CN114869877B

CN114869877B - Small molecule compound and application thereof as LAG3 protein inhibitor

Info

Publication number: CN114869877B
Application number: CN202210096008.7A
Authority: CN
Inventors: 高坤; 李航鹰; 李亚
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2022-01-26
Filing date: 2022-01-26
Publication date: 2024-05-31
Anticipated expiration: 2042-01-26
Also published as: CN114869877A

Abstract

The invention relates to the application field of small organic molecule compounds, in particular to a small molecule compound and application thereof as LAG3 protein inhibitor. The compound 5,6,7,8,3',4' -hexamethoxyflavanone can inhibit the combination of LAG3 protein and MHC II protein, and the IC ₅₀ value is 1.54 mu M, so that the compound can be used for treating tumors, and a new idea is provided for developing novel anticancer drugs.

Description

Small molecule compound and application thereof as LAG3 protein inhibitor

Technical Field

The invention relates to the application field of small organic molecule compounds, in particular to a small molecule compound and application thereof as LAG3 protein inhibitor.

Background

Lymphocyte activation gene 3 (LAG-3 or CD 223) is a type I transmembrane protein, an inhibitory receptor within a targeted tumor microenvironment that is expressed on the cell surface of activated cd4+ and cd8+ T cells and a subset of NK and dendritic cells, which upon activation by its ligand can inhibit proliferation, activation, effector function and homeostasis of CD4 and CD 8T cells, promoting immune escape of tumor cells. LAG-3 is closely related to CD4, while CD4 is a T helper activated accessory receptor. Both molecules have 4 extracellular Ig-like domains and need to bind their ligands (major histocompatibility complex (MHC) class II) to exert their functional activity. Unlike CD4, LAG-3 is expressed only on the cell surface of activated T cells, and its cleavage from the cell surface terminates LAG-3 signaling. FGL1 is a liver secreted protein, a major LAG-3 functional ligand independent of MHC-II.

Patent CN110720039a discloses a method of treating a tumor in a human patient, the method comprising (i) identifying the patient as having a LAG-3 positive tumor and (ii) administering to the patient a PD-1 pathway inhibitor, a PD1 pathway inhibitor and immune checkpoint inhibitor combination, a LAG-3 inhibitor and PD-1 pathway inhibitor combination, or an anti-CTLA 4 antibody. Patent CN113825527a discloses a method for treating or inhibiting the growth of cancer, comprising selecting a patient suffering from cancer and administering a therapeutically effective amount of a LAG-3 inhibitor in combination with a therapeutically effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody or antigen-binding fragment thereof). In certain embodiments, administration of the PD-1 inhibitor enhances the efficacy of the LAG-3 inhibitor (e.g., an anti-LAG-3 antibody or antigen-binding fragment thereof) in inhibiting cancer growth. The medicine taking LAG-3 as a target spot mainly treats cancers and autoimmune diseases, is an immune escape mechanism and has guiding significance for the design of cancer immunotherapy. However, no document currently discloses that the compound 5,6,7,8,3',4' -hexamethoxyflavanone is capable of inhibiting LAG3 protein binding to MHC ii protein or LAG3 protein binding to FGL1 protein. Therefore, developing a small molecule inhibitor for inhibiting the combination of LAG3 and MHC-II can provide a new idea for cancer immunotherapy and improve the cure rate of cancer.

Disclosure of Invention

In view of the above problems and the deficiencies of the prior art, it is a primary object of the present invention to provide the use of the compound 5,6,7,8,3',4' -hexamethoxyflavanone as LAG3 protein inhibitor.

Preferably, the compound 5,6,7,8,3',4' -hexamethoxyflavanone inhibits LAG3 protein binding to MHC ii proteins.

The second object of the invention is to provide the application of the compound 5,6,7,8,3',4' -hexamethoxyflavanone in preparing the medicine for treating LAG3 positive tumor, wherein the structural formula of the compound is shown as the formula (I):

The third object of the invention is to provide application of 5,6,7,8,3',4' -hexamethoxyflavanone in preparing medicines for preventing LAG3 positive tumors, wherein the structural formula of the compound is shown as formula (I):

Preferably, the tumor is a malignancy selected from the group consisting of liver cancer, