CRTC2 lactylation specific polypeptide inhibitor and application thereof
By designing a CRTC2 lactation-specific peptide inhibitor, 2K-peptide-2#, to inhibit HBV transcription, the problem of existing anti-HBV drugs being unable to clear cccDNA and causing relapse is solved, providing a new anti-HBV treatment option.
Patent Information
- Application Number
- CN202511151960.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-18
- Publication Date
- 2025-11-07
AI Technical Summary
Existing anti-HBV drugs such as NAs and interferons are difficult to clear covalently closed circular DNA (cccDNA) in the liver after long-term use, and relapse is common after drug withdrawal. They also have adverse reactions. There is an urgent need to develop new therapies that target cccDNA, viral protein assembly, or host immune regulation.
A CRTC2 lactation-specific peptide inhibitor, 2K-peptide-2#, was designed and synthesized. By inhibiting the lactation modification of CRTC2, it intervenes in the HBV transcription process and reduces HBV replication and expression.
It effectively inhibits the lactation modification of CRTC2 and significantly reduces HBV transcription levels, providing a new anti-HBV therapeutic target and drug with potential clinical application value.
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Figure CN120904296A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the technical field of biological medicine, and relates to a polypeptide inhibitor and application thereof in pharmacy. BACKGROUND
[0002] The 2024 Global Hepatitis Report shows that there are about 254 million people infected with Hepatitis B virus (HBV) worldwide. After chronic infection caused by HBV, there is a high risk of developing cirrhosis and liver cancer. Currently, anti-HBV drugs are mainly divided into two categories: nucleos(t)ide analogues (NAs) and interferons. NAs such as entecavir and tenofovir can effectively inhibit viral replication by inhibiting HBV polymerase activity, significantly improving liver inflammation and fibrosis, but long-term medication is required to maintain efficacy, and it is difficult to eliminate liver covalently closed circular DNA (cccDNA), and it is easy to relapse after drug withdrawal. Interferons, including ordinary interferons and pegylated interferons, have both antiviral and immunomodulatory effects, and some patients can achieve HBsAg clearance or seroconversion, but the response rate is only about 30%, and there are adverse reactions such as fever, bone marrow suppression, and thyroid function abnormalities, the treatment course is fixed but the applicable population is limited. Both of the above two types of drugs cannot completely eradicate HBV, therefore, it is urgent to develop new therapies and new drugs targeting cccDNA, viral protein assembly or host immune regulation.
[0003] Polypeptide inhibitors have unique advantages of high affinity, strong specificity and targeting of "undruggable" targets (such as protein-protein interaction interfaces) in the field of biomedicine. In the development of innovative drugs, they have been successfully applied in anti-tumor (such as targeting MDM2 / p53 pathway), anti-virus (HIV fusion inhibitor) and metabolic disease treatment (GLP-1 receptor agonist derived peptide), and multiple varieties have been approved by FDA for listing. As molecular probes, polypeptide inhibitors have realized precise analysis of target protein function in functional genomics and signal pathway research. In addition, by specifically interfering with pathological targets (such as abnormal kinases), they have become a key tool for exploring disease mechanisms. However, there are few reports on the regulation of polypeptide inhibitors on HBV. Exploring the regulation of polypeptide inhibitors on HBV may provide a new treatment for antiviral therapy.
[0004] Hepatitis B virus (HBV) infection causes metabolic reprogramming in the liver. Among them, the change of glucose metabolic pathway is a relatively classic metabolic remodeling. Under the condition of HBV infection, hepatocytes rely on glycolysis pathway to produce energy, accompanied by a large amount of lactic acid production. Recent studies have shown that lactic acid can mediate lactylation modification and regulate the basic biological functions, pathogenic mechanisms and host-virus interactions of viruses such as PRRSV, KSHV, HCMV and HSV-1 under the condition of infection. Therefore, further exploration of the regulatory effect of lactylation on HBV has important clinical significance for the development of new targeted therapy strategies.
