Use of carnosic acid as an erap1 inhibitor for the preparation of a medicament for the treatment of ankylosing spondylitis

Abstract

The application relates to the field of biotechnology, and particularly relates to the application of carnosic acid as an ERAP1 inhibitor in the preparation of a drug for treating ankylosing spondylitis of an autoimmune disease. The application first discovers that a monomer compound carnosic acid from a natural product source can be used as an ERAP1 direct targeting inhibitor in the preparation of a drug for regulating an endogenous antigen processing and presentation pathway and ankylosing spondylitis of a related autoimmune disease. The carnosic acid can be specifically combined in an ERAP1 active site through direct high-activity targeting, and the carnosic acid inhibits the activity of the ERAP1, is the highest in activity in the world, and has high selectivity in targeting the ERAP1 active site. The carnosic acid reverses the ankylosing spondylitis molecular pathological phenotypes caused by abnormal ERAP1 activity, including endoplasmic reticulum stress, reducing an extra amount of HLA-B27 molecules displayed on a cell surface, reducing the proportion of the HLA-B27 molecules incorrectly assembled on the cell surface, and maintaining normal antigen presentation functions.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to the use of oxalic acid as an ERAP1 inhibitor in the preparation of drugs for the treatment of ankylosing spondylitis, an autoimmune disease. Background Technology

[0002] HLA-B27-related autoimmune diseases, such as ankylosing spondylitis, severely impact patients' quality of life and ultimately lead to disability and loss of earning capacity. To date, the pathogenesis of HLA-B27-mediated diseases remains unclear. The most mainstream view holds that the misfolding property of the HLA-B27 molecule, leading to endoplasmic reticulum stress and its incorrect presentation to the cell surface, is a key factor in mediating these diseases. This view is supported by a wealth of experimental data, ranging from molecular to clinical data. However, there are currently no highly effective and specific interventions targeting this mechanism.

[0003] With advancements in genetics and immunology, it has become increasingly clear that while HLA-B27 is the undisputed most relevant molecule for ankylosing spondylitis (AS), the resulting autoimmune diseases are influenced and regulated by multiple factors and genes. Therefore, endoplasmic reticulum aminopeptidase 1 (ERAP1) has attracted increasing attention from researchers. It is the second most important molecule after MHC class I in its association with various serious autoimmune diseases, including ankylosing spondylitis. ERAP1 belongs to the M1 zinc metallopeptidase-oxytocin subfamily. Its main function is to cleave endogenous N-terminal elongated peptide precursors in the endoplasmic reticulum during the processing and presentation of endogenous antigenic peptides. These precursors are then loaded into the antigenic peptide grooves of MHC class I molecules, presenting them to the cell surface to activate CD8+ T cells or NK cells, triggering the corresponding immune response. In recent years, numerous experimental results have demonstrated that excessive ERAP1 activity exacerbates HLA-mediated autoimmune phenotypes. For example, highly active ERAP1-SNPs increase ER stress caused by HLA-B27 molecule misfolding and assembly, and increase the number of HLA free heavy chains (FHC) and HLA dimers (HD) on the cell surface, leading to the onset or severity of AS. In fact, the strong genetic association between ERAP1 and HLA influencing this large class of autoimmune diseases known as "MHC-I opathy" is also evident at the molecular level. ERAP1 and HLA molecules, as core molecules, are jointly located in the endoplasmic reticulum (ERR) and participate in the processing and presentation of endogenous antigens. ERAP1, as a key "editor" in the final cleavage of mature antigen peptide profiles in the ER, as mentioned earlier, directly affects the correctness and efficiency of HLA molecule assembly, thus influencing this class of diseases. On the other hand, this crucial role of ERAP1 also affects the efficacy of tumor immunotherapy. Numerous studies in recent years have shown that excessive ERAP1 cleavage of antigenic peptides disrupts tumor neoantigen peptides, preventing HLA-I molecules from presenting sufficient tumor antigen peptides and thus hindering the activation of CD8+ T cells and NK cells, leading to tumor immune escape. Therefore, ERAP1 has become a key target protein for various severe autoimmune diseases, including ankylosing spondylitis (AS), and tumor immunity. Inhibition of ERAP1 function will significantly reduce disease phenotypes associated with MHC-I lesions, such as the excessive release of cytokines like IL-17 and TNF-α mediated by endoplasmic reticulum stress and misfolded HLA molecules on the cell surface, as well as inhibit tumor immune escape.

[0004] Given the crucial role of highly active ERAP1 in major autoimmune diseases such as AS and in tumor immune escape, coupled with the existence of another subtype of endoplasmic reticulum aminopeptidase in humans, ERAP2, which has a different substrate preference and activity than ERAP1, the development of highly active and selective ERAP1 inhibitors has become a significant pharmaceutical research endeavor. However, current research shows that most traditional inhibitors that completely target the active site are peptide-like compounds. While possessing strong inhibitory activity at the nanomolar level, they lack selective inhibitory activity against ERAP1 and structural novelty, such as the phosphorus-containing amino acid derivative DG013A and its series of derivatives. As a more recent approach, inhibitors targeting ERAP1 allosteric sites exhibit strong selectivity, but their activity is generally limited, such as arylsulfonamides, benzofurans, 3,4-diaminobenzoic acid derivatives, and the natural product chlorine. Therefore, novel inhibitors possessing high activity, high selectivity, novel structure, and druggability have become a key focus of research. Summary of the Invention

[0005] The purpose of this application is to provide the application of the natural product sarsaparilla oleracea as a novel and highly effective ERAP1-targeting inhibitor in the preparation of drugs that regulate the processing and presentation pathway of endogenous antigens and for the treatment of ankylosing spondylitis.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0007] This invention provides the application of oxalic acid in the preparation of endoplasmic reticulum aminopeptidase ERAP1 inhibitors.

[0008] Furthermore, in the above technical solution, the natural product sarsaparilla acid is used as a targeted inhibitor of ERAP1 in regulating ERAP1 activity and physiological function.

[0009] Furthermore, in the above technical solution, the natural product oxalic acid obtained by screening for targeting the ERAP1 catalytic site can significantly inhibit the activity of ERAP1. The high activity of oxalic acid in targeting and inhibiting ERAP1 is reflected in the fact that it is the compound with the highest selectivity for ERAP1 / ERAP2 targeting the ERAP1 active site worldwide. Oxalic acid does not exhibit inhibitory activity against the homologous protein ERAP2 or the proteases IRAP, Aps, MMP-1, Trypsin, and Furin. Simultaneously, due to its natural product origin, it possesses good medicinal value.

[0010] In the above technical solution, oxalic acid targets the catalytic domain of ERAP1 and has a strong direct interaction with ERAP1. The M1 zinc metallopeptidase oxytocin subfamily has high conservation in its functional structure, and oxalic acid has good target selectivity for proteins inside and outside this family.

[0011] This invention also provides the application of the natural product sarsaparilla acid in the preparation of drugs for diseases caused by endoplasmic reticulum stress due to abnormal ERAP1 activity.

[0012] Furthermore, in the above technical solution, ursolic acid is used as an ERAP1 inhibitor in the preparation of drugs for treating ankylosing spondylitis.

[0013] Furthermore, in the above technical solution, sarsaparilla oxalate inhibits ERAP1 activity, thereby regulating the endogenous antigen peptide processing and presentation pathway, a key factor in the pathogenesis of ankylosing spondylitis.

[0014] Furthermore, in the above-mentioned technical solution, sarsaparilla acid significantly inhibited the activation of the epigenetic endoplasmic reticulum stress-related pathway, a key disease molecule induced by ERAP1, in an HLA-B27-mediated ankylosing spondylitis model, including the downregulation of the expression levels of proteins such as CANX, BIP, and CHOP.

[0015] Furthermore, in the above-mentioned technical solution, sarsaparilla oxalate inhibits the endoplasmic reticulum stress response caused by HLA-B27 molecule misfolding due to abnormal ERAP1 activity and the pathological phenotype mediated by HLA-B27 misassembly and presentation of misfolded molecules, thereby maintaining normal antigen presentation and reducing the amount of extra HLA molecules displayed on the cell surface in the HLA-B27 mediated model.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This application is the first to discover that the monomeric compound succinate, derived from a natural product, can be used as a direct-targeting inhibitor of ERAP1 in the preparation of drugs that regulate the endogenous antigen processing and presentation pathway and related autoimmune diseases, such as ankylosing spondylitis. This invention reveals that succinate can specifically and directly and highly actively bind to the active site of ERAP1, inhibiting ERAP1 activity. This is manifested in its lack of inhibitory activity against the homologous protein ERAP2 and representative homologous proteases, thus exhibiting high ERAP1 / ERAP2 selective inhibitory activity. It is the compound with the highest activity and selectivity currently available worldwide that targets the active site of ERAP1. Furthermore, due to its natural product origin, it possesses good medicinal value; it exhibits strong direct interaction with ERAP1 both in vivo and in vitro, reversing the molecular pathological phenotype of ankylosing spondylitis caused by abnormal ERAP1 activity, including endoplasmic reticulum stress response and reducing the amount of additional HLA-B27 molecules displayed on the cell surface, while also reducing the proportion of misassembled HLA-B27 molecules on the cell surface, maintaining normal antigen presentation function.

[0018] This invention marks the first discovery of ergosaccharide, a food-derived natural product, as a highly active and selective ERAP1-targeting inhibitor. It is the most active compound worldwide that targets the ERAP1 active site with the highest selectivity. This research provides a novel drug candidate for ankylosing spondylitis. Attached Figure Description

[0019] Figure 1 For protein validation, screening process and chemical structure of oxalic acid;

[0020] A. Purified ERAP1 and ERAP2;

[0021] B. Enzyme activities of ERAP1 and ERAP2;

[0022] C. Guidelines for the entire screening process, including virtual screening and physical screening;

[0023] Chemical structure of compound D 3-23.

[0024] Figure 2 The effect of succinic acid on the hydrolysis of ERAP1 substrates;

[0025] A. The IC50 of the compound sarsaparilla oxalate on the hydrolysis of L-AMC by ERAP1 50 test;

[0026] B. The IC50 of compound sarsaparilla acid on ERAP2 hydrolysis of R-AMC 50 test;

[0027] C. The IC50 of the compound sarsaparilla acid on the ERAP1-hydrolyzed natural long peptide substrate analog peptide VAFKARAF 50 test;

[0028] D. The inhibitory activity of the compound sarsaparilla acid at 50 μM against trypsin, furin, APs, MMP1, ERAP2, IRAP and ERAP1;

[0029] E. Michaelis-Menten analysis of L-pNA hydrolyzed by ERAP1 in the presence of 10, 20 or 30 μM sarsaparilla acid;

[0030] F. Lineweaver–Burk inhibition curves at different 3-23 concentrations.

[0031] Figure 3 To verify the direct binding of erapoxetine to ERAP1 at the molecular and cellular levels;

[0032] A. The affinity of oxalic acid for binding to ERAP1 was analyzed by BLI;

[0033] B. Representative Western blots show the thermal stability of ERAP1 after thermal stimulation in the presence of oxalic acid.

[0034] C. Quantitative analysis of the thermal stability of ERAP1 in three independent experiments, n=3±SEM, the stability of ERAP1 in the presence of oxalic acid is represented by ΔTm;

[0035] D. Representative Western blots show the thermal stability of ERAP1 after heat stimulation at 60°C in the presence of different concentrations of sarsaparilla acid;

[0036] E. CETSA ITDRF showed that the stability of ERAP1 at 60°C was dose-dependent; OC 50 =9.3μM, n=3±SEM, normalized with tubulin.

[0037] Figure 4 Structural properties of the ERAP1-sarsaparilla acid complex;

[0038] A. Optimized structure of ERAP1(3MDJ) after docking with oxalic acid; blue and purple represent the α-helix and β-sheet of the ERAP1 protein, respectively; yellow bars represent oxalic acid; green bars represent the surrounding amino acid residues most relevant to the interaction with oxalic acid (O: red, N: blue, S: orange); pink and green dashed lines represent the interaction bonds between the ERAP1 protease and oxalic acid;

[0039] A two-dimensional diagram of the interaction between B.ERAP1 and oxalic acid; triangles represent amino acid residues surrounding oxalic acid, labeled red (negatively charged), purple (positively charged), green (hydrophobic), and blue (polar) according to their side chain chemical properties; the colored bands between the residues and oxalic acid represent pockets; pink arrows represent hydrogen bond interactions;

[0040] Comparison of active site residues of CD.ERAP1 (PDB ID: 3MGQ, green) with oxalic acid and Bestatin (PDB ID: 3MGQ, orange) or DG014 (PDB ID: 6MGQ, purple); blue-purple bars represent Bestatin, gray bars represent DG014 (O: red, N: blue, S: orange); E. Detection of inhibitory activity of mutant ERAP1.

[0041] Figure 5 A comparison of the binding modes of arugula acid with ERAP1 and ERAP2;

[0042] A. Surface representation of the complex of arugula acid and ERAP1 / 2 (PDB ID: 3MDJ / 7SH0); ERAP1 is shown in gray, and ERAP2 is shown in cyan; the binding of arugula acid to ERAP1 is represented by yellow bars, and the binding to ERAP2 is represented by pink bars; zinc ions are represented by gray spheres, and the key homologous differential amino acid residues located at the "gate" of the binding site are Ser316 (ERAP1, blue) / Pro333 (ERAP2, red);

[0043] Close-up of important residues in B.ERAP1 / ERAP2 that interact with erucic acid, shown in rods (C: ERAP1 is green, ERAP2 is cyan, O: red, N: blue), and the GAMEN ring is shown as a white sphere;

[0044] Sequence alignment of homologous amino acids in C.ERAP1 and ERAP2;

[0045] Figure 6 To regulate the endoplasmic reticulum stress response mediated by ERAP1-mediated HLA-B27 misfolding using oxalic acid; A. Whole-cell extracts of HEK293T cells overexpressing ERAP1 and HLA-B27 were subjected to SDS-PAGE, followed by Western blotting of ERAP1 and representative ER stress proteins CANX, BIP and Chop, with tubulin as a loading control;

[0046] Quantitative analysis of B.CANX; n = 3 ± SEM; *p < 0.05; **p < 0.01;

[0047] Quantitative analysis of C.BIP; n = 3 ± SEM; *p < 0.05; **p < 0.01;

[0048] Quantitative analysis of D. Chop; n = 3 ± SEM; *p < 0.05; **p < 0.01.

[0049] Figure 7 Succinic acid reduces disease-associated phenotypes by inhibiting ERAP1, which affects the presentation of endogenous antigenic peptides in living cells.

[0050] AB. SDS-PAGE analysis was performed on endoplasmic reticulum extracts from HEK293T cells overexpressing ERAP1 and HLA-B27, followed by Western blotting of HLA, with TAP-1 as an internal control; each bar plot shows the mean of n = 3 ± SEM; *p < 0.05; **p < 0.01;

[0051] CF. SDS-PAGE analysis was performed on cell membrane extracts from HEK293T cells overexpressing ERAP1 and HLA-B27, followed by Western blotting of HLA and β2m, with ATPase as an internal control; each bar plot shows the mean of n = 3 ± SEM; *p < 0.05; **p < 0.01;

[0052] G. Immunofluorescence localization of HLA expression on the cell membrane; blue indicates DAPI staining, and green indicates HLA staining.

[0053] Whole-cell immunofluorescence quantification of H.HLA expression; blue represents DAPI staining, and green represents HLA staining. Detailed Implementation

[0054] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0055] In the following embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0056] The compound library is derived from a commercial compound library from MCE.

[0057] Example 1: High-throughput drug screening targeting ERAP1

[0058] This study utilized the natural product libraries of MedChem Expression and COCONUT, along with the crystal structure of ERAP1 (PDB ID: 3MDJ), for virtual screening. First, LeDock was used as a rapid screening tool to initially screen 200,000 compounds, identifying those with LeDock scores below -6 kcal / mol. This screening was repeated to eliminate unstable and false positive results. Subsequently, AutoDock Vina was used for fine-tuning, ultimately identifying 3250 natural product molecules with Vina scores less than -7.6 kcal / mol for physical screening experiments. All compounds used for physical screening were derived from MCE. High-purity ERAP1 and ERAP2 proteins (such as...) were also used. Figure 1 As shown in A), and a reasonable enzyme reaction system (such as...) Figure 1 (As shown in B) was used for entity screening. Several candidate compounds were identified through experiments and calculations. To ensure the accuracy of the screening results, these compounds were re-screened and tested three times, as follows: Figure 1 As shown in C, after two rounds of virtual screening and three rounds of re-screening and testing, we obtained sarsaparilla acid, a compound with inhibitory potential against ERAP1.

[0059] The structural formula of sarsaparilla acid (numbered 3-23 in this study) is as follows:

[0060]

[0061] Example 2: Determination of the specificity and inhibition mechanism of oxaloolase activity in rat tails

[0062] Inhibitor screening was performed using 96-well plates. Each well contained 100 μL of reaction solution, including 25 mM Tris-150 M mNaCl buffer (pH 8.0), 2 μg of ERAP1 or ERAP2 protein, and substrate (final concentration 1.6 μM L-pNA for ERAP1, K-pNA for ERAP2) to initiate the reaction. Absorbance at 405 nm was monitored using a Thermo Scientific Varioskan Flash multi-plate reader. To measure enzyme kinetics, substrate concentration gradients were set at 0 μM, 0.08, 0.16 μM, 0.32 μM, 0.48 μM, 0.64 μM, 0.8 μM, 1.2 μM, 1.6 μM, 2.0 μM, 2.4 μM, and 3.2 μM, and compound concentration gradients at 0 μM, 10 μM, 20 μM, and 30 μM. The sample was incubated at 37°C for 15 minutes in a 25 mM Tris 150 M NaCl buffer solution (pH 8.0), and quenched with 1% formic acid. The absorbance at 405 nm was monitored using a Thermo Scientific Varioskan Flash multi-mode microplate reader. Results are as follows: Figure 2 A shows that the inhibitory activity of oxalic acid on ERAP1 is IC50. 50 The value is 10.73 μM. (For example...) Figure 2 B shows that the inhibitory activity of oxalic acid on ERAP2 is IC50. 50 The value is much greater than 200 μM. For example... Figure 2 C showed that oxalic acid inhibited the degradation of another long peptide substrate by ERAP1 by 16.98 μM. Select the results of the inhibitory activity assay, such as... Figure 2 D. Raphanusolic acid exhibits little or no inhibitory activity against other proteases in the ERAP1 family, demonstrating good selective inhibitory ability. The initial rate of the enzymatic reaction was tested and plotted; the time to reach the maximum reaction rate of ERAP1 was prolonged with the addition of inhibitors, such as... Figure 2 As shown in E. Simultaneously, in the double reciprocal plot, the inhibition curves of different concentrations of 3-23 have the same Y-intercept, intersecting at different positions on the X-axis, indicating that changes in inhibitor concentration lead to changes in Km of ERAP1. For example... Figure 2 As shown in F, the analysis shows that if the Vmax value remains unchanged and Km increases, then the way 3-23 inhibits ERAP1 activity is competitive inhibition.

[0063] Example 3: Determination of the affinity of oxalic acid for ERAP1 binding

[0064] 1. BLI (Biomembrane Interference Assay): The test compound was diluted to an appropriate concentration (1-100 μM) using 25 mM Tris 150 M NaCl buffer. ERAP1 protein was immobilized on a biosensor coated with nickel triacetate (Ni-NTA, Fortebio, USA). The biosensor tip was immersed in wells containing a series of dilutions and allowed to bind (200 seconds), followed by a dissociation step (150 seconds). 25 mM Tris 150 M NaCl containing 1% DMSO (Sigma, USA) was used as a control. KD values ​​were fitted using Dataanalysis90 software via Global (Full) cohort analysis. Results are as follows: Figure 3 As shown in Figure A, oxalic acid has a strong affinity for ERAP1 protein, reaching 9.05 μM.

[0065] 2. CETSA and CETSA-ITDRF (Protein Thermal Migration Series Assays): HEK293 cells were seeded in 6-well plates, with 500,000 cells per well. When the cells reached 60-80% saturation, the plasmid overexpressing ERAP1 (pcDNA3.1-ERAP1) was transfected into the cells using a Lipo2000 transfector. After 6 hours, the culture medium was changed, and after 24 hours, oxalic acid was added to a final concentration of 50 μM (1% final DMSO), and the cells were incubated for 4 hours. Cells were then collected and aliquoted (30 μL per condition) into 10 0.2 mL PCR microtubes. The tubes were then briefly heated to a temperature range of 50°C to 80°C for 5 minutes using a PCR thermal cycler, followed by cooling to room temperature for 3 minutes. After adding 20 μL of lysis buffer containing a mixture of commercially available protease inhibitors (SW105-02, Sevenbio) (100X) to each PCR tube, protein extraction was performed by three freeze / thaw cycles in liquid nitrogen. Insoluble proteins were separated by centrifugation (20000g, 20 min, 4℃), and 30 μL of supernatant corresponding to soluble proteins was stored for Western blotting. Quantification of the Western blots was performed using ImageJ software. Three independent experiments were conducted for each condition. The CETSA-ITDRF experiment was performed as described in the CETSA experiment, except that cells were incubated with different drug concentrations for 4 h, cells were collected, heat-shocked at 60℃ for 5 min, and then proteins were extracted and analyzed by Western blotting as described above. The results are as follows. Figure 3As shown in the BE, ERAP1 protein degraded with increasing temperature after heating from 50°C to 80°C for 5 min, while the protein co-incubated with the compound succinic acid showed significantly less degradation with increasing temperature. This demonstrates that succinic acid can effectively stabilize ERAP1 in complex cellular systems and has a direct targeting effect on ERAP1. Furthermore, ITDRF was used to measure the maximum half-full occupancy concentration (OC). 50 The result is OC. 50 =9.3 μM, proving that the compound sarsaparilla acid has a concentration-dependent effect, and half of its stability can be achieved at a concentration of 9.3 μM.

[0066] Example 4: Study on the interaction mode between oxalic acid and ERAP1

[0067] To investigate the competitive inhibition mode of arbutin against the target ERAP1 and the mechanism of its strong selectivity for ERAP1 / ERAP2, theoretical calculation methods such as the structure of the ERAP1-arbutin molecular docking complex, interaction energy calculation, and molecular dynamics simulation, as well as amino acid point mutagenesis experiments, were employed in this study. Figure 4 AB analysis revealed that succinic acid occupies the catalytic center position of ERAP1, which is centered around the "GAMEN loop." It forms a stable hydrogen bond network with surrounding important residues Gln181, Glu183, Ser316, Ala318, Met319, and the catalytic residue Glu320. Simultaneously, it forms hydrophobic interactions with His160, Pro184, and His353. Notably, the two hydroxyl groups of succinic acid simultaneously form hydrogen bonds with Glu183, with an interaction energy as high as -16.45 kcal / mol, making it the core of the entire interaction system. Figure 4 CD analysis revealed significant differences between the ERAP1-sarstilbene complex structure and the resolved crystalline structures of Bestatin, a classic broad-spectrum aminopeptidase inhibitor commonly used as a positive control but lacking selective ERAP1 / ERAP2 inhibitory activity, and DG014, a highly active but low-selective phosphorylated tripeptide transition state analog. The main difference lies in the fact that sarstilbene binds primarily to the outer and upper parts of the ERAP1 catalytic pocket, while the other two non-selective inhibitors bind mainly to the lower and inner parts. Figure 4 E shows that the reliability of our model and inferences was verified by site-directed mutagenesis experiments on some key amino acid residues in the active pocket of ERAP1 bound to oxalic acid.

[0068] To investigate the structural mechanism underlying the significant difference in ERAP1 / ERAP2 selectivity of arugula oxalate, we performed docking and optimization of arugula oxalate with ERAP2 (PDB ID: 7SH0) in the same manner, followed by superposition analysis with the ERAP1-arugula oxalate complex described earlier. The results are as follows... Figure 5 AC analysis revealed a certain angular deviation in the binding of succinic acid to ERAP1 and ERAP2, with its binding to ERAP1 being more biased towards the outer S1'-pocket. In contrast, when succinic acid binds to ERAP2, its conformation is significantly more downward and inward, resulting in its overall position being concentrated between the S1 and S1'-pockets. This is likely an important structural mechanism underlying its good ERAP1 / ERAP2 selectivity.

[0069] Example 5: Determination of the regulatory effect of succinate on endoplasmic reticulum stress response induced by ERAP1 overexpression in an HLA-B27-mediated ankylosing spondylitis model.

[0070] HEK293 cells were seeded in 6-well plates, 500,000 cells per well. When the cells reached 60-80% confluency, plasmids expressing ERAP1 (pcDNA3.1-ERAP1), HLA-B27 (pcDNA3.1-HLA-B27α), and β2m (pcDNA3.1-β2m) were transfected into the cells using a Lipo2000 transfection machine. The culture medium was changed after 6 hours, and total protein was extracted after 48 hours. The primary antibody used was anti-β-Tubulin (10094-1-AP, Pro). Proteins were collected using Proteintech antibodies against Flag (ab205606, Abcam), BIP (ab108613, Abcam), Chop (66741-1-lg, Proteintech), and CANX (66903-1-lg, Proteintech). Secondary antibodies included goat anti-rabbit (66903-1-lg, Proteintech) and goat anti-mouse (RGAM001, Proteintech). Immunoblotting analysis was performed. In simple terms, protein concentration was determined using a BCA kit after collection. 10 μg of cell protein from each group was separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane via electroblotting. The PVDF membrane was blocked with 5% skim milk and incubated overnight at 4°C with primary antibody. After washing, the membrane was co-incubated with secondary antibody for 2 hours, and protein expression was detected using a chemiluminescence analyzer. Finally, the protein blots were quantified using ImageJ software. The results are shown below. Figure 6 As shown, overexpression of ERAP1 in HLA-B27-mediated ankylosing spondylitis model cells can induce an increase in endoplasmic reticulum stress marker proteins—a strong molecular phenotype associated with the disease—proving that endoplasmic reticulum stress is caused by ERAP1 overexpression. However, the addition of oxalic acid significantly reduced endoplasmic reticulum stress proteins, demonstrating that oxalic acid can inhibit the endoplasmic reticulum stress response induced by ERAP1.

[0071] Example 6: Determination of the effect of succinate on the reversal of disordered presentation of key molecular phenotypic antigens in an HLA-B27-mediated ankylosing spondylitis model.

[0072] HEK293 cells were seeded in 10 mm dishes, with 3.5 million cells per dish. When the cells reached 60-80% confluency, plasmids expressing E1, HLA-B27, and β2m were transfected into the cells via PEI. The culture medium was changed after 6 hours. After 48 hours, membrane proteins (C500049, Sangon Biotech) and endoplasmic reticulum proteins (BB-36051, Bestbio) were extracted. Primary antibodies included anti-ATPase (55187-1-AP, Proteintech), anti-TAP-1 (68412-1-lg, Proteintech), and anti-HLA. Class I (15240-1-AP, Proteintech), anti-β2m antibody (66207-1-lg, Proteintech), and secondary antibodies (goat anti-rabbit (RGAR001, Proteintech) and goat anti-mouse (RGAM001, Proteintech)) were used for Western blotting analysis, as shown above. Protein concentration was determined using a BCA kit after collection. 10 μg of cell protein from each group was separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane by Western blotting. The PVDF membrane was blocked with 5% skim milk and incubated overnight at 4°C with primary antibody. The membrane was then washed and co-incubated with secondary antibody for 2 hours. Protein expression was detected using a chemiluminescence analyzer. Finally, the protein blots were quantified using ImageJ software. Results are shown below. Figure 7 As shown in Figure AB, transfection with ERAP1 in an HLA-B27-mediated ankylosing spondylitis model significantly increased the expression of HLA-B27 molecules, a key pathogenic molecule for ankylosing spondylitis, in the endoplasmic reticulum. Conversely, the addition of the compound oxalic acid significantly reduced the expression of HLA-B27 molecules. The entire presentation process was precisely analyzed by detecting the HC epitopes of HLA on the cell membrane surface and the expression level of β2m. Figure 7 CF results showed that ERAP1 overexpression in the HLA-B27-mediated ankylosing spondylitis model led to a large increase in HLA molecules on the cell surface, present in a very high HC ratio. This indicates that the majority of the additional HLA molecules presented to the cell surface are in the FHC and HD forms of HLA-B27. Currently, the most important understanding of the molecular phenotype of ankylosing spondylitis is the presence of a significantly higher proportion of FHC and HD HLA-B27 molecules in the synovial cells or related cells of the spinal joints of patients compared to healthy individuals, a phenomenon consistent with its pathogenic mechanism. The addition of oxalic acid can reduce the number of HLA-HC molecules while simultaneously decreasing the HC ratio, increasing the proportion of correctly assembled HLA ternary complex molecules, and reducing the proportion of misassembled HLA molecules. This helps maintain the rationality of the endogenous antigen presentation pathway, and immunofluorescence experiments also corroborate this phenomenon. Figure 7GH results showed that the addition of succinic acid 3-23 significantly reduced the expression of HLA on the cell membrane surface, proving that 3-23 can reduce the number of HLA molecules overexpressed on the cell surface caused by ERAP1.

[0073] The most widely accepted theories regarding the pathogenesis of ankylosing spondylitis are: ① the endoplasmic reticulum stress theory mediated by HLA-B27 misfolding; ② the theory that HLA-B27 misassembly leads to a significant increase in the number / proportion of abnormal HLA molecules on the cell surface; and ③ the theory of potential gut microbiota dysbiosis as a pathogenic factor. However, the ③ gut microbiota theory remains controversial. Therefore, we have demonstrated from the perspectives of both ① and ②, the two most crucial pathogenic models, that oxalic acid can reverse the ERAP1-mediated molecular pathological model of ankylosing spondylitis, demonstrating strong scientific validity and application potential.

Claims

1. Application of oxalic acid as an ERAP1 inhibitor in the preparation of drugs for the treatment of ankylosing spondylitis.

2. The application according to claim 1, characterized in that, Succinic acid inhibits ERAP1 activity and regulates the processing and presentation pathways of endogenous antigenic peptides.

Images