WO2023024351A1

WO2023024351A1 - Use of ggpp binding and allosteric activation of human fbp1 in preparation of anti-hepatocellular carcinoma drug

Info

Publication number: WO2023024351A1
Application number: PCT/CN2021/139760
Authority: WO
Inventors: 方雷; 章竞子; 李朝军; 赵越
Original assignee: 南京大学
Priority date: 2021-08-27
Filing date: 2021-12-20
Publication date: 2023-03-02
Also published as: CN113694072A; CN113975285A

Abstract

Provided is the use of GGPP binding and allosteric activation of human FBP1 in the preparation of an anti-hepatocellular carcinoma drug。 The amino acid sequence of the human FBP1 protein is as shown in SEQ ID No. 1. A direct binding protein action network of GGPP in a normal liver is established, and a plurality of GGPP binding proteins involved in the regulation of hepatic glycolipid metabolism are screened and verified. The GGPP specifically binds to FBP1 and up-regulates the enzyme activity of FBP1 to promote gluconeogenesis, and can inhibit the migration of hepatocytes and hepatoma cells. The GGPP can regulate the molecular mechanism of the occurrence and development of hepatocellular carcinoma by means of sensitizing the metabolic balance of a target protein FBP1-coupled glycolipid, which can be applied to the treatment of hepatocellular carcinoma. GGPP regulates the metabolic balance of a hepatic glycolipid by means of specifically and directly binding to a target protein FBP1 thereof, and provides a new potential target, drug and treatment method for the treatment of HCC.

Description

Application of GGPP binding and allosterically activating human FBP1 in the preparation of anti-hepatocellular carcinoma drugs

technical field

The invention relates to the field of biotechnology, in particular to the application of geranylgeranyl pyrophosphate GGPP in combination with and allosterically activating human FBP1 in the preparation of anti-hepatocellular carcinoma drugs.

Background technique

The regulation of glucose and lipid metabolism homeostasis is the basis for maintaining the body's life activities. In my country, due to changes in lifestyle and dietary structure in recent decades, the glucose and lipid metabolism patterns of some populations have changed accordingly, and the incidence of metabolic syndrome such as obesity and diabetes has increased significantly. As the central organ of metabolism, the liver bears the brunt of the long-term disturbance of glucose and lipid metabolism, leading to chronic liver diseases such as non-alcoholic fatty liver disease (NAFLD) and liver fibrosis (Liver fibrosis). Recent research results have shown that, in addition to viral hepatitis and alcoholic liver disease, chronic liver diseases such as metabolic syndrome and NAFLD are also important causes of primary liver cancer. At the same time, more and more attention has been paid to the prevention and treatment of cancer by controlling the glucose and lipid metabolism of cancer cells and using various strategies to specifically block the energy source of cancer cells. Therefore, the homeostasis of glucose and lipid metabolism plays an important role in the occurrence, development, prevention, early diagnosis and treatment of primary liver cancer.

As the main type of primary liver cancer, hepatocellular carcinoma (Hepatocellular carcinoma, HCC) is one of the most common malignancies in vivo worldwide, and it is also one of the most common causes of tumor-related death. In addition to surgical treatment, for patients with advanced liver cancer, the multi-kinase inhibitor Sorafenib (Sorafenib) is the only drug approved by many countries for the systemic treatment of advanced HCC, but it is expensive and has special adverse reactions. Effectiveness is also debatable. In addition, in terms of new drug development, clinical trials of other molecularly targeted agents such as Sunitinib, Brivanib, and Linifanib for the treatment of liver cancer have all obtained a series of negative results. And "new use of old drugs", in the process of high-investment and high-risk drug research and development, because it can greatly shorten the research and development time, cost and clinical application cycle, it has become a new trend in drug research and development. As the most common type of lipid-lowering prescription drugs in clinical practice, statins are used by a wide range of people and are mainly used in the treatment and prevention of hyperlipidemia associated with metabolic syndrome and various cardiovascular diseases. A large number of recent clinical statistics show that statins can reduce the risk of death of HCC and other cancer patients, suggesting the possibility of HCC as a new indication for Statins. However, the role of statins in the prevention and treatment of HCC remains uncertain. This may be related to the fact that statins inhibit the hydroxymethylglutaryl-CoA reductase (3-hydroxy-3-methylglutaryl-CoA reductase, HMGCR) located in the upstream of the mevalonate pathway, resulting in a variety of important downstream functions. Small metabolic molecules with biological functions, such as farnesyl pyrophosphate (FPP), squalene (Squalene), cholesterol (Cholesterol), geranylgeranyl pyrophosphate (GGPP) and ubiquinone ( Ubiquinone) and other synthetic hindered (as shown in Figure 1).

