Gly-His-Ser-Asp-Phe-Leu-Gln-Lys-Met-Asp-Asp), is used to sequentially couple each Fmoc-protected amino acid to the peptide resin. The amount of amino acid added in a single batch is 2.5 to 4.0 molar equivalents of the initial resin loading, the total amount of condensing agent is 3 to 4 times the molar amount of amino acids, and the amount of the base N,N-diisopropylethylamine is 6 to 8 times the molar amount of amino acids. Each coupling reaction takes 1 to 1.5 hours. After each coupling reaction, ninhydrin is used to confirm complete reaction; if a blue color appears, the coupling is repeated once. (2) The target compound was obtained by pyrolysis with a cleavage reagent, freeze-drying, and preparative reversed-phase high-performance liquid chromatography purification. The specific steps are as follows: (2-1) The crude peptide resin obtained in step (1) was placed in a cleavage reagent for treatment. The cleavage reagent was prepared by trifluoroacetic acid (TFA), triisopropylsilane (TIS) and double-distilled water in a volume ratio of 95:2.5:2.5. After cleavage by shaking at room temperature in the dark for 2 h, the resin was removed by filtration. Most of the trifluoroacetic acid was removed by rotary evaporation under reduced pressure. The remaining liquid was slowly added dropwise to pre-cooled diethyl ether to precipitate the precipitate. After standing in an ice bath, the precipitate was collected by centrifugation and freeze-dried to obtain crude peptide. (2-2) Dissolve the crude peptide precipitate obtained in step (2-1) in a water-acetonitrile mixed solution containing 0.1% (v / v) trifluoroacetic acid, and filter it through a 0.22 μm microporous membrane to remove insoluble particles; (2-3) The filtered solution was purified by gradient elution using a reversed-phase high-performance liquid chromatography (RP-HPLC) column. The target peak components were collected, combined, and freeze-dried to finally obtain the high-purity target compound CC1.

[0039] Identified by mass spectrometry and RP-HPLC column analysis, the mass spectrum of CC1 is as follows: Figure 4 As shown.

[0040] Example 3: The interfering short peptide CC1 can specifically bind to the CRLF1 protein. This embodiment employs surface plasmon resonance (SPR) technology to detect the interaction between gradient concentrations of CC1 short peptides and CRLF1 protein. The procedure includes multi-cycle kinetic analysis using a Biacore 1K instrument and a CM5 chip; CRLF1 protein is immobilized in channel 2, with channel 1 serving as a blank reference; and HBS-EP is used for... + The flow rate was 30 μL / min for the running buffer, the CC1 binding and dissociation times were 120 s each, and the glycine solution was regenerated for 30 s. A CC1 short peptide injection gradient of 1.5625~200 nM was set to acquire the sensing curve.

[0041] like Figure 5 As shown, during the 0-120 s binding phase after sample introduction, CC1 binds to CRLF1 protein in a concentration-dependent manner, with the binding response value increasing synchronously with the increase of CC1 concentration. After elution with the 120 s start-up buffer, the signal rapidly declines, leaving a stable binding residue. Using a 1:1 binding model, the equilibrium dissociation constant KD was calculated to be 2.65 × 10⁻⁶. -9 M, nanomolar affinity confirmed that CC1 and CRLF1 proteins have a specific and highly stable binding, specifically blocking the binding of CLCF1 to CRLF1, thereby inhibiting the secretion of CLCF1 into the extracellular space.

[0042] Example 4: Verification of the analgesic effect of short peptide CC1 1. Establish a SNI-induced neuropathic pain model. The SNI is established in the same way as in Example 1.

[0043] 2. Determination of analgesic effect after single-dose administration On day 5 after SNI modeling, the model mice were given a single dose of compound CC1 (2 μL, 500 μM) synthesized in Example 2 via a pre-placed cannula in the DRG region. An equal volume of PBS was used as a negative control. Changes in the mechanical pain threshold of the mice were detected using Von Frey fibers before administration (baseline, BL) and at 0.5, 2, 4, 6, 24, and 36 h after administration. Figure 6 A).

[0044] On day 5 after SNI modeling, the mechanical pain threshold of mice in the PBS control group remained at a very low level, indicating that the neuropathic pain phenotype was stable. Figure 6 As shown in Figure B, compared with the PBS group, local administration of CC1 via DRG rapidly increased the mechanical pain threshold in mice. A significant increase in pain threshold was observed 0.5 h after administration, reaching a peak analgesic effect at 2 h, followed by a slow, time-dependent decline. Statistical analysis showed that the mechanical pain threshold in the CC1-treated group was significantly higher than that in the PBS group at 0.5, 2, 4, and 6 h after administration. A significant analgesic effect was still observed at 24 h after administration, and there was no significant difference in pain threshold between the two groups at 36 h. These results confirm that local targeted delivery of the interfering short peptide CC1 via DRG can rapidly, efficiently, and time-sensitively reverse SNI-induced mechanical hyperalgesia.

[0045] Furthermore, this embodiment determined the long-acting analgesic effect of the CC1 short peptide in vivo. For example... Figure 7 As shown in A, a mouse model of SNI neuropathic pain was first constructed. PBS solvent (Vehicle) or 100 μM CC1 short peptide (flow rate 0.25 μL / h) was continuously administered via a micro-osmotic pump within the DRG for 14 consecutive days. The mechanical withdrawal threshold of the mice was detected on days 3, 5, 7, 10, and 14 after modeling. Von Frey mechanical pain assessment results are as follows: Figure 7 As shown in B, the mechanical withdrawal threshold of mice in the Vehicle group was significantly and continuously reduced after SNI modeling, forming a stable mechanical analgesia. Intrathecal administration of CC1 short peptide significantly increased the mechanical withdrawal threshold of mice from the 3rd day after surgery, and maintained an analgesic effect throughout the 14-day dosing cycle. The differences between the groups at each time point were all statistically significant. The above results indicate that the short peptide CC1 can provide long-term relief from neuropathic pain induced by nerve damage.

Claims

1. Interfering short peptide CC1, characterized in that, The amino acid sequence is shown below: Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Gly-Gly-His-Ser-Asp-Phe-Leu-Gln-Lys-Met-Asp-Asp.

2. The use of the interfering short peptide CC1 as an active ingredient in the preparation of a drug for treating neuropathic pain, as described in claim 1.

3. The application according to claim 2, characterized in that, The neuropathic pain mentioned is peripheral neuropathic pain.

4. The application according to claim 3, characterized in that, The peripheral neuropathic pain mentioned is SNI-induced neuropathic pain.

5. The application according to claim 2, characterized in that, The neuropathic pain is spontaneous pain induced by the human CLCF1-CRLF1 complex protein.

6. A drug for treating neuropathic pain, characterized in that, It contains the interfering short peptide CC1 as described in claim 1.

7. The drug according to claim 6, characterized in that, The drug also includes pharmaceutical excipients.

8. The medicament according to claim 7, characterized in that, The drug is an injectable or oral preparation.

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