[0005] CRTC2 (CREB-regulated transcription coactivator 2) is a key coactivator involved in transcriptional regulation of CREB (cAMP response element-binding protein), which not only plays an important role in metabolism and cancer, but also has been found to be directly involved in host immune response and viral replication in recent years. SUMMARY
[0006] The purpose of the present application is to investigate the application value of polypeptide inhibitors in anti-HBV, so as to develop effective new drugs for anti-HBV.
[0007] Through research, the present application provides the following technical solutions:
[0008] 1. A CRTC2 lactylation-specific polypeptide inhibitor, the amino acid sequence of which is YGRKKRRQRRRPRKFSEKIALQKQRQAEE (SEQ ID No. 2).
[0009] 2. The use of the CRTC2 lactylation-specific polypeptide inhibitor in the preparation of an anti-HBV drug.
[0010] Further, the anti-HBV drug is an anti-HBV transcription drug.
[0011] The present application researches and finds that CRTC2 can be subjected to lactylation modification, and the level of lactylation modification of CRTC2 is significantly up-regulated in the HBV infection state and promotes HBV transcription; the present application further identifies a key site of CRTC2 subjected to lactylation modification, and according to the site and structure thereof, three competitive polypeptide inhibitors 2K-peptide-1# (the sequence is shown as SEQ ID No. 1), 2K-peptide-2# (the sequence is shown as SEQ ID No. 2), and 2K-peptide-3# (the sequence is shown as SEQ ID No. 3) are designed and synthesized, wherein 2K-peptide-2# is verified by experiments to be able to effectively reduce the level of lactylation modification of CRTC2 and inhibit the transcription of HBV in vitro and in vivo, and can be used for preparing an anti-HBV drug.
[0012] The present application has the beneficial effects that the present application provides a CRTC2 lactylation specific polypeptide inhibitor and application thereof in preparing an anti-HBV drug, and provides a new target and new drug for hepatitis B treatment. BRIEF DESCRIPTION OF DRAWINGS
[0013] Figure 1 It is shown that the level of CRTC2 lactylation is significantly increased in the HBV infection state. Among them, A is an exogenous immunoprecipitation (IP) experiment and a Western blotting experiment of a pan-lactylation antibody (Pan-Kla) after Anti-Flag enrichment of Flag-CRTC2 plasmid transfection of HBV stably expressed HepAD38 cells; B is an exogenous IP experiment and a Western blotting experiment of Pan-Kla after Anti-Flag enrichment of Flag-CRTC2 plasmid transfection of HBV infected HepG2-NTCP cells; C is an endogenous IP experiment and a Western blotting experiment of Pan-Kla after Anti-CRTC2 enrichment of HBV stably expressed HepAD38 cells; D is an endogenous IP experiment and a Western blotting experiment of Pan-Kla after Anti-CRTC2 enrichment of HBV infected HepG2-NTCP cells.
[0014] Figure 2The lysines (Lys, K) at positions 25 and 30 of the CRTC2 protein, K25 and K30, are key sites for lactylation modification thereof. Among them, A-C are liquid chromatography-tandem mass spectrometry (LC-MS / MS) identification of K25, K30, K56 as possible lactylation modification sites of CRTC2; D is site mutation technology combined with IP experiment and Pan-Kla Western blotting experiment to verify that K25 and K30 are key sites for lactylation modification of CRTC2.
[0015] Figure 3 The CRTC2 lactylation modification promotes HBV transcription. Among them, A is the verification of the CRTC2 knockdown and overexpression effect in HBV stably expressed HepAD38 cells, i.e., HepAD38-Tet(-) cells; B is the detection of HBV Total RNAs by real-time fluorescence quantitative PCR (qRT-PCR) in HepAD38-Tet(-) cells; C is the detection of HBV 3.5 kb RNA by qRT-PCR in HepAD38-Tet(-) cells; in A-C, sgNC represents no knockdown of endogenous CRTC2 expression, sgCRTC2 represents knockdown of endogenous CRTC2 expression, sgCRTC2+WT represents overexpression of CRTC2 wild type after knockdown of endogenous CRTC2 expression, and sgCRTC2+2KR represents overexpression of CRTC2 double-site combined mutant 2KR after knockdown of endogenous CRTC2 expression.