The body's response to the deletion of these metabolic small molecules is extremely complex, which may be one of the important reasons why statins have significant differences in the therapeutic effects of different cancer types and even different individual patients. To elucidate the role of these small molecules in the mevalonate metabolic pathway in the occurrence and development of HCC is of great significance for the application of statins in the treatment of HCC and the further development of related treatment options. Figure 1 is a diagram of the mevalonate pathway.

Among them, geranylgeranyl pyrophosphate (GGPP) is one of the important metabolic small molecules downstream of the mevalonate pathway, and is another important metabolic molecule of farnesyl pyrophosphate (FPP) besides cholesterol. product (as shown in Figure 1). The main functions of FPP and GGPP have been reported as substrates to participate in an important class of protein fatty acylation modification - prenylation modification, by changing the hydrophobicity of some specific proteins to make their localization and activation in the membrane system. The activity changes and regulates downstream signaling pathways to exert specific functions.

Geranylgeranyl pyrophosphate synthase 1 (GGPPS1) is a synthetase responsible for catalyzing the conversion of FPP to GGPP in vivo. In our laboratory, Ggpps1-specific knockout mice in different organs and tissues were used to study the effects of relative changes in FPP and GGPP levels on body functions. At present, the explanation of the phenotype of Ggpps1 knockout mice in the present invention focuses on the changes in the protein prenylation modification pattern after the FPP/GGPP balance is disrupted, that is, the loss of geranylation modification, and the excessive farnesylation activation of specific proteins Changes in downstream signaling pathways resulted in a series of phenotypes, such as cardiac hypertrophy in adult mice, reproductive impairment in male mice, and developmental disorders in oocytes in female mice. Analysis of skeletal muscle-specific Ggpps1 knockout heterozygous mice found that GGPPS1 loss triggers high-fat-induced systemic insulin resistance, confirming that GGPPS1 is involved in the regulation of lipid metabolism. Studies on liver-specific Ggpps1 knockout mice have shown that Ggpps1 knockout reduces the level of GGPP in the liver of mice, and also alleviates the fat accumulation caused by high-fat food in the liver, suggesting that GGPP plays an important role in the balance of glucose and lipid metabolism in the liver. Regulatory effect. More importantly, liver-specific Ggpps1 knockout mice are more likely to form primary liver cancer when undergoing DEN chemical mutagenesis; and clinical detection of liver cancer samples from HCC patients found that the expression level of GGPP synthetase GGPPS1 was correlated with the expression level of HCC. The degree of malignancy was positively correlated, and the prognosis of HCC patients with high expression of GGPPS1 was better, suggesting that GGPPS1 and GGPP may protect the liver through compensatory up-regulation during the progression of HCC progression. At the same time, Clarivate Analytics' Metadrug software analysis also showed that GGPP has significant anti-tumor activity. These results suggest that GGPP plays an important role in the regulation of glucose and lipid metabolism in the liver and the occurrence and development of liver cancer, but the direct target proteins regulated by it and the corresponding mechanism of action are still poorly understood.

FBP1 is a key rate-limiting enzyme in the process of gluconeogenesis, and it has also been proved to be an important tumor suppressor protein. Many studies have reported that the deletion, mutation and decreased expression of FBP1 can lead to the occurrence and development of various cancers such as hepatocellular carcinoma, clear cell renal cell carcinoma, non-small cell lung cancer, and breast cancer. In HCC, the absence or reduction of FBP1 may affect the Warburg effect, leading to glucose and lipid metabolism disorders and abnormal accumulation of lipids, providing a large amount of material and energy for cancer cells, and accelerating the occurrence and development of HCC. Statistical data also show that patients with high FBP1 expression levels in HCC have significantly better prognosis, which makes FBP1 a potential predictive marker for HCC prognosis.

At present, there is a lack of application of a geranylgeranyl pyrophosphate GGPP that binds to and allosterically activates human FBP1 in the preparation of anti-hepatocellular carcinoma drugs.

Contents of the invention

In order to solve the deficiencies in the prior art, the present invention provides an application of geranylgeranyl pyrophosphate GGPP in combination with and allosterically activating human FBP1 in the preparation of anti-hepatocellular carcinoma drugs.

In order to achieve the purpose of the above invention, the technical scheme adopted in the present invention is as follows: the application of geranylgeranyl pyrophosphate GGPP of the present invention in combination with and allosterically activating human FBP1 in the preparation of anti-hepatocellular carcinoma drugs, said geranylgeranyl pyrophosphate GGPP The chemical formula of geranyl pyrophosphate GGPP is as shown in formula (I):

The amino acid sequence of the human source FBP1 protein is shown in SEQID No.1.

Further, the geranylgeranyl pyrophosphate GGPP specifically binds to FBP1 and up-regulates the enzyme activity of FBP1 to promote gluconeogenesis and inhibit the migration of hepatic parenchymal cells and liver cancer cells.

Further, the geranylgeranyl pyrophosphate GGPP sensitizes its target protein FBP1 to couple the balance of glucose and lipid metabolism, thereby regulating the molecular mechanism of the occurrence and development of hepatocellular carcinoma, and acting on hepatocellular carcinoma.

