A small molecule inhibitor of palmitoyl transferase zdhhc8 and applications thereof

Abstract

The application discloses a small-molecule inhibitor of palmitoyltransferase zDHHC8 and application thereof. The application finds, through screening verification, a specific small-molecule inhibitor PF-670462 for palmitoyltransferase zDHHC8, the small-molecule inhibitor can effectively inhibit a zDHHC8-GPX4 palmitoylation modification signal axis and promote the sensitivity of tumor cells to ferroptosis. + The PF-670462 alone can effectively enhance the T cell-mediated tumor cell ferroptosis sensitivity and promote tumor immune infiltration, thereby inhibiting the growth and metastasis of tumors; meanwhile, the PF-670462 can enhance the tumor treatment effect of an immune checkpoint inhibitor, and the synergistic combination of the PF-670462 and an anti-PD-1 antibody can greatly enhance the anti-tumor effect, thereby improving the anti-tumor effect of ICB treatment and CAR-T cell therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a small molecule inhibitor of palmitoyltransferase zDHHC8 and its application. Background Technology

[0002] Tumor immunotherapy, particularly immune checkpoint inhibitor (ICB) therapy and chimeric antigen receptor (CAR) T-cell therapy, has revolutionized cancer treatment, ushering in a new era. The fundamental molecular mechanism by which most existing cancer immunotherapies exert their anti-tumor effects is through the restoration or enhancement of specific CD8+ receptors. + Effector function of T cells. CD8 + T cells can induce various types of cell death in tumor cells, including apoptosis, pyroptosis, necroptosis, and ferroptosis. Ferroptosis is a recently discovered form of programmed cell death characterized by lipid peroxidation. Ferroptosis depends on a balance between pro-feroptosis cellular activity and the ferroptosis defense system. When the ferroptosis defense system cannot withstand pro-feroptosis stress, lipid peroxidation is induced, triggering ferroptosis and inhibiting cell growth. Conversely, inhibiting ferroptosis promotes cell survival. The solute carrier family 7 member 11 / glutathione / glutathione peroxidase 4 (SLC7A11 / GSH / GPX4) pathway is considered the most important cellular defense system against ferroptosis. SLC7A11 is the systemic xc-transporting subunit, primarily responsible for cysteine ​​uptake. Cystine can be reduced to cysteine ​​intracellularly, serving as a raw material for glutathione synthesis. GPX4 utilizes glutathione to eliminate lipid peroxides, thereby protecting cells from ferroptosis. Inducing ferroptosis in tumor cells has been proven to be an effective method for enhancing anti-tumor immune responses. However, traditional ferroptosis inducers, such as RSL3, IKE, and ML162, not only induce ferroptosis in tumor cells but also inevitably lead to ferroptosis in immune cells within the tumor microenvironment, including cytotoxic CD8 cells. + T cell death significantly weakens anti-tumor immune responses. Therefore, selectively inducing ferroptosis in tumor cells is crucial for enhancing anti-tumor immune responses. Previous studies have shown that CD8+... + T cells can induce ferroptosis in tumor cells by secreting the cytokine IFN-γ, providing a feasible strategy for selectively inducing ferroptosis in tumor cells. However, tumor cells often develop resistance to ferroptosis through various pathways, thereby weakening CD8. + The anti-tumor effect is mediated by T cells (including CD8T cells after ICB treatment). Therefore, exploring the anti-ferroptosis mechanism of tumor cells is particularly important for finding drugs that effectively sensitize ICB treatment.

[0003] The applicant's previous research indicated that the palmitoyltransferase zDHHC8 can palmitoylate the ferroptosis core pathway molecule GPX4, thereby promoting ferroptosis resistance in tumor cells. Furthermore, the creation of zDHHC8-deficient cell lines through gene editing demonstrated that targeting zDHHC8 effectively enhances tumor cell sensitivity to ferroptosis and promotes anti-tumor immune responses. The study also found that zDHHC8 is highly expressed in various tumors, including pancreatic cancer, colorectal cancer, and melanoma. Therefore, zDHHC8 is a crucial target for effectively promoting anti-tumor immunotherapy. However, no specific small-molecule inhibitors of zDHHC8 have been reported to date. Summary of the Invention

[0004] The purpose of this invention is to overcome the above-mentioned defects and deficiencies in the prior art and to provide the application of CK1δ / ε inhibitor PF-670462 in the preparation of an inhibitor of palmitoyltransferase zDHHC8.

[0005] A second objective of this invention is to provide the use of the CK1δ / ε inhibitor PF-670462 in formulations that enhance the sensitivity of tumor cells to ferroptosis.

[0006] A third objective of this invention is to provide the application of the CK1δ / ε inhibitor PF-670462 in the preparation of potentiators for immune checkpoint inhibitors.