[0016] Figure 4 The polypeptide inhibitor 2K-peptide-2# can effectively inhibit CRTC2 lactylation. Among them, A is the structure of CRTC2 and the CRTC2 lactylation inhibition targeting sequences contained in three polypeptide inhibitors 2K-peptide-1#, 2K-peptide-2#, and 2K-peptide-3#; B is the CCK-8 evaluation of the cytotoxicity of the three polypeptide inhibitors; and C is the detection of the effect of the three polypeptide inhibitors in inhibiting CRTC2 lactylation in HepAD38-Tet(-) cells.
[0017] Figure 5 The sequences of the polypeptide inhibitor 2K-peptide-2# and its mutant 2KR-peptide-2# are shown.
[0018] Figure 6This study demonstrates the effectiveness of the peptide inhibitor 2K-peptide-2# in inhibiting HBV transcription in a cell model. A shows the efficacy of 2K-peptide-2# and its mutant 2KR-peptide-2# in inhibiting CRTC2 lactation in HepAD38-Tet(-) cells; B shows HBV total RNAs detected by qRT-PCR in HepAD38-Tet(-) cells; and C shows HBV 3.5kb RNA detected by qRT-PCR in HepAD38-Tet(-) cells.
[0019] Figure 7 This study demonstrates the effectiveness of the peptide inhibitor 2K-peptide-2# in inhibiting HBV transcription in animal models. A shows the efficacy of 2K-peptide-2# and its mutant 2KR-peptide-2# in inhibiting CRTC2 lactation levels in HBV-infected mouse liver tissue; B shows HBV total RNAs detected by qRT-PCR in HBV-infected mouse liver tissue; and C shows HBV 3.5kb RNA detected by qRT-PCR in HBV-infected mouse liver tissue. Detailed Implementation
[0020] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the preferred embodiments of this invention are described in detail below. Experimental methods in the preferred embodiments that do not specify specific conditions are generally performed according to conventional conditions in the art or conditions recommended by the reagent manufacturer.
[0021] Example 1. Differences in CRTC2 lactation modification levels before and after HBV infection
[0022] HepAD38 cells were cultured under tetracycline-free conditions to induce stable HBV expression, constructing the HBV-expressing cell model HepAD38-Tet(-); HepG2-NTCP cells were infected with HBV viral particles to construct an HBV infection model. Subsequently, HepAD38 cells with and without HBV expression were cultured separately. Figure 1 A) HepG2-NTCP cells with / without HBV infection ( Figure 1 B) The Flag-CRTC2 plasmid (obtained by cloning the CRTC2 encoding gene into the pSEB-3Flag vector) was transfected into cells. The level of lactation modification of Flag-tagged CRTC2 (CRTC2-Flag) was detected using an anti-Flag antibody-enriched exogenous IP assay and a Pan-Kla Western blotting assay. Furthermore, in HepAD38 cells with / without HBV expression (… Figure 1C) HepG2-NTCP cells with / without HBV infection Figure 1 D) Endogenous IP experiment with Anti-CRTC2 antibody and Western blotting experiment with Pan-Kla to detect the level of CRTC2 lactylation modification.
[0023] The results show that the level of CRTC2 lactylation modification is up-regulated after HBV expression or infection compared with no HBV expression or infection Figure 1 ).
[0024] Example 2. Identification of key sites of CRTC2 lactylation modification
[0025] Huh7 cells were infected with recombinant adenovirus AdGFP and AdHBV1.3, respectively, and the obtained cell samples were entrusted to Hangzhou Jingjie Biological Technology Co., Ltd. to identify the possible lactylation modification sites of CRTC2 by LC-MS / MS technology. Based on the information of ion mass-to-charge ratio shift, sequence correspondence, and auxiliary ion peak, etc., the possible lactylation modification sites of CRTC2 were finally determined to be K25 Figure 2 A), K30 Figure 2 B), and K56 Figure 2 C).