Furthermore, a GGPP probe with a Biotin tag was synthesized, and the GGPP-binding protein in primary mouse liver parenchymal cells was enriched using a biotin-streptavidin affinity purification system and detected by mass spectrometry. Multiple GGPP potential target proteins related to glucose and lipid metabolism, fructose-1,6-bisphosphatase 1 in gluconeogenesis pathway, hepatic pyruvate kinase in glycolysis pathway and carnitine palmitoyl involved in lipid oxidation Transferase 1.

Further, the GGPP specifically binds to FBP1 and up-regulates the enzymatic activity of FBP1 to promote gluconeogenesis, reverse the Warburg effect of tumors, and thereby inhibit the occurrence and development of various tumors.

Further, the anti-tumor drug is an anti-hepatocellular carcinoma drug.

Beneficial effects: the present invention adopts the high-throughput chemical proteomics method to establish the direct binding protein network of geranylgeranyl pyrophosphate GGPP in normal liver for the first time, and screens and verifies multiple proteins involved in the regulation of liver glucose and lipid metabolism. GGPP binding protein. From molecular level, cellular level, animal models and clinical cases, etc., the molecular mechanism that GGPP regulates the balance of glucose and lipid metabolism in the liver through specific and direct combination with its target protein FBP1, and then regulates the occurrence and development of HCC has been elucidated, and provides a basis for HCC. Therapeutics provide new potential targets, drugs and treatments.

Compared with the prior art, the present invention has the following advantages: the further study of the present invention on the interaction between geranylgeranyl pyrophosphate GGPP and FBP1 found that GGPP can specifically bind to FBP1 and up-regulate the enzyme activity of FBP1 to promote gluconeogenesis growth, and inhibit the migration of hepatic parenchymal cells and liver cancer cells; with the help of molecular docking, FBP1 mutants and cryo-electron microscopy experiments, the present invention confirmed the binding mode and key sites of GGPP and FBP1 and the mechanism of allosteric activation of FBP1. It will further elucidate the molecular mechanism of GGPP regulating the occurrence and development of hepatocellular carcinoma by sensitizing its target protein FBP1 to couple the balance of glucose and lipid metabolism, and provide potential drugs and targets for the treatment of hepatocellular carcinoma.

Description of drawings

Figure 1 is a diagram of the mevalonate pathway.

Fig. 2 is a molecular diagram of Biotin-GGPP (BGPP) with a biotin label of the present invention.

Fig. 3 is a technical roadmap for screening metabolic small molecule GGPP target proteins of the present invention.

Fig. 4 is an affinity purification diagram of the metabolic small molecule GGPP target protein of the present invention.

Fig. 5 is a bioinformatics analysis diagram of the GGPP potential direct binding protein of the present invention. A-C. Gene Ontology analysis of GGPP-binding proteins: including analysis of biological processes, molecular functions, and cellular components; D. KEGG pathway analysis of GGPP-binding proteins; E. Functional classification of GGPP-binding proteins; F. Regulation of glucose metabolism in GGPP-binding proteins Protein-protein interaction network analysis of key enzymes in lipid metabolism.

Fig. 6 is a graph of FPP/GGPP ratio and phenotype of liver-specific Ggpps1 knockout mice of the present invention. A. The ratio changes of FPP/GGPP and FOH/GGOH in primary liver parenchymal cells of liver-specific Ggpps1-knockout mice; B. Liver morphology induced by normal diet and high-fat induction of wild-type and liver-specific Ggpps1-knockout mice; C. wild-type HE staining of liver slices with type and liver-specific Ggpps1 knockout, the vacuoles shown in WT HFD group are lipid droplets; D. Oil red staining of liver slices with wild-type and liver-specific Ggpps1 knockout, red dots show liver fat drop.

Fig. 7 is the GGPP-binding protein FBP1 peptide and the secondary spectrum identified by the affinity purification mass spectrometry of the present invention. A. The GGPP-binding protein FBP1 peptide identified by two affinity purification mass spectrometry; B. The second-order spectrum of the FBP1 peptide DFDPAINEYLQR2+ (m/z=740.85).

Figure 8 is a Western blot and MST verification diagram of multiple GGPP-binding proteins of the present invention. A. Western blot verification of GGPP binding proteins such as FBP1, ACADL and ACSL1; B. Verification of the binding of FBP1, ACADL and ACSL1 to GGPP; C. Verification of the binding of FBP1, ACADL and ACSL1 to BGPP.

Fig. 9 is a competitive binding experiment and SPR and BLI verification diagram of the GGPP-binding protein FBP1 of the present invention. A. Competitive binding experiment of GGPP binding protein FBP1; B. SPR verification of GGPP and FBP1 binding; C. BLI verification of GGPP and FBP1 binding.