[0007] The fourth objective of this invention is to provide the application of the CK1δ / ε inhibitor PF-670462 in combination with an anti-PD-1 antibody in the preparation of anti-tumor drugs.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] Previous research results of this invention indicate that targeting zDHHC8 can effectively promote the ferroptosis sensitivity of tumor cells, thereby improving CD8+. +T-cell-mediated anti-tumor immunotherapy is a key area of ​​research. However, there are currently no specific small molecule inhibitors for zDHHC8. Therefore, this invention aims to develop a specific small molecule inhibitor targeting the zDHHC8 protein. Based on AlphaFold's prediction of the zDHHC8 protein core domain, this invention first used molecular docking to perform high-throughput screening of approximately 220,000 small molecule compounds. Further screening and validation of the top 5% of commercially available small molecules revealed PF-670462 as the most specific zDHHC8 inhibitor. Simultaneously, in vivo and in vitro experiments demonstrated that PF-670462 effectively promotes CD8 T-cell-mediated anti-tumor immunity, enhances the therapeutic effect of immune checkpoint inhibitors, and exhibits a significant synergistic anti-tumor effect with anti-PD-1 antibodies. In summary, this invention developed the first small molecule inhibitor of zDHHC8, PF-670462, and demonstrated that this small molecule inhibitor can downregulate the palmitoylation level of GPX4 protein and increase the sensitivity of tumor cells to ferroptosis by binding to and promoting the degradation of zDHHC8 protein via the lysosomal pathway. PF-670462 treatment can promote CD8... + T cell-mediated anti-tumor immunity selectively promotes CD8 + T-mediated tumor cell ferroptosis provides an effective strategy. PF-670462 alone can effectively enhance CD8+. + T cell-mediated tumor cell ferroptosis sensitivity and promotion of tumor immune infiltration, thereby inhibiting tumor growth and metastasis, and the synergistic combination of PF-670462 and anti-PD-1 antibody can significantly enhance the anti-tumor effect, providing a potential strategy for improving the efficacy of tumor immunotherapy.

[0010] Therefore, the present invention first provides the application of the CK1δ / ε inhibitor PF-670462 in the preparation of an inhibitor of palmitoyltransferase zDHHC8.

[0011] This invention provides the use of the CK1δ / ε inhibitor PF-670462 in formulations that enhance the sensitivity of tumor cells to ferroptosis.

[0012] Furthermore, the iron death is CD8. + T-mediated ferroptosis in tumor cells. This is because PF-670462 is used to increase the resistance of tumor cells to CD8+. + Application in formulations that enhance the sensitivity of tumor cells to T-mediated ferroptosis.

[0013] Furthermore, the tumor is selected from one or more of cervical cancer, breast cancer, colon cancer, lung cancer, liver cancer, or melanoma.

[0014] This invention provides the application of the CK1δ / ε inhibitor PF-670462 in the preparation of potentiators for immune checkpoint inhibitors.

[0015] This invention demonstrates that the use of immune checkpoint inhibitors alone has little effect on the progression of B16-F10 tumors, while the synergistic combination of PF-670462 and immune checkpoint inhibitors significantly enhances the anti-tumor effect. These findings suggest that PF-670462 can enhance the therapeutic effect of immune checkpoint inhibitors, and the combined use of PF-670462 and immune checkpoint inhibitors may be a potential strategy for improving the efficacy of immunotherapy.

[0016] Furthermore, the immune checkpoint inhibitor is an anti-PD-1 antibody.

[0017] This invention provides the application of the CK1δ / ε inhibitor PF-670462 in combination with an anti-PD-1 antibody in the preparation of antitumor drugs. The positive effect of PF-670462 on tumor immune responses suggests that it may enhance the therapeutic effect of immune checkpoint inhibitors. Furthermore, this invention investigates that the use of anti-PD-1 antibody alone has little effect on the progression of melanoma, while the synergistic combination of PF-670462 and the anti-PD-1 antibody significantly enhances the antitumor effect.

[0018] Furthermore, the tumor is a melanoma.

[0019] The present invention provides a pharmaceutical composition containing the CK1δ / ε inhibitor PF-670462 and an anti-PD-1 antibody.

[0020] Furthermore, the pharmaceutical composition contains an effective dose of PF-670462 and anti-PD-1 antibody; preferably, the mass ratio of PF-670462 to anti-PD-1 antibody in the pharmaceutical composition is 1 to 20:2.

[0021] Preferably, the effective dose of PF-670462 in the pharmaceutical composition is 10-20 mg / kg, and the effective dose of anti-PD-1 antibody is 200 μg.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention first provides the application of the CK1δ / ε inhibitor PF-670462 in the preparation of inhibitors for palmitoyltransferase zDHHC8. Through screening and validation, this invention discovered a specific small molecule inhibitor for palmitoyltransferase zDHHC8, PF-670462. This small molecule inhibitor effectively inhibits the zDHHC8-GPX4 palmitoylation modification signaling axis, promoting the sensitivity of tumor cells to ferroptosis, and providing a selective pathway for promoting CD8+. +T-mediated tumor cell ferroptosis provides an effective strategy. PF-670462 alone can effectively enhance CD8+. + T-cell-mediated tumor cell ferroptosis sensitivity and promote tumor immune infiltration, thereby inhibiting tumor growth and metastasis; at the same time, PF-670462 can enhance the tumor treatment effect of immune checkpoint inhibitors, and the synergistic combination of PF-670462 and anti-PD-1 antibody can significantly enhance the anti-tumor effect, thereby improving the anti-tumor effect of ICB therapy (such as anti-PD-1 antibody therapy) and CAR-T cell therapy. Attached Figure Description

[0024] Figure 1 The mechanism of zDHHC8 palmitoylation modification of GPX4 and its resistance to ferroptosis was studied; among which Figure 1 a) The palmitoylation modification level of GPX4-HA was detected by acylbiotin replacement assay (ABE) 36 hours after transfecting HEK293T cells with GPX4-HA plasmid, and simultaneously transfecting empty vector plasmid, zDHHC8-Flag wild-type (WT) or its C134S enzyme activity mutant plasmid; b) The palmitoylation modification level of GPX4 protein was detected by ABE after ZDHHC8 knockout in HT-1080 cells; c) Cell viability was detected after stable overexpression of WT and C134S enzyme activity mutant ZDHHC8 in control HT-1080 cells and ZDHHC8 knockout HT-1080 cells, respectively, and treatment with different concentrations of RSL3 for 24 hours; d) The mRNA expression level of ZDHHC8 in various cancers was analyzed using TCGA dataset and GTEx database; e) The mRNA expression level of ZDHHC8 in cutaneous melanoma (SKCM) was analyzed using TCGA dataset and GTEx database; f) Real-time... PCR analysis was used to determine the mRNA expression level of ZDHHC8 in cancerous and normal tissues of SKCM patients, and the unpaired Students' t-test was used for statistical analysis. g represents the palmitoylation modification expression level of GPX4 protein in cancerous and adjacent tissues of SKCM patients analyzed by ABE method. h-j show images (h), tumor growth curve (i), and tumor weight statistics of mice after establishing a xenograft model using the shRNA system to construct a control and Zdhhc8 knockdown B16-F10 cell lines, respectively. k represents the effect of Zdhhc8 knockdown on lipid peroxidation levels in B16-F10 xenograft tumor cells assessed by BODIPY-C11 staining. l represents the flow cytometry analysis of CD8+ in the tumor microenvironment. + T cell proportion; m~o represent the IFN-γ concentration in the tumor microenvironment analyzed by flow cytometry. + (m), TNFα + (n) and GZMB+ (o) of CD8 + T cell percentage; p represents mouse survival curve; the above statistical analysis used the unpaired students' t-test ( Figure 1 (e, f) One-way ANOVA ( Figure 1 (j~o) and the log-rank test (Mantel-Cox test) (j~o) Figure 1 p) indicates no significant difference, and ns indicates no significant difference.

[0025] Figure 2 The results of high-throughput screening of zDHHC8 candidate inhibitors; among which Figure 2 Image a shows the structure of ZDHHC8 from the AlphaFold protein structure database; image b shows the cell viability assay of HT-1080 cells after treatment with candidate small molecule compounds for molecular docking and DMSO or RSL3 (0.05 μM) for 24 hours; image c shows the PI staining of HT-1080 cells after treatment with PF-670462 and DMSO or RSL3 (0.05 μM) for 10 hours, followed by flow cytometry detection of PI. + Cell percentage; d represents the lipid peroxidation level of HT-1080 cells after 10 hours of simultaneous treatment with PF-670462 and DMSO or RSL3 (0.05 μM) by BODIPY-C11 staining; e-g represent the cell viability of HT-1080 cells after 24 hours of treatment with specified concentrations of RSL3 (e), ML162 (f), or IKE (g), with or without PF-670642 (10 μM) and ferrostatin-1 (Fer-1) (2 μM); h represents the cell viability of various tumor cells after 24 hours of simultaneous treatment with PF-670462 and DMSO or RSL3 (0.05 μM). Cell viability assay; i-j represent cell viability assays of HT-1080 cells after simultaneous treatment with a specified concentration of RSL3 and SR-3029(i) or LH-846(j) for 24 hours; k represents the construction of control (shCtrl) and CK1δ and CK1ε knockdown (shCK1δ / ε) HT-1080 cell lines using the shRNA system, with Western blotting used to detect the protein expression levels of CK1δ and CK1ε to verify the successful construction of the knockdown cell lines; l represents the cell viability assays of shCtrl and shCK1δ / ε HT-1080 cell lines after treatment with a specified concentration of RSL3 for 24 hours; statistical analysis was performed using two-way ANOVA (…). Figure 2 (c, d)

[0026] Figure 3 To investigate the effect of PF-670462 on zDHHC8; among which, Figure 3In Figures a and b, HT-1080 cells were treated with a specified concentration of PF-670462 for 24 hours, followed by detection of ZDHHC8 mRNA (a) and protein expression (b) levels using real-time quantitative PCR and Western blotting, respectively. Figure c shows HEK293T cells transfected with the ZDHHC8-Flag plasmid for 24 hours, pretreated with PF-670462 (10 μM) for 8 hours, then treated with actinomycin (CHX, 100 mg / mL) for a specified time, and finally detected by Western blotting of zDHHC8-Flag protein expression levels. Figure d shows HEK293T cells transfected with the ZDHHC8-Flag plasmid for 24 hours, pretreated with PF-670462 (10 μM) for 8 hours, then treated with either the proteasome inhibitor MG132 (10 μM) or the lysosomal inhibitor Baf, respectively. Treatment with A1 (20 nM) for 12 hours, and finally detection of zDHHC8-Flag protein expression level by Western blotting; e represents control (Mock) and ZDHHC8 knockout (ZDHHC8). KO HT-1080 cell lines were treated with PF-670462 (10 μM) for 12 hours, followed by ABE reaction, and the palmitoylation modification level of GPX4 protein was detected by Western blotting; f represents the control (Mock) and ZDHHC8 knockout (ZDHHC8). KO The viability of HT-1080 cell lines was assessed after 24 hours of co-treatment with specified concentrations of RSL3 and DMSO or PF-670462 (10 μM); statistical analysis was performed using one-way ANOVA. Figure 3 In a), ns indicates no significant difference.

[0027] Figure 4 To verify the results of PF-670462 specifically inhibiting zDHHC8; among which, Figure 4Image a shows HEK293T cells transfected with ZDHHC family member plasmids for 24 hours, treated with different concentrations of PF-670462 for 16 hours, and then their protein expression levels were detected by Western blotting; image b is a heatmap showing the protein expression levels of zDHHC family members after 16 hours of treatment with different concentrations of PF-670462; image c shows HEK293T cells transfected with ZDHHC8-Flag and ZDHHC8(NTD)-Flag plasmids for 36 hours, pretreated with DMSO or PF-670462 (10 μM) for 3 hours, and then their protein expression levels were detected by cell thermostability assay. The expression level of zDHHC8 was shown in Figure d. After HEK293T cells were transfected with ZDHHC5-Flag, ZDHHC9-Flag and ZDHHC20-Flag plasmids for 36 hours, they were pretreated with DMSO or PF-670462 (10 μM) for 3 hours, and then the protein expression level was detected by cell thermostability assay. The sequence diagram of the zDHHC8 truncated mutant was shown in Figure e. The interaction between the full-length zDHHC8, the N-terminal (core region) zDHHC8 or the C-terminal (disordered region) domain zDHHC8 and PF-670462 was analyzed by surface plasmon resonance (SPR) technology.

[0028] Figure 5 To investigate the in vivo toxic side effects of PF-670462; among which, Figure 5 In Figure a, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CR), and creatine kinase-MB (CK-MB) were measured after treatment with PF-670462 at doses of 10 mg / kg and 20 mg / kg (a); in Figure b, hematoxylin and eosin (H&E) staining analysis was performed on tissues collected from the heart, liver, spleen, lung, and kidney. Statistical analysis was performed using one-way ANOVA. Figure 5 In a), ns indicates no significant difference.

[0029] Figure 6 This study investigates the effects and mechanisms of PF-670462 on tumor progression; among which, Figure 6 Images a through c in the middle section show tumor images (a), tumor growth curves (b), and tumor weight statistics in a mouse xenograft model treated with PF-670462, respectively. Image d shows the effect of PF-670462 on lipid peroxidation levels in B16-F10 xenograft tumor cells, assessed by BODIPY-C11 staining. Image e shows flow cytometry analysis of CD8+ in the tumor microenvironment. + T cell proportion; f~h represent the IFN-γ concentration in the tumor microenvironment analyzed by flow cytometry. + (f), TNFα + (g) and GZMB+ (h) CD8 + T cell proportion; (i-l) are lung images (i), nodule number (j), hematoxylin & eosin (H&E) staining analysis (k), and survival curve (l) of mice with melanoma lung metastasis model treated with PF-670462, respectively; m-n are tumor images (m) and tumor weight statistics (n) of mice with B16-F10 xenograft treated with PF-670462 (10 mg / kg) combined with the ferroptosis inhibitor Liproxstatin-1 (10 mg / kg), respectively; Statistical analysis was performed using unpaired students' t-test ( Figure 6 (c~h,j), one-way ANOVA ( Figure 6 (n) and the log-rank test (Mantel-Cox test) ( Figure 6 (l).

[0030] Figure 7 To synergistically promote CD8 by PF-670462 + Research results on T-mediated anti-tumor immune responses; among which, Figure 7 In the middle, a to c represent the removal of peripheral CD8+ from C57BL / 6 mice using anti-CD8α antibody, respectively. + The flowchart (a), tumor image display (b), and tumor weight statistics (c) of the operation for detecting the anti-tumor effect of PF-670462 after T-cell treatment (n=6); d~f are respectively the Rag2 of T cells and B cells with defects treated with PF-670462. - / - Images of B16-F10 xenografts in mice (n=5) are shown in (d), a schematic diagram of the procedure (e), and tumor weight statistics (f); g~h are schematic diagrams of the procedure (g) and mouse survival curves (h) respectively, used to explore the anti-tumor effect of the combination of PF-670462 and anti-PD1 antibody in a B16-F10 xenograft model constructed in C57BL / C mice (n=12); statistical analysis was performed using two-way ANOVA. Figure 7 (c, f) and the log-rank test (Mantel-Cox test) ( Figure 7 In the interval h), ns indicates no significant difference. Detailed Implementation

[0031] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0032] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0033] PF-670462: Molecular formula: C 19 H 20 FN5·2HCl, CAS No.: 950912-80-8, has the following structural formula:

[0034] Purchased from TargetMol.

[0035] Example 1

[0036] The following research was conducted in the early stages of this invention:

[0037] HEK293T cells were transfected with the GPX4-HA plasmid, and simultaneously transfected with the empty vector plasmid, wild-type zDHHC8-Flag (WT), or its C134S enzyme activity mutant plasmid. After 36 hours, the palmitoylation modification level of GPX4-HA was detected by the acylbiotin replacement assay (ABE). The palmitoylation modification level of GPX4 protein was detected by ABE after ZDHHC8 knockout in HT-1080 cells. WT and the C134S enzyme activity mutant ZDHHC8 were stably overexpressed in control HT-1080 cells and ZDHHC8 knockout HT-1080 cells, respectively. Cell viability was detected after treatment with different concentrations of RSL3 for 24 hours. The mRNA expression level of ZDHHC8 in various cancers was analyzed using the TCGA dataset and GTEx database. The mRNA expression level of ZDHHC8 in cutaneous melanoma (SKCM) was analyzed using the TCGA dataset and GTEx database. Real-time PCR was used to analyze the mRNA expression level of ZDHHC8 in cancerous tissues and normal tissues of SKCM patients. ABE assay was used to analyze the palmitoylation modification expression level of GPX4 protein in cancerous tissues and adjacent normal tissues of SKCM patients.

[0038] Furthermore, a control and Zdhhc8 knockdown B16-F10 cell line were constructed using the shRNA system and seeded into C57BL / 6J cells to establish a xenograft model. Tumor size was monitored starting on day 6. Some mice (n=6) were euthanized on day 15, and tumors were collected. Another group of mice (n=12) were used for continuous tumor growth observation, with significant ulceration on the tumor surface or a diameter exceeding 1.5 cm as the endpoint for mortality plotting survival curves. The effect of Zdhhc8 knockdown on lipid peroxidation levels in B16-F10 xenograft cells was assessed using BODIPY-C11 staining. Flow cytometry was used to analyze CD8+ in the tumor microenvironment. + T cell proportion. Flow cytometry analysis of IFNγ in the tumor microenvironment. + TNFα + and GZMB + CD8 + T cell ratio.

[0039] The results are as follows Figure 1 As shown, the results indicate that the palmityltransferase zDHHC8 can palmitoylate the ferroptosis core pathway molecule GPX4, thereby promoting ferroptosis resistance in tumor cells. Furthermore, the construction of a zDHHC8-deficient cell line through gene editing demonstrated that targeting zDHHC8 effectively enhances the sensitivity of tumor cells to ferroptosis and promotes anti-tumor immune responses. The study also found that zDHHC8 is highly expressed in various tumors, including pancreatic cancer, colorectal cancer, and melanoma. Therefore, this invention suggests that zDHHC8 is a crucial target for effectively promoting anti-tumor immunotherapy; targeting zDHHC8 can effectively enhance the ferroptosis sensitivity of tumor cells, thereby improving CD8+. + T-cell-mediated anti-tumor immunotherapy.

[0040] However, no specific small molecule inhibitors for zDHHC8 have been found to date. Therefore, this invention develops a specific small molecule inhibitor targeting the zDHHC8 protein.

[0041] Example 2

[0042] 1. High-throughput screening of small molecule compounds based on molecular docking and zDHHC8 binding:

[0043] The molecular structure of zDHHC8 was obtained from the AlphaFold protein structure database website, and its core structure was optimized using the Protein Preparation Wizard panel by modifying bond order, adding hydrogen atoms, distributing charges, and predicting protonation state (pH 7.0). Figure 2 (a) This invention selected approximately 220,000 compounds from Enamine for screening. First, these compounds were protonated and desalted using the Epik program at pH 7.0 ± 2.0 to generate tautomers while maintaining their original atomic chirality. To ensure conformational diversity of small molecules during virtual screening, a maximum of 32 conformations were generated for each small molecule. A stepwise strategy was employed during screening, with protein structures set to rigid and small molecules to flexible, while other parameters remained at default values. Docking results were energy-optimized, and the top 10% of small molecules were retained for subsequent MMGBSA binding free energy calculations. Furthermore, this invention used AdmetSAR 3.0 (http: / / lmmd.ecust.edu.cn / admetsar3 / index.php) to further filter the virtual screening results, based on ADMET properties and referencing Lipinski, Pfizer, and GSK drug formation rules to obtain the final screening results. Ultimately, this invention selected the top 5% of commercially available 53 small molecule compounds for further screening. Figure 2(b)

[0044] 2. Cell viability screening revealed that PF-670462 is potentially the most effective zDHHC8 inhibitor.

[0045] Previous results of this invention have shown that zDHHC8 deficiency increases the sensitivity of tumor cells to ferroptosis. Therefore, this invention aims to develop a zDHHC8 small molecule inhibitor that can effectively promote the sensitivity of tumor cells to ferroptosis. Based on this background, this invention screened 53 candidate small molecules using the MTT assay, a method for detecting cell viability.

[0046] Specifically, this invention describes the cell viability assay of HT-1080 cells after 24 hours of simultaneous treatment with molecular docking candidate small molecule compounds and DMSO or RSL3 (0.05 μM). HT-1080 cells were treated with PF-670462 and DMSO or RSL3 (0.05 μM) for 10 hours, followed by pyridine iodide (PI) staining, and then flow cytometry was used to detect PI. + Cell proportions. Lipid peroxidation levels in HT-1080 cells were detected by BODIPY-C11 staining after 10 hours of simultaneous treatment with PF-670462 and DMSO or RSL3 (0.05 μM). Cell viability was assessed after 24 hours of treatment with specified concentrations of RSL3, ML162, or IKE, with or without PF-670642 (10 μM) and ferrostatin-1 (Fer-1) (2 μM). Cell viability was also assessed after 24 hours of simultaneous treatment with PF-670462 and DMSO or RSL3 (0.05 μM) using various tumor cell lines. HT-1080 cell viability was also assessed after 24 hours of simultaneous treatment with specified concentrations of RSL3 and SR-3029 or LH-846. A control (shCtrl) and CK1δ and CK1ε knockdown (shCK1δ / ε) HT-1080 cell lines were constructed using the shRNA system. Western blotting was used to detect the protein expression levels of CK1δ and CK1ε to verify the successful construction of the knockdown cell lines. Cell viability was assessed in the shCtrl and shCK1δ / ε HT-1080 cell lines after treatment with specified concentrations of RSL3 for 24 hours.

[0047] The results showed that one of the candidate small molecule compounds for molecular docking, named PF-670462, could effectively promote RSL3-induced ferroptosis in HT-1080 cells. Figure 2(b-e). Subsequently, this invention employed other ferroptosis inducers such as ML162 and IKE, and conducted experiments on various human and mouse-derived tumor cell lines, including HT-1080, confirming that PF-670462 can indeed increase the sensitivity of tumor cells to ferroptosis. Figure 2 (f~h). Furthermore, considering previous studies have shown that PF-670462 is an inhibitor of CK1δ / ε, this invention investigated the role of CK1δ / ε in ferroptosis. Through experiments using other small molecule inhibitors of CK1δ / ε (SR-3029 and LH-846) and constructing CK1δ / ε knockdown cell lines, this invention found that inhibiting CK1δ / ε does not alter the sensitivity of tumor cells to ferroptosis (f~h). Figure 2 Therefore, this invention concludes that the target of PF-670462 in promoting the ferroptosis sensitivity of tumor cells is not CK1δ / ε.

[0048] 3. The target of PF-670462 in promoting the ferroptosis sensitivity of tumor cells was identified as zDHHC8.

[0049] The experimental procedure is as follows:

[0050] (1) HT-1080 cells were treated with a specified concentration of PF-670462 for 24 hours, and then the mRNA and protein expression levels of ZDHHC8 were detected by real-time quantitative PCR and Western blotting, respectively.

[0051] (2) After transfecting HEK293T cells with ZDHHC8-Flag plasmid for 24 hours, they were pretreated with PF-670462 (10 μM) for 8 hours, followed by treatment with actinomycin (CHX, 100 mg / mL) for a specified time. Finally, the expression level of zDHHC8-Flag protein was detected by Western blotting.

[0052] (3) HEK293T cells were transfected with ZDHHC8-Flag plasmid for 24 hours and then pretreated with PF-670462 (10 μM) for 8 hours. Subsequently, they were treated with the proteasome inhibitor MG132 (10 μM) or the lysosome inhibitor Baf A1 (20 nM) for 12 hours. Finally, the expression level of zDHHC8-Flag protein was detected by Western blotting.

[0053] (4) Control (Mock) and ZDHHC8 knockout (ZDHHC8) KO The HT-1080 cell line was treated with PF-670462 (10 μM) for 12 hours and then subjected to ABE reaction. The level of palmitoylation modification of GPX4 protein was detected by Western blotting.

[0054] (5) Control (Mock) and ZDHHC8 knockout (ZDHHC8) KO Cell viability was measured after HT-1080 cell lines were treated with a specified concentration of RSL3 and either DMSO or PF-670462 (10 μM) for 24 hours.

[0055] To confirm that zDHHC8 is the target of PF-670462 in promoting tumor cell ferroptosis sensitivity, this invention first examined the effect of PF-670462 treatment on zDHHC8. The results showed that PF-670462 treatment significantly downregulated the protein expression level of zDHHC8 without affecting zDHHC8 mRNA levels. Figure 3 (a, b) This suggests that PF-670462 promotes the degradation of zDHHC8. Further CHX assays and degradation pathway inhibitor intervention experiments confirmed that PF-670462 mainly promotes the lysosomal degradation of zDHHC8. Figure 3 (c, d). Subsequent ABE experiments confirmed that PF-670462 treatment significantly downregulated GPX4 palmitoylation modification levels while reducing zDHHC8 protein expression in wild-type cells; however, in ZDHHC8 knockout (ZDHHC8... KO In cells, PF-670462 did not downregulate the palmitoylation modification level of GPX4 protein. Figure 3 (e). It was also found in ZDHHC8 KO PF-670462 in cells lost its ability to promote ferroptosis in tumor cells. Figure 3 (f). The above results fully demonstrate that PF-670462 enhances the sensitivity of tumor cells to ferroptosis by targeting zDHHC8.

[0056] 4. Confirmation of selective inhibition of zDHHC family members by PF-670462:

[0057] The experimental procedure is as follows:

[0058] (1) HEK293T cells were transfected with plasmids of ZDHHC family members for 24 hours and then treated with different concentrations of PF-670462 for 16 hours. The protein expression levels were then detected by Western blotting. The protein expression levels of zDHHC family members after 16 hours of treatment with different concentrations of PF-670462 are shown in the heatmap.

[0059] (2) HEK293T cells were transfected with ZDHHC8-Flag and ZDHHC8(NTD)-Flag plasmids for 36 hours, and then pretreated with DMSO or PF-670462 (10 μM) for 3 hours. The protein expression level was then detected by cell thermostability assay.

[0060] (3) HEK293T cells were transfected with ZDHHC5-Flag, ZDHHC9-Flag and ZDHHC20-Flag plasmids for 36 hours, and then pretreated with DMSO or PF-670462 (10 μM) for 3 hours. The protein expression level was then detected by cell thermostability assay.

[0061] (4) The interaction between the full-length zDHHC8, N-terminal (core region) zDHHC8 or C-terminal (disorder region) structural domain zDHHC8 and PF-670462 was analyzed by surface plasmon resonance (SPR) technology.

[0062] Because the screening in this invention is based on the core domain of the zDHHC8 molecule, and the core domains of zDHHC family members are highly conserved, PF-670462 may have off-target effects on other zDHHC family members. Therefore, this invention first examined the effect of PF-670462 on the protein expression levels of zDHHC family members, and the results showed that PF-670462 had the most significant effect on reducing zDHHC8 expression. Figure 4 (a, b) This suggests that PF-670462 has a certain selectivity for the zDHHC8 protein. Furthermore, this invention used CETSA experimental methods to detect the binding of PF-670462 to the full-length zDHHC8, the N-terminal domain (core structure) of zDHHC8, zDHHC5 which is highly similar to the overall structure of zDHHC8, and the structurally resolved zDHHC9 and zDHHC20. The results showed that PF-670462 interacts most strongly with the full-length zDHHC8. Figure 4 (c, d) further suggest the selective effect of PF-670462 on zDHHC8. To further elucidate the high selectivity of PF-670462 for zDHHC8, this invention employed surface plasmon resonance (SPR) experiments to detect the binding of PF-670462 with the entire zDHHC8 length, the zDHHC8-N-terminal domain (core structure), and the zDHHC8-C-terminal domain (disorder region). The results show that the interaction between PF-670462 and the entire zDHHC8 length is the strongest, and the binding with the zDHHC8-N-terminal domain is stronger than that with the zDHHC8-C-terminal domain (…). Figure 4 (e, f) This suggests that the zDHHC8-N-terminal domain is the structural basis for the binding of zDHHC8 to PF-670462, the zDHHC8-C-terminal domain facilitates its binding to PF-670462, and the integrity of the zDHHC8 structure is a necessary condition for the specific binding of PF-670462 to zDHHC8.

[0063] Example 3

[0064] 1. In vivo experiments have confirmed that PF-670462 has no obvious toxic side effects.

[0065] To investigate whether PF-670462 can safely function as a small molecule inhibitor for tumor treatment in vivo, this invention administered intraperitoneal injections of 10 mg / kg and 20 mg / kg to normal mice (n=3) five times consecutively, with each injection spaced two days apart. After treatment, the mice were euthanized, and tissue samples were collected for further analysis. This invention measured serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (CR), and creatine kinase isoenzyme (CK-MB), and performed histological analysis on samples from the heart, liver, spleen, lung, and kidney. Specifically, hematoxylin and eosin (H&E) staining was performed on heart, liver, spleen, lung, and kidney tissues. The results showed that PF-670462 did not cause significant toxicity to normal tissues. Figure 5 (a, b)

[0066] 2. In vivo experiments confirmed the effect of PF-670462 on anti-tumor immunity.

[0067] Next, this invention investigated the effect of PF-670462 on tumor growth in xenografted mice of the melanoma cell line B16-F10. The specific experimental methods are as follows:

[0068] (1) 2×10 5 B16-F10 cells were seeded in C57BL / 6 mice to establish a xenograft model. Starting on day 6, PF-670462 was administered every two days (n=6) in either the control solvent or at a dose of 10 mg / kg, and tumor volume was recorded for five consecutive days. Tumors were collected after euthanasia on day 15, photographed, and tumor growth curves and weights were calculated. The effect of PF-670462 on lipid peroxidation levels in B16-F10 xenograft tumor cells was assessed using BODIPY-C11 staining. Flow cytometry was used to analyze CD8+ in the tumor microenvironment. + T cell ratio; flow cytometry analysis of IFNγ in the tumor microenvironment + (f), TNFα + (g) and GZMB + (h) CD8 + T cell ratio.

[0069] (2) Inject 2×10 via tail vein injection 5B16-F10 cells were used to construct a melanoma lung metastasis model. On day 4, mice were randomly divided into groups and treated with either the control solvent or PF-67046 at a dose of 10 mg / kg (n=6). The drugs were administered every two days. Some mice were euthanized on day 17, and their lungs were collected for photography. The number of nodules was recorded, and hematoxylin and eosin (H&E) staining analysis was performed. Other mice were continuously observed, and survival curves were plotted with a 20% decrease in body weight as the endpoint.

[0070] (3) Effect of PF-670462 (10 mg / kg) combined with ferroptosis inhibitor Liproxstatin-1 (10 mg / kg) on ​​the growth of B16-F10 xenografts in mice (n=6), tumor images were taken and tumor weight was counted.

[0071] The results showed that treatment with PF-670462 significantly inhibited tumor growth. Figure 6 (a~c) and simultaneously enhanced the lipid peroxidation level of B16-F10 cells within the tumor ( Figure 6 (d). Further analysis of immune cell infiltration in the tumor showed that PF-670462 treatment significantly increased CD8+. + T cell infiltration ( Figure 6 (e). Furthermore, IFNγ was observed in tumors treated with PF-670462. + TNFα + and GZMB + CD8 + The number of T cells increased significantly. Figure 6 (f~h). By constructing a B16-F10 metastatic tumor model, we observed that PF-670462 significantly reduced the number and size of B16-F10 metastatic pulmonary nodules (f~h). Figure 6 (i~k), and prolonged the overall survival of mice ( Figure 6 More importantly, the ferroptosis inhibitor Liproxstatin-1 (Lipro-1) can reverse the antitumor effect of PF-670462. Figure 6 The presence of m and n further supports the mechanism by which PF-670462 inhibits tumor immune escape by enhancing the sensitivity of tumor cells to ferroptosis.

[0072] Previous studies have shown that CD8 infiltrates tumors + T cells are the main immune cells mediating ferroptosis in tumor cells. Therefore, this invention investigates whether the antitumor effect of PF-670462 depends on CD8. + T cells. The specific experimental method is as follows:

[0073] (1) Using anti-CD8α antibody to clear peripheral CD8 in C57BL / 6 mice+ The anti-tumor effect of PF-670462 after T-cell therapy was detected (n=6), tumor images were taken, and tumor weight was measured.

[0074] (2) PF-670462 in T cells and B cells with Rag2 deficiency - / - The effect of mice on the growth of B16-F10 xenografts (n=5), tumor images were taken and the tumor weight was counted.

[0075] (3) The anti-tumor effect of PF-670462 (10 mg / kg) combined with anti-PD-1 antibody (200 μg / mouse) was investigated in the B16-F10 xenograft model constructed by C57BL / C mice (n=12), and the survival curve of mice was plotted with anti-IgG antibody as isotype control.

[0076] By using anti-CD8α antibodies to deplete peripheral cytotoxic CD8 in vivo + T cells, this invention found that the tumor growth retardation induced by PF-670462 treatment was reversed ( Figure 7 (a-c) This indicates that the therapeutic effect of PF-670462 depends on the cytotoxic CD8+ infiltrating within the tumor. + T cells. Furthermore, in Rag2 - / - In mice, PF-670462 lost its anti-tumor effect, but supplementation with CD8... + T-cell therapy restored the anti-tumor effect, while B-cell supplementation failed to achieve the same effect. Figure 7 These results (d-f) clearly demonstrate that PF-670462 can effectively enhance CD8. + T cell-mediated tumor cell ferroptosis sensitivity and promotes tumor immune infiltration, thereby inhibiting tumor growth and metastasis.

[0077] The positive effect of PF-670462 on tumor immune response suggests that it may enhance the therapeutic effect of immune checkpoint inhibitors. In a B16-F10 xenograft model, compared with the isotype control group treated with anti-IgG antibody (200 μg / animal), it was observed that anti-PD-1 antibody alone (200 μg / animal) had little effect on the progression of B16-F10 tumors, while the synergistic combination of PF-670462 (10 mg / kg) and anti-PD-1 antibody (200 μg / animal) significantly enhanced the anti-tumor effect. Figure 7 These findings suggest that the combined use of PF-670462 and anti-PD-1 antibodies may be a potential strategy to improve the efficacy of immunotherapy.

[0078] In summary, this study reveals that the small molecule compound PF-670462 specifically inhibits the function of zDHHC8, thereby reducing the palmitoylation level of GPX4 and enhancing the resistance of tumor cells to CD8+ in antitumor immunotherapy. + Sensitivity of T cell-mediated tumor cell ferroptosis.

Claims

1. Application of CK1δ / ε inhibitor PF-670462 in the preparation of inhibitors of palmitoyltransferase zDHHC8.

2. Application of CK1δ / ε inhibitor PF-670462 in the preparation of formulations that promote the sensitivity of tumor cells to ferroptosis.

3. The application according to claim 2, characterized in that, The iron death is CD8. + T-mediated ferroptosis in tumor cells.

4. The application according to claim 2, characterized in that, The tumor is selected from one or more of cervical cancer, breast cancer, colon cancer, lung cancer, liver cancer, or melanoma.

5. Application of CK1δ / ε inhibitor PF-670462 in the preparation of potentiators for immune checkpoint inhibitors.

6. The application according to claim 5, characterized in that, The immune checkpoint inhibitor is an anti-PD-1 antibody.

7. Application of CK1δ / ε inhibitor PF-670462 in combination with anti-PD-1 antibody in the preparation of antitumor drugs.

8. The application according to claim 8, characterized in that, The tumor is melanoma.

9. A pharmaceutical composition, characterized in that, It contains the CK1δ / ε inhibitor PF-670462 and an anti-PD-1 antibody.

10. The pharmaceutical composition according to claim 9, characterized in that, The mass ratio of the CK1δ / ε inhibitor PF-670462 to the anti-PD-1 antibody in the pharmaceutical composition is 1 to 20:2.

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