Application of CD300LF small-molecule inhibitor

Abstract

The invention relates to application of a CD300LF small-molecule inhibitor, and belongs to the technical field of medicines. The CD300LF small-molecule inhibitor comprises temopril, and the small-molecule inhibitor temopril taking CD300LF as a target spot is screened from a small-molecule drug library approved by FDA through computer high-throughput virtual drug screening in combination with biological activity screening. The invention reveals that temopril can be used as a CD300LF small-molecule inhibitor, is specifically combined with CD300LF, induces tumor-related macrophages to be converted from an M2 phenotype to an M1 phenotype, enhances the phagocytosis of the macrophages on tumor cells, and shows remarkable inhibition capability on tumor growth in an in-vivo experiment for the first time. In addition, it is found that after temopril and an immune checkpoint inhibitor are combined for use, a remarkable synergistic anti-tumor effect can be generated. The temopril can be used as a potential immunotherapy drug, and is expected to provide an experimental basis and a candidate drug for clinical treatment and prevention of cancers.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a CD300LF small molecule inhibitor. Background Technology

[0002] In recent years, the role of the tumor microenvironment in tumor progression has received widespread attention. Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the tumor microenvironment, and TAMs have been shown to be closely associated with poor patient prognosis. Within the tumor microenvironment, TAMs are generally considered to be M2-like phenotypes with both pro-tumorigenic and immunosuppressive functions. These M2-like TAMs promote tumor proliferation by secreting growth factors, support tumor development by inducing angiogenesis, and promote tumor metastasis by remodeling the extracellular matrix. Furthermore, M2-like TAMs highly express PD-L1, secrete inhibitory cytokines, competitively consume essential nutrients, and inhibit CD8. + The function of T cells is one of the key factors in resistance to PD-1 / PD-L1 immune checkpoint inhibitor therapy.

[0003] CD300LF (also known as MAIR-V) is a type I transmembrane glycoprotein initially discovered as the primary physiological receptor for norovirus, mediating viral entry into cells. The CD300LF structure contains two immunoreceptor tyrosine inhibitory motifs and one immunoreceptor-based tyrosine switch motif. CD300LF's function extends beyond viral infection; it participates in multiple other biological processes, including ligand binding to regulate the activation, inhibition, or apoptosis of immune cells. CD300LF is primarily expressed on human monocytes, activating the PI3K / Akt pathway in monocytes and driving macrophages towards preferential M2 polarization, accompanied by PD-L1 upregulation. This signaling can be inhibited using CD300LF monoclonal antibodies. In recent years, with advancements in sequencing technology, researchers have been elucidating the role of CD300LF in cancer development and progression. Multiple studies have shown that CD300LF is upregulated in various tumors and is associated with patient prognosis. Inhibiting CD300LF can significantly weaken the growth ability of tumors. These findings reveal the potential application value of CD300LF blockade in tumor immunotherapy. Currently, there are no approved or reported small molecule compounds that can specifically inhibit CD300LF activity. Summary of the Invention

[0004] The purpose of this invention is to provide the use of a CD300LF small molecule inhibitor in the preparation of drugs for the prevention and / or treatment of cancer.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Application of CD300LF small molecule inhibitors in the preparation of formulations or drugs that regulate the functional phenotypic transition of macrophages.

[0006] Furthermore, the CD300LF small molecule inhibitor is temocapil, and the molecular formula of temocapil is C3. 23 H 28 N2O5S2, the structural formula is shown in formula (1); Equation (1).

[0007] Furthermore, the macrophages mentioned are tumor-associated macrophages, and the regulation of macrophage functional phenotype conversion refers to promoting the conversion of macrophages from the M2 phenotype to the M1 phenotype.

[0008] Furthermore, one or more uses selected from the following group: (1) Increase the proportion of macrophages with the M1 phenotype; (2) Increase CD8 + The proportion of T cells; (3) Regulate the immune microenvironment of tumor tissue; (4) Prevention and / or treatment of tumors; The tumor in question is one rich in macrophage infiltration and / or overexpressing CD300LF protein.

[0009] Furthermore, the tumors mentioned include, but are not limited to, squamous cell carcinoma of the head and neck, breast cancer, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, lung cancer, stomach cancer, liver cancer, kidney cancer, bladder cancer, prostate cancer, or esophageal squamous cell carcinoma.