Fig. 10 is a graph showing that FBP1 enzyme activity is significantly up-regulated after GGPP of the present invention binds to FBP1. A. The combination of GGPP and FBP1 does not affect the formation of FBP1 homotetramer; B. The combination of GGPP and FBP1 significantly up-regulates the enzyme activity of FBP1, while FPP has no activation effect.

Fig. 11 is a graph showing the change in the tetramer structure of FBP1 after GGPP binds to FBP1 revealed by the negative staining electron microscope of the present invention. A-B. Negative staining electron microscopy overall results of FBP1 tetramer after FBP1 and GGPP added; C-D. Negative staining electron microscopy collection of different conformations of FBP1 and GGPP-FBP1 particles after magnification; E-F. Negative staining electron microscopy of FBP1 and GGPP-FBP1 particles 3D reconstruction of conformation; G-J. 3D reconstruction reveals conformational and turn changes of the FBP1 tetramer upon GGPP binding.

Fig. 12 is a diagram of the X-ray crystal diffraction of the present invention revealing that GGPP binding is located in the middle cavity of the FBP1 tetramer.

Fig. 13 is a diagram showing that the FBP1-R50 site mutation of the present invention inhibits the activity of FBP1 in response to GGPP stimulation to inhibit liver cancer cells. A-B. Mutation of R50 reduces the ability of FBP1 to inhibit the viability and proliferation of hepatoma cell Huh7 in response to GGPP stimulation; C-D. Mutation of R50 reduces the ability of FBP1 to inhibit the viability and proliferation of hepatoma cell HepG2 in response to GGPP stimulation.

Fig. 14 is a graph showing the response to GGPP stimulation of FBP1 enzyme activity inhibited by the mutation of the GGPP binding site of the human FBP1 protein of the present invention. A. GGPP binding site mutation does not affect the formation of FBP1 homotetramer; B. GGPP binding site mutation inhibits FBP1 enzyme activity in response to GGPP stimulation.

Fig. 15 is a graph showing that the GGPP precursor GGOH of the present invention can reverse the occurrence and development of mouse hepatocellular carcinoma. A. Timeline of high-fat diet and chemical mutagenesis to create mouse primary hepatocellular carcinoma model; B. Normal control group, high-fat diet and chemical mutagenesis mouse primary hepatocellular carcinoma group and GGOH replenishment The reversal situation; C-E.B The comparison of the malignancy of the three groups of mouse hepatocellular carcinoma and the statistical analysis of the number of tumors and the size of the tumor.

Fig. 16 is a graph showing that GGPP treatment of the present invention significantly inhibits the viability and proliferation of liver cancer cells. A. GGPP treatment significantly inhibited the viability and proliferation of liver cancer cells Huh7; B. GGPP treatment significantly inhibited the viability and proliferation of liver cancer cells HepG2.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings, in which several embodiments of the present invention are shown, but the present invention can be realized in different forms, and is not limited to the text described On the contrary, these embodiments are provided to make the disclosure of the present invention more thorough and comprehensive.

The application of the geranylgeranyl pyrophosphate GGPP of the present invention in binding and allosterically activating human FBP1 in the preparation of anti-hepatocellular carcinoma drugs, the chemical formula of the geranylgeranyl pyrophosphate GGPP is as formula (I) Shown:

The geranylgeranyl pyrophosphate GGPP specifically binds to FBP1, up-regulates the enzyme activity of FBP1, promotes gluconeogenesis, and inhibits the migration of hepatic parenchymal cells and liver cancer cells.

The geranylgeranyl pyrophosphate GGPP sensitizes its target protein FBP1 to couple the balance of glycolipid metabolism, thereby regulating the molecular mechanism of the occurrence and development of hepatocellular carcinoma, and acts on hepatocellular carcinoma.

Synthesized GGPP probe with Biotin tag, used biotin-streptavidin affinity purification system to enrich GGPP-binding protein in primary mouse liver parenchymal cells and detected by mass spectrometry, and screened out multiple GGPP-binding proteins GGPP potential target proteins related to lipid metabolism, fructose-1,6-bisphosphatase 1 in gluconeogenesis pathway, liver pyruvate kinase in glycolysis pathway and carnitine palmitoyltransferase 1 involved in lipid oxidation.

The GGPP specifically binds to FBP1 and up-regulates the enzyme activity of FBP1 to promote gluconeogenesis, reverse the Warburg effect of tumors, and thereby inhibit the occurrence and development of various tumors.

The antitumor drug is an anti-hepatocellular carcinoma drug.

Example 1

The GGPP probe with Biotin tag was used to enrich the GGPP-binding protein in primary mouse liver parenchymal cells by using the biotin-streptavidin affinity purification system and detected by mass spectrometry, and screened out multiple glycolipids Metabolism-related GGPP potential target proteins, such as fructose-1,6-bisphosphatase 1 (Fructose-1,6-bisphosphatase, FBP1) in the gluconeogenesis pathway, liver pyruvate kinase (Liver pyruvate kinase in the glycolysis pathway , LPK) and carnitine palmitoyl transferase 1 (Carnitine palmitoyl transferase 1, CPT1) involved in lipid oxidation, etc. Among them, FBP1 has attracted attention due to its significant binding to GGPP (as shown in Figures 5-8), its important role in the regulation of glucose and lipid metabolism, and its correlation with various cancers.

1. Establish a technical route for the enrichment and identification of metabolic small molecule GGPP direct binding proteins

1.1 Design and synthesis of Biotin-GGPP probe

In order to enrich the proteins that directly bind to GGPP in the liver, the present invention synthesized a biotin-labeled GGPP probe (BGPP, Figure 2). In the BGPP molecule, biotin is added to the C3 position of the long-chain carbon skeleton of the GGPP molecule with a biotin label to ensure the flexibility of the front hydrophobic long chain and the back-end pyrophosphate of GGPP and can be exposed to bind with it protein interaction. Fig. 2 is a molecular diagram of Biotin-GGPP (BGPP) with a biotin label of the present invention.

Described GGPP (Chinese name: geranylgeranyl pyrophosphate) chemical formula is as shown in formula (I):

1.2 General technical route for protein screening of small metabolic molecules or small drug molecules

The present invention establishes a general technical route for screening metabolic small molecules or small drug molecular target proteins (Fig. 3). In order to identify target proteins that directly bind to and be regulated by GGPP in the liver, we synthesized GGPP molecules with biotin tags, and treated isolated and cultured 8-week-old C57BL/ 6J male mouse primary liver parenchymal cells. After the cells were lysed to extract the total protein, the GGPP-binding protein was affinity-purified by streptavidin magnetic beads, and mass spectrometry was analyzed by 2D LC-MS/MS, and then combined with the non-labeled proteomics quantification and biological analysis based on the number of spectra. The probable GGPP-interacting proteome in primary hepatic parenchymal cells of adult mice was obtained by letter analysis. Through this technical route, the present invention can screen the direct binding proteins of important metabolic small molecules or small drug molecules in the human body or in cells, and provide targets and theoretical basis for explaining the biological activities and molecular mechanisms of these small molecules. Fig. 3 is the screening technical route of the metabolic small molecule GGPP target protein of the present invention; Fig. 4 is the affinity purification diagram of the metabolic small molecular GGPP target protein of the present invention.

In the present invention, the primary liver parenchymal cells of 8-week-old C57BL/6J male mice are firstly separated, divided into three parts and cultured respectively. When adding small molecule drugs for experimental grouping, two negative controls were set up: the addition (Biotin&GGPP) group and the (BGPP&8 times GGPP) competition group, respectively, and the addition of BGPP group to treat the isolated and cultured mouse primary liver parenchyma cell. Four hours after drug addition, the primary hepatic parenchymal cells of the three groups of mice were lysed to extract the total protein (Input), and affinity purification was carried out by adding streptavidin magnetic beads to obtain the possible protein in primary hepatic parenchymal cells of adult mice GGPP-acting protein (AP). After silver staining of the binding protein obtained by the affinity purification of Biotin-GGPP, it was found that the Biotin-GGPP group had multiple distinct bands compared with the Biotin group (Figure 4), indicating that GGPP may indeed have a specific binding protein. Therefore, the present invention carried out further LC-MS/MS mass spectrometry analysis on these proteins. Fig. 5 is the bioinformatics analysis diagram of the GGPP potential direct binding protein of the present invention; Gene Ontology analysis of A-C.GGPP binding protein: including biological process, molecular function and cell component analysis; KEGG pathway analysis of D.GGPP binding protein; E. Functional classification of GGPP-binding proteins; F. Protein-protein interaction network analysis of key enzymes regulating glucose metabolism and lipid metabolism in GGPP-binding proteins.

After searching the database of the raw mass spectrometry data, 259 and 479 proteins were detected in the Biotin control group and Biotin-GGPP treatment group, respectively. The data of the two groups were integrated for relative quantitative comparison of Label Free, and the relative abundance was increased by more than 1.5 times in the Biotin-GGPP group compared with the Biotin control group, and the number of detected peptides was greater than 1 as the standard, and a total of 211 potential GGPP-binding proteins were obtained. indivual. Bioinformatics analysis was performed on these 211 proteins, including Gene Ontology analysis (Figure 5A-C), KEGG Pathway analysis (Figure 5D), protein function classification analysis (Figure 5E) and protein-protein interaction network analysis (Figure 5F) , the results showed that these proteins were heavily involved in various metabolic processes, especially fatty acid metabolism and glycolysis/gluconeogenesis pathways. These glucose and lipid metabolism-related proteins such as CPT1α and FBP1 can be used as candidate proteins for subsequent exploration of the mechanism of GGPP regulation of liver metabolic diseases and hepatocellular carcinoma (Table 1). The key enzymes regulating liver glucose and lipid metabolism in the GGPP direct binding protein of the present invention are as shown in Table 1:

Test example 1

Construction and phenotype analysis of liver-specific Ggpps1 knockout mice

In order to study the effect of GGPP deletion on liver lipid metabolism, the present invention successfully constructed Ggpps1 ^-/- Mx1-Cre liver-specific induction knockout mice, and carried out related phenotype analysis. In primary liver cells of liver-specific knockout Ggpps1 mice, metabolomics mass spectrometry detection showed that the ratios of FPP/GGPP and FOH/GGOH were significantly increased (Figure 6A), while the decrease of GGPP content and the increase of FPP content High significantly inhibited the accumulation of triglyceride (TG) in liver-specific Ggpps1 knockout mice, making the knockout mice insensitive to high-fat diet induction and not showing obvious fatty liver phenotype (Fig. 6B-D). Fig. 6 is the FPP/GGPP ratio and phenotype of the liver-specific Ggpps1 knockout mouse of the present invention; A. the ratio change of FPP/GGPP and FOH/GGOH in primary liver parenchymal cells of the liver-specific Ggpps1 knockout mouse; B. Liver morphology of wild-type and liver-specific Ggpps1 knockout induced by normal diet and high fat; C. HE staining of liver sections of wild-type and liver-specific Ggpps1 knockout, the vacuoles shown in WT HFD group are lipid droplets; D. Oil red staining of wild-type and liver-specific Ggpps1 knockout liver sections, red dots show lipid droplets in the liver.

Test example 2

GGPP participates in the occurrence and development of hepatocellular carcinoma by binding to FBP1 to regulate liver glucose and lipid metabolism

FBP1 is a key rate-limiting enzyme in the process of gluconeogenesis, and it has also been proved to be an important tumor suppressor protein. Many studies have reported that the deletion, mutation and decreased expression of FBP1 can lead to the occurrence and development of various cancers such as hepatocellular carcinoma, clear cell renal cell carcinoma, non-small cell lung cancer, and breast cancer. In stem cell carcinoma HCC, the deletion or reduction of FBP1 may affect the Warburg effect, leading to glucose and lipid metabolism disorders and abnormal accumulation of lipids, providing a large amount of material and energy for cancer cells, and accelerating the occurrence and development of HCC. Statistical data also show that patients with high FBP1 expression level in HCC have significantly better prognosis, which makes FBP1 a potential predictive marker for HCC prognosis.

Clinically, the detection of HCC patient samples found that the expression level of GGPPS1, the synthetase of GGPP, was positively correlated with the malignancy of HCC, and the prognosis of HCC patients with high expression of GGPPS1 was better, suggesting that GGPPS1 and GGPP may compensate for the progression of HCC progression. Sexual upregulation to protect the liver. Therefore, it is speculated that GGPP may affect the balance of glucose and lipid metabolism by binding to FBP1 and up-regulating its activity, and participate in the occurrence and development of HCC. Therefore, the present invention clarifies the molecular mechanism of GGPP regulating the balance of glucose and lipid metabolism through specific and direct binding with its target protein FBP1, and then regulating the occurrence and development of HCC from multiple levels such as molecular level, cellular level, animal models and clinical cases. , and provide new potential targets, drugs and therapeutic methods for the treatment of HCC.

1 Secondary spectrum of GGPP-binding protein FBP1 peptide

In two repeated affinity purification experiments using BGPP as a probe, the present invention identified human FBP1 protein, the number of peptides identified by protein spectrum was 7 and 5, respectively, and the overall coverage of human FBP1 protein was 28.7% % and 26% (Fig. 7A). The b-series and y-series product ion peaks of the FBP1 peptide DFDPAINEYLQR ²⁺ (m/z=740.85) perfectly cover the entire peptide (Fig. 7B), indicating that the present invention is indeed identified in the BGPP-binding protein Arrived at FBP1. Figure 7 is the GGPP-binding protein FBP1 peptide and its secondary spectrum identified by affinity purification mass spectrometry of the present invention; A. The situation of the GGPP-binding protein FBP1 peptide identified by two affinity purification mass spectrometry; B. FBP1 peptide DFDPAINEYLQR ²⁺ (m/z=740.85) Secondary Spectrum.

2 Western blot and MST verification of GGPP-binding protein FBP1

By Western blot, the present invention verified FBP1, ACADL and ACSL1 in BGPP-binding proteins: Compared with the negative control group of Biotin&GPPP, the AP samples of BGPP group contained significantly enriched FBP1, ACADL and ACSL1 proteins (Figure 8A). At the same time, in order to further verify the direct combination of glucose and lipid metabolism-related proteins such as FBP1, ACADL and ACSL1 with GGPP, the present invention expressed and purified the fusion protein with the EGFP tag using the E. ), which further confirmed that FBP1, ACADL and ACSL1 proteins can specifically and directly bind to GGPP, and the dissociation constant KD value of FBP1 and GGPP binding is 250nM (Fig. 8B-C). Figure 8 is a Western blot and MST verification diagram of multiple GGPP binding proteins of the present invention; Western blot verification of GGPP binding proteins such as A.FBP1, ACADL and ACSL1; verification of the combination of B.FBP1, ACADL and ACSL1 with GGPP; C. Validation of FBP1, ACADL and ACSL1 binding to BGPP.

3 Competitive binding experiments, SPR and BLI of GGPP binding to FBP1

FBP1, as a candidate molecule for subsequent biological function research, was further verified in various ways for GGPP binding. GGPP competition binding experiments showed that when high doses of free GGPP molecules existed, the binding between BGPP and human FBP1 protein decreased in a dose-dependent manner. When the concentration of free GGPP reached 40 μM (8 times that of the BGPP probe), the binding between BGPP and FBP1 was almost completely destroyed, demonstrating that BGPP specifically binds directly to human FBP1 protein ( FIG. 9A ). Surface plasmon resonance (SPR) (Fig. 9B) and molecular interaction (BLI) (Fig. 9C) experiments also showed that the FBP1 recombinant protein purified from E. coli could specifically bind to GGPP in vitro. The K _D value of the dissociation constant for the combination of FBP1 and GGPP detected by SPR was 262 nM ( FIG. 9B ). Fig. 9 is the competitive binding experiment of the GGPP binding protein FBP1 of the present invention and the SPR and BLI verification diagram; A. The competitive binding experiment of the GGPP binding protein FBP1; the SPR verification of the combination of B.GGPP and FBP1; the combination of C.GGPP and FBP1 BLI validation.

4 GGPP binding up-regulates FBP1 enzyme activity; Figure 10. GGPP combined with FBP1 significantly up-regulated FBP1 enzyme activity; A. GGPP combined with FBP1 did not affect the formation of FBP1 homotetramer; B. GGPP combined with FBP1 significantly up-regulated FBP1 enzyme activity, while FPP has no activating effect.

FBP1 is a key rate-limiting enzyme in gluconeogenesis. Therefore, it is of great significance to study whether GGPP binding to FBP1 affects its enzymatic activity. In order to clarify the biological function of the direct combination of GGPP and FBP1, the present invention further detected the effect of GGPP on the activity of FBP1. In vitro experiments confirmed that GGPP does not affect the formation of FBP1 homotetramers (Figure 10A), but it can significantly up-regulate the activity of FBP1 in a dose-dependent manner (Figure 10B), while FPP similar in structure to GGPP does not have this effect, which indicates that GGPP binds FBP1 and regulates its activity with specificity. However, the FBP1 activity inhibitor AMP reported in the literature can reduce the activity of FBP1 in a dose-dependent manner, which proves the effectiveness of the detection system.

5 Characterization of the binding mode of GGPP and FBP1; Figure 11 shows the negative staining electron microscope of the present invention reveals the structural changes of FBP1 tetramer after GGPP binds to FBP1; A-B. The overall negative staining electron microscope result of FBP1 tetramer after FBP1 and GGPP added; C-D .Negative stain electron microscopy acquisition of different conformations of FBP1 and GGPP-FBP1 particles after magnification; E-F. 3D reconstruction of the conformation of FBP1 and GGPP-FBP1 particles under negative stain electron microscopy; G-J. 3D reconstruction reveals the structure of FBP1 tetramer after GGPP binding Conformation and turn angle changes.

In order to elucidate the molecular mechanism of GGPP activating FBP1 activity, the present invention analyzed the conformational changes of FBP1 homologous protein tetramers after GGPP was added by cryo-electron microscopy. It was shown that binding of GGPP narrows the angle between the upper and lower FBP1 dimers in the FBP1 tetramer, causing a deflection of around 6 degrees (Fig. 11). After the addition of GGPP, the relative position of the two dimers in the FBP1 tetramer can be better stabilized and the relative angle between them can be reduced. Further X-ray crystallography results showed that the GGPP molecule specifically binds to the middle cavity of the FBP1 tetramer, and the amino acid residues of FBP1 that directly interact with it include R50, S46, P189 and A190 (Figure 12). This combination of GGPP is like a "door bolt", which prevents the rotation of the upper and lower dimers in the FBP1 tetramer to the inactive R state, thereby keeping the FBP1 tetramer in the active state to a large extent. The T state of the state, upregulates the activity of FBP1. Fig. 12 is a diagram of the X-ray crystal diffraction of the present invention revealing that GGPP binding is located in the middle cavity of the FBP1 tetramer.