[0026] According to the above identification results, the recombinant plasmids of CRTC2 single-site mutation (K25R, K30R, K56R), double-site combined mutation (K25R & K30R, i.e. 2KR), and triple-site combined mutation (K25R & K30R & K56R, i.e. 3KR) were constructed according to the construction method of Flag-CRTC2 plasmid. The Flag-CRTC2 plasmid and the above CRTC2 mutant plasmids were transfected into HEK293 cells, and the exogenous IP experiment with Anti-Flag antibody and Western blotting experiment with Pan-Kla were used to detect the lactylation modification level of CRTC2-Flag and Flag-tagged CRTC2 mutants (K25R-Flag, K30R-Flag, K56R-Flag, 2KR-Flag, 3KR-Flag). The results showed that the lactylation modification level of K25R & K30R double-site combined mutant (2KR-Flag) was significantly lower than that of wild-type CRTC2 (CRTC2-Flag), but there was no significant difference compared with K25R & K30R & K56R triple-site combined mutant (3KR-Flag), indicating that K25 and K30 are the key sites of CRTC2 lactylation modification Figure 2 D).
[0027] Example 3. Effect of CRTC2 lactylation modification on HBV transcription
[0028] The CRTC2 encoding gene was cloned into the pAdTrack-TO4 vector to obtain a recombinant adenovirus vector, and HEK293 cells were transfected to package the recombinant adenovirus AdCRTC2 mediating CRTC2 expression; the recombinant adenovirus Ad2KR containing the CRTC2 double-site combined mutant 2KR encoding gene was constructed by the same method; after knocking down the endogenous CRTC2 expression in HepAD38-Tet(-) cells using the CRISPR-Cas9 gene editing system, the cells were cultured for 24 hours, and then infected with the recombinant adenovirus AdGFP overexpressing GFP (as a control), the recombinant adenovirus AdCRTC2 overexpressing CRTC2 wild type, and the recombinant adenovirus Ad2KR overexpressing CRTC2 double-site combined mutant 2KR, respectively; 48 hours later, the cell samples were collected, half of each group was used for protein extraction and Western blotting detection of CRTC2 knockdown and overexpression effect (A), and the remaining half was subjected to total RNA extraction, reverse transcription, and qRT-PCR detection of HBV Total RNAs (B), HBV 3.5kb RNA level (C). Figure 3 A), the remaining half was subjected to total RNA extraction, reverse transcription, and qRT-PCR detection of HBV Total RNAs (B), HBV 3.5kb RNA level (C). Figure 3 B). Figure 3 C).
[0029] It was found that compared with overexpression of CRTC2 double-site combined mutant 2KR, overexpression of CRTC2 wild type significantly promoted the expression of HBV Total RNAs and HBV 3.5kb RNA, indicating that the lactylation modification of CRTC2 significantly promoted the transcriptional regulation of HBV (D). Figure 3
[0030] Example 4. Design and screening of CRTC2 lactylation-specific polypeptide inhibitors
[0031] The mechanism of polypeptide inhibitors in intervening protein post-translational modification includes: binding to the active site or specific domain of the enzyme involved in protein post-translational modification, preventing the normal binding of the enzyme to the substrate through steric hindrance or chemical interaction, thereby inhibiting the progress of the modification reaction. Therefore, according to the site and structure of CRTC2 lactylation modification, we designed and synthesized three competitive polypeptide inhibitors: 2K-peptide-1#, 2K-peptide-2#, and 2K-peptide-3#, which all have an amino acid sequence composed of two parts: a cell-penetrating sequence YGRKKRRQRRR + a CRTC2 lactylation inhibition targeting sequence. Among them, the CRTC2 lactylation inhibition targeting sequences contained in 2K-peptide-1#, 2K-peptide-2#, and 2K-peptide-3# are shown in Figure 4 A.
[0032] The cytotoxicity of the three polypeptide inhibitors was detected by a cytotoxicity detection kit (Cell Counting Kit-8, CCK-8), and the drug concentration was determined to be 10 μM Figure 4 B) According to the above drug concentration, the three polypeptide inhibitors were used to treat HepAD38-Tet(-) cells, and endogenous IP experiments and Pan-Kla Western blotting experiments were performed using Anti-CRTC2 antibody to detect the effectiveness of the three polypeptide inhibitors in interfering with CRTC2 lactylation. The results are shown in Figure 4 C, the polypeptide inhibitor 2K-peptide-2# can effectively inhibit CRTC2 lactylation.
[0033] Example 5. Effect of CRTC2 lactylation-specific polypeptide inhibitors on HBV transcription in a cell model
[0034] To verify the physiological function of the polypeptide inhibitor 2K-peptide-2#, a mutant 2KR-peptide-2# (amino acid sequence shown in SEQ ID No. 4) was designed and synthesized according to the sequence of 2K-peptide-2# as a negative control. Figure 5
[0035] 2K-peptide-2# and 2KR-peptide-2# were used to treat HepAD38-Tet(-) cells at a concentration of 10 μM, and after 48 h, half of the sample in each group was used for protein extraction and endogenous IP experiments and Pan-Kla Western blotting experiments using Anti-CRTC2 antibody to detect the lactylation modification level of CRTC2 Figure 6 A), and the remaining half of the sample was subjected to total RNA extraction, reverse transcription, and qRT-PCR to detect HBV Total RNAs Figure 6 B), HBV 3.5 kb RNA level Figure 6 C).
[0036] The results showed that compared with the blank control group (vehicle) and the negative control group (2KR-peptide-2#), the CRTC2 lactylation modification level of HepAD38-Tet(-) cells treated with 2K-peptide-2# was reduced, and the HBV Total RNAs and HBV 3.5 kb RNA levels were significantly decreased Figure 6 ).
[0037] Example 6. Effect of CRTC2 lactylation-specific polypeptide inhibitors on HBV transcription in an animal model
[0038] A mouse model of HBV infection was established by tail vein injection of AAV-HBV in C57BL / 6 mice. Seven days after injection, the model mice were randomly divided into three groups: a blank control group (vehicle), a 2K-Peptide-2# group, and a negative control group (2KR-Peptide-2#). Mice in each group received the drug via intraperitoneal injection at a dose of 10 mg / kg / day for a total of 10 days. All mice were then sacrificed, and liver tissue samples were collected. A suitable amount of liver tissue was taken, and proteins were extracted. Endogenous IP assays using Anti-CRTC2 antibody and Pan-Kla Western blotting assays were performed to detect the lactation modification level of CRTC2. Figure 7 A), and normalized the concentrations between samples according to the internal control. An appropriate amount of liver tissue samples were also taken for total RNA extraction, reverse transcription, and qRT-PCR detection of HBV Total RNAs. Figure 7 B) HBV 3.5kb RNA level ( Figure 7 C).
[0039] The results showed that compared with the blank control group (vehicle) and the negative control group (2KR-peptide-2#), HBV-infected mice treated with 2K-peptide-2# showed a decrease in CRTC2 lactation modification levels, and a significant decrease in HBV total RNAs and HBV 3.5kbRNA levels. Figure 7 ).
[0040] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A CRTC2 lactylation-specific polypeptide inhibitor, with an amino acid sequence of YGRKKRRQRRRPRKFSEKIALQK QRQAEE. 2.Use of the CRTC2 lactylation-specific polypeptide inhibitor of claim 1 in the preparation of an anti-HBV drug.
3. Use according to claim 2, characterized in that: The anti-HBV drug is an anti-HBV transcription drug.