[0010] Medications used for the prevention and / or treatment of cancer include the CD300LF small molecule inhibitor temopril or a pharmaceutically acceptable salt, pharmaceutically acceptable carrier, or excipient thereof.

[0011] Furthermore, the carrier or excipient is selected from fillers, diluents, stabilizers, sustained-release matrices, liposomes, or polymeric nanoparticles.

[0012] Furthermore, the dosage form of the drug for the prevention and / or treatment of tumors is any pharmacologically acceptable dosage form, including but not limited to tablets, granules, capsules, powders, pills, oral liquids, powders for injection, injections, transdermal patches, gels, ointments, sustained-release formulations, or nanoformulations.

[0013] Furthermore, the dosage of the drug used for the prevention and / or treatment of tumors is any pharmacologically acceptable dosage.

[0014] The application of CD300LF small molecule inhibitors and immune checkpoint inhibitors in the preparation of synergistic drugs for the prevention and / or treatment of tumors, wherein the CD300LF small molecule inhibitor is temopril, and the immune checkpoint inhibitor is a PD-1 antibody, including but not limited to anti-mouse PD-1 (CD279)-In vivo, pembrolizumab, nivolumab, and serplulimab.

[0015] Drugs for the prevention and / or treatment of tumors, the active ingredients of which include a CD300LF small molecule inhibitor and an immune checkpoint inhibitor, wherein the CD300LF small molecule inhibitor is temopril and the immune checkpoint inhibitor is a PD-1 antibody, including but not limited to anti-mouse PD-1 (CD279)-in vivo, pembrolizumab, nivolumab, and serplulimab.

[0016] Application of temopril as a small molecule inhibitor of CD300LF.

[0017] The beneficial effects of this invention are: This invention, through computer-aided screening and in vitro / in vivo activity experiments, has for the first time identified and obtained temopril, a small molecule inhibitor of CD300LF. Temopril exhibits a strong binding affinity for CD300LF, effectively inducing TAMs to polarize from an M2-like phenotype to an M1-like phenotype, and enhancing macrophage-mediated phagocytosis of tumor cells in vitro.

[0018] Temopril inhibited tumor growth in in vivo experiments with no significant toxic side effects on organs. Mechanistically, temopril treatment reprogrammed tumor microenvironments (TAMs), changing the tumor microenvironment from an immunosuppressive, tumor-promoting state to an immune-activated, anti-tumor state. This suggests that temopril is a potential modulator of tumor immunotherapy. Furthermore, the combination of temopril and immune checkpoint inhibitors enhanced the anti-tumor effects of the inhibitors, potentially providing an experimental basis for clinical treatment and prevention of cancer.

[0019] This invention discloses the use of temopril or its pharmaceutically acceptable salts, pharmaceutically acceptable carriers or excipients, and analogues in the prevention and / or treatment of cancers caused by high expression of CD300LF. Attached Figure Description

[0020] Figure 1 A technical roadmap for screening CD300LF small molecule inhibitors; Figure 2Figure 1 shows experimental data on the affinity and binding mode of CD300LF small molecule inhibitors to CD300LF. Figure 3 Figure showing experimental data on macrophage phenotypic transformation induced by CD300LF small molecule inhibitor; Figure 4 Figure showing experimental data on the effect of CD300LF small molecule inhibitors on macrophage-mediated phagocytosis of tumor cells; Figure 5 Figure 1: Experimental data on the inhibition of tumor growth in a mouse model of head and neck squamous cell carcinoma SCC7 by temopril at doses of 2 mg / kg and 6 mg / kg; Figure 6 This is a graph showing experimental data on the synergistic inhibition of tumor growth in a 4T1 in situ model of breast cancer by the combination of temopril and PD-1 antibody. Detailed Implementation

[0021] The present invention will be further described below with reference to the embodiments and accompanying drawings.

[0022] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained commercially or prepared by existing methods.

[0023] Statistical analysis Between-group differences were obtained through unpaired comparison. t The analysis was performed using a test. Unless otherwise stated, all data are expressed as the mean standard error (SEM) of the mean. Significant differences are indicated by *. p <0.05,** p <0.01, *** p <0.001.

[0024] Example 1 I. Virtual Screening of CD300LF Small Molecule Inhibitors This study utilizes a combination of computer-generated virtual screening and bioactivity screening to identify CD300LF small molecule compounds, thereby exploring their therapeutic potential. The screening workflow is as follows: Figure 1As shown. The protein crystal structure of CD300LF (PDB Code: 6C6Q) was obtained by searching the Protein Data Bank (PDB) database. The protein structure was prepared and optimized using Molecular Operating Environment (MOE) software, and potential docking pockets were defined based on the ligand binding region and standard druggable cavity size. A structure-based, computer-aided high-throughput virtual screening strategy was used to screen small molecule inhibitors of CD300LF. After molecular docking, the Unique Filter was used to select the optimal binding conformation for each compound from 1763 molecules in an FDA-approved small molecule drug library, ranking the compounds based on binding affinity (S score), molecular weight, hydrophobicity, and docking mode. The top 10 compounds were selected for further investigation.

[0025] II. Evaluation of the affinity and binding mode between small molecule inhibitors and CD300LF The binding affinity between the small molecule and human CD300LF protein (hCD300LF) was determined by micro-thermophoresis (MST). Recombinant hCD300LF-His protein (Sinochem Biotech, 16034-H08H, Beijing, China) was labeled with a second-generation RED-tris-NTA dye (MO-L018, nano temperature Technologies GmbH, Germany). The small molecule compound was serially diluted in PBST containing 0.05% Tween-20. Equal volumes of the dye-labeled hCD300LF-His protein solution were mixed with the serially diluted small molecule solution and incubated at room temperature for 5 minutes to obtain a mixture. The mixture was then loaded into capillaries, and the dissociation constant (K0) was determined using a nanotemperature instrument (nano temperature 151 Technologies GmbH, Germany). D ).

[0026] Affinity test results of small molecule compounds with hCD300LF protein are as follows: Figure 2 As shown, the dissociation constant (K) between glafenin hydrochloride and hCD300LF protein is... D The dissociation constant (K2) of nifekalan hydrochloride and hCD300LF protein was 2.948 ± 2.171 μM. D The dissociation constant (K2) of temopril with hCD300LF protein was 0.527 ± 0.342 (μM). D The effective concentration (μM) was 0.800 ± 0.688, indicating that all three small molecule compounds exhibited good binding activity with CD300LF.

[0027] Molecular docking analysis was performed using MOE to simulate the potential binding modes of candidate molecules with CD300LF. Residues of CD300LF are shown in blue, and the CD300LF backbone is shown in light gray. Grafenin hydrochloride, nifecalanol hydrochloride, and temopril are represented by green, cyan, and red rods, respectively. Hydrogen bonds are represented by cyan dashed lines.

[0028] The results showed that gelafenimine hydrochloride formed hydrogen bonds with R42, E49, and M100 residues of CD300LF; nifekalan hydrochloride interacted with R42 and G96 residues of CD300LF via hydrogen bonds; and temopril formed hydrogen bonds with R42, S43, K94, L97, and D98 residues of CD300LF. This demonstrates the potential of these candidate small molecules as lead compounds for further development.

[0029] Example 2 macrophage polarization First, mouse bone marrow-derived macrophages (BMDMs) were cultured for 5 days in the presence of 20 ng / mL granulocyte-macrophage colony-stimulating factor (GM-CSF, 315-03-100UG, PeproTech, USA). On the third day, fresh GM-CSF-containing medium was added. On the fifth day, BMDMs were stimulated for 48 hours with 20 ng / mL interleukin-4 (IL-4, 214-14-100UG, PeproTech, USA) and 20 ng / mL interleukin-13 (IL-13, 210-13-100UG, PeproTech, USA) to induce M2 polarization. Simultaneously, a candidate small molecule compound was added to a final concentration of 50 μM, and the cells were incubated for a total of 48 hours. Cells were stained with anti-CD11b (M1 / 70, Thermo Fisher Scientific, USA) and anti-F4 / 80 antibody (BM8, Thermo Fisher Scientific, USA) to identify macrophages, stained with anti-CD86 antibody (GL1, Thermo Fisher Scientific, USA) to detect M1-like macrophages, and stained with anti-CD206 antibody (MR6F3, Thermo Fisher Scientific, USA) to detect M2-like macrophages. The proportions of M2 and M1 macrophages were quantitatively analyzed, and intergroup comparisons were performed.

[0030] The results are as follows Figure 3 As shown, blocking CD300LF with 50 μM candidate small molecule compounds significantly attenuated IL-4-induced M2 polarization. Flow cytometry analysis revealed that gelafenimine hydrochloride, nifekalan hydrochloride, and temopril all promoted macrophage polarization from M2-like to M1-like, manifested as increased CD86 expression and decreased CD206 expression. Figure 3 The control group used PBS solution containing DMSO.

[0031] Example 3 Phagocytosis of tumor cells by macrophages SCC7 tumor cells labeled with fluorescein diacetate succinimide ester (CFSE, ThermoFisher Scientific, USA) and M2 macrophages were co-cultured at a 4:1 ratio in hypoadsorbed 96-well plates for 4 hours in the presence of 50 μM small molecule compound. Cells were then harvested, stained with anti-CD11b and anti-F4 / 80 antibodies, and analyzed by flow cytometry (BD Bioscience, USA). Phagocytosis was quantified as F4 / 80. + CFSE + Cells account for 80% of the total F4. + Percentage of cells.

[0032] The results are as follows Figure 4 As shown, compared with the control group (PBS solution containing DMSO), only macrophages treated with temopril showed a significantly enhanced phagocytic capacity for SCC7 tumor cells. Statistical analysis showed that temopril significantly increased CFSE. + CD11b + F4 / 80 + The proportion of cells. These results indicate that temopril can effectively activate macrophages, reverse the M2-like pro-tumor phenotype, and enhance macrophage-mediated tumor cell phagocytosis.

[0033] Example 4 Temopril inhibits tumor growth in a head and neck squamous cell carcinoma SCC7 model SPF-grade female C3H mice (6-8 weeks old) used for in vivo anti-tumor studies were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed under sterile conditions throughout the entire process, with free access to food and water. All cages, feed, bedding, and water used were autoclaved and sterilized, and the housing environment met the requirements for medical laboratory animal facilities.

[0034] 1×10 6 SCC7 cells were subcutaneously injected into the right back of 6-8 week old female SPFC3H mice. Tumor volume and body weight were monitored every other day. Tumors were recorded when they reached approximately 80-100 mm in size. 3 Mice were re-randomized and administered drugs according to tumor volume. Mice in the treatment group received daily intraperitoneal injections of temopril at doses of 2 mg / kg or 6 mg / kg, while mice in the control group received daily intraperitoneal injections of the same volume of PBS solution containing DMSO. Tumor volume was calculated using the formula: V = 1 / 2 × a (length) × b (width) × c (height). On day 26, mice were euthanized, and tumor, spleen, and lymph node tissues were harvested for subsequent analysis.

[0035] from Figure 5It can be seen that temopril effectively inhibited tumor growth; compared with the control group, the SCC7 tumor-bearing mice in the temopril group showed a significant reduction in tumor volume. No significant difference was detected between the 2 mg / kg and 6 mg / kg dose groups.

[0036] Example 5 In vivo antitumor experiment of temopril combined with PD-1 antibody in the treatment of mouse orthotopic tumor-bearing 4T1 model SPF-grade female BALB / c mice (6-8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The animals were housed under sterile conditions throughout the entire process, with free access to food and water. All cages, feed, bedding, and water used were autoclaved and sterilized, and the housing environment met the requirements for medical laboratory animal facilities.

[0037] The antitumor efficacy of temopril was further evaluated by establishing a PD-1 non-responsive 4T1 mouse model. In a clean bench sterilized with ultraviolet irradiation, 5×10⁻⁶ mol / L temopril was mixed... 4 4T1 tumor single-cell suspension was inoculated into the fourth mammary fat pad of BALB / c female mice via in situ tumor bearing. Tumor formation was closely monitored after injection. Tumor volume and body weight were monitored every other day. Tumors were identified when they reached approximately 30-50 mm in size. 3 Mice were re-randomized and administered drugs according to tumor volume. The groups were: control group (PBS solution containing DMSO), IgG group, temopril group, PD-1 antibody group (Anti-mousePD-1(CD279)-InVivo, clone number: RMP1-14), and temopril combined with PD-1 antibody treatment group. The PD-1 antibody was the experimentally tested PD-1 antibody in mice (RMP1-14, Selleck). For the temopril group, each mouse received 2 mg / kg temopril intraperitoneally once daily. The control group received PBS solution containing DMSO, with the injection frequency and timing consistent with the temopril group. For the PD-1 antibody group, each mouse received 10 mg / kg of the experimentally tested PD-1 antibody in mice (RMP1-14, Selleck) intraperitoneally every three days. The IgG group received 10 mg / kg IgG per mouse, with the injection frequency and timing consistent with the PD-1 antibody group. Mice in the temopril combined with PD-1 antibody treatment group were injected intraperitoneally with 2 mg / kg temopril once daily, and intraperitoneally with 10 mg / kg of the experimental PD-1 antibody every three days. The mice were euthanized on day 29.

[0038] from Figure 6It can be seen that both temopril and PD-1 antibody monotherapy exerted anti-tumor effects. Compared with the use of either drug alone, the synergistic treatment of temopril and PD-1 antibody produced a more significant inhibitory effect on tumor growth. These results indicate that in PD-1 antibody-resistant tumor models, the combined use of temopril and immune checkpoint inhibitors (ICIs) can significantly improve the efficacy of immunotherapy, highlighting their potential to overcome immune tolerance and sensitize tumors to immunotherapy.

[0039] Other embodiments of this disclosure will readily occur to those skilled in the art upon consideration of the specification and practice of the disclosure herein. This disclosure is intended to cover any variations, uses, or adaptations of this disclosure that follow the general principles of this disclosure and include common knowledge or customary techniques in the art not disclosed herein. The specification and examples are to be considered exemplary only, and the true scope and spirit of this disclosure are indicated by the claims.

[0040] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of patent protection of the present invention shall be determined by the claims. Similarly, any equivalent structural changes made based on the content of the present invention's specification shall also be included within the scope of protection of the present invention.

Claims

1. Application of CD300LF small molecule inhibitors in the preparation of formulations or drugs that regulate the functional phenotypic transition of macrophages.

2. The application of the CD300LF small molecule inhibitor according to claim 1, characterized in that, The CD300LF small molecule inhibitor is temopril, and the structural formula of temopril is shown in formula (1); Equation (1).

3. The application of the CD300LF small molecule inhibitor according to claim 1, characterized in that, The macrophages mentioned are tumor-associated macrophages, and the regulation of macrophage functional phenotype conversion refers to promoting the conversion of macrophages from the M2 phenotype to the M1 phenotype.

4. The application of the CD300LF small molecule inhibitor according to claim 1, characterized in that, Also selected from one or more of the following groups of uses: (1) Increase the proportion of macrophages with the M1 phenotype; (2) Increase CD8 + The proportion of T cells; (3) Regulate the immune microenvironment of tumor tissue; (4) Prevention and / or treatment of tumors; The tumor in question is one rich in macrophage infiltration and / or overexpressing CD300LF protein.

5. The application of the CD300LF small molecule inhibitor according to claim 4, characterized in that, The tumors mentioned include, but are not limited to, squamous cell carcinoma of the head and neck, breast cancer, colon cancer, cervical cancer, ovarian cancer, endometrial cancer, lung cancer, stomach cancer, liver cancer, kidney cancer, bladder cancer, prostate cancer, or esophageal squamous cell carcinoma.

6. A drug for the prevention and / or treatment of tumors, characterized in that, The active ingredient contains a CD300LF small molecule inhibitor.

7. The medicament for preventing and / or treating tumors according to claim 6, characterized in that, The CD300LF small molecule inhibitor is temopril.

8. The application of CD300LF small molecule inhibitors and immune checkpoint inhibitors in the preparation of synergistic drugs for the prevention and / or treatment of tumors, characterized in that, The CD300LF small molecule inhibitor is temopril, and the immune checkpoint inhibitor is a PD-1 antibody, including but not limited to anti-mouse PD-1 (CD279)-In vivo, Pembrolizumab, Nivolumab, and Serplulimab.

9. A drug for the prevention and / or treatment of tumors, characterized in that, The active ingredients include a CD300LF small molecule inhibitor and an immune checkpoint inhibitor. The CD300LF small molecule inhibitor is temopril, and the immune checkpoint inhibitor is a PD-1 antibody. The PD-1 antibody includes, but is not limited to, anti-mouse PD-1 (CD279)-in vivo, pembrolizumab, nivolumab, and serplulimab.

10. Application of temopril as a small molecule inhibitor of CD300LF.

Images