According to the results of the above structure experiments, the present invention constructed FBP1 mutants and observed their ability to form FBP1 tetramers and changes in enzyme activity. The results showed that the mutation of the GGPP binding site of human FBP1 protein hardly affected the formation of FBP1 tetramer (Figure 14A), but significantly inhibited the response of FBP1 enzyme activity to GGPP stimulation (Figure 14B), confirming that these sites are important The association of FBP1 and GGPP is critical. Fig. 14 is a graph showing that the mutation of the GGPP binding site of the human FBP1 protein of the present invention inhibits the FBP1 enzymatic activity in response to GGPP stimulation; A.GGPP binding site mutation does not affect the formation of FBP1 homotetramer; B.GGPP binding The site mutation inhibits the response of FBP1 enzymatic activity to GGPP stimulation.

6 The combination of GGPP and FBP1 regulates the occurrence and development of hepatocellular carcinoma

In view of the important regulatory function of GGPP on the activity of FBP1 and the important function of FBP1 itself as a tumor suppressor protein, the present invention studies the function of the combination of GGPP and FBP1 in inhibiting the occurrence and development of mouse hepatocellular carcinoma. The results showed that when using high-fat food and chemical mutagenesis to create mouse primary hepatocellular carcinoma models, wild-type mice would form liver cancer (Figure 15A-B); Significantly inhibited the occurrence and development of mouse liver cancer (Fig. 15C-E). Figure 15 is a graph showing that GGOH of the present invention can reverse the occurrence and development of mouse hepatocellular carcinoma; A. the time line of high-fat food and chemical mutagenesis to manufacture the mouse primary hepatocellular carcinoma model; B. normal control group, high-fat Food and chemical mutagenesis mice primary hepatocellular carcinoma group and the reversal after GGOH replenishment; C-E.B The comparison of the malignancy of the three groups of mouse hepatocellular carcinoma and the statistical analysis of the number and size of tumors.

Similarly, in the liver cancer cell lines Huh7 and HepG2, GGPP treatment can also significantly inhibit the viability and proliferation of liver cancer cells ( FIG. 16 ). When FBP1-WT and FBP1-R50N were transferred into Huh7 or HepG2, the viability of these two cells had no significant change; however, when FBP1-WT and FBP1-R50N were transferred to GGPP treatment at the same time, the FBP1-WT/GGPP group was better than that of FBP1 -R50N/GGPP was significantly reduced, indicating that the FBP1-R50 site is critical for its response to GGPP stimulation (Figure 13). Figure 16 is a graph showing that GGPP treatment of the present invention significantly inhibits the viability and proliferation of liver cancer cells; A. GGPP treatment significantly inhibits the viability and proliferation of liver cancer cells Huh7; B. GGPP treatment significantly inhibits the viability and proliferation of liver cancer cells HepG2. Figure 13. FBP1-R50 site mutation inhibits FBP1 in response to GGPP stimulation to inhibit the viability of liver cancer cells; A-B. The mutation of R50 reduces the ability of FBP1 to inhibit the viability and proliferation of liver cancer cell Huh7 in response to GGPP stimulation; C-D. The mutation of R50 reduces The ability of FBP1 to inhibit the viability and proliferation of hepatoma cell HepG2 in response to GGPP stimulation.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have For various changes and improvements, the protection scope of the present invention is defined by the appended claims, description and their equivalents.

Claims

Geranylgeranyl pyrophosphate GGPP binding and allosteric activation of human fructose-1, 6-bisphosphatase 1 FBP1 in the preparation of anti-hepatocellular carcinoma drugs, characterized in that: the chemical formula of the GGPP such as the formula ( I) Shown:

The amino acid sequence of the human source FBP1 protein is shown in SEQID No.1.
The application according to claim 1, characterized in that: the geranylgeranyl pyrophosphate GGPP specifically binds to FBP1 and up-regulates the enzymatic activity of FBP1 to promote gluconeogenesis, and inhibits the growth of liver parenchymal cells and liver cancer cells. migrate.
The application according to claim 2, characterized in that: said geranylgeranyl pyrophosphate GGPP is a molecule that regulates the occurrence and development of hepatocellular carcinoma by sensitizing its target protein FBP1 and coupling the balance of glycolipid metabolism. mechanism, which can be applied to the treatment of hepatocellular carcinoma.
The application according to claim 3, characterized in that: synthesize the GGPP probe with the Biotin label, and utilize the biotin-streptavidin affinity purification system to enrich the GGPP binding in the mouse primary liver parenchymal cells Proteins were detected by mass spectrometry, and multiple potential target proteins of GGPP related to glucose and lipid metabolism, fructose-1,6-bisphosphatase 1 in the gluconeogenesis pathway, liver pyruvate kinase in the glycolysis pathway and participating Lipid-oxidizing carnitine palmitoyltransferase 1.
The application according to claim 1 or 4, characterized in that: the GGPP specifically binds to FBP1 and up-regulates the enzymatic activity of FBP1 to promote gluconeogenesis, reverse the Warburg effect of tumors, and thereby inhibit the occurrence and development of various tumors.
The use according to claim 1 or 4, characterized in that: the anti-tumor drug is an anti-hepatocellular carcinoma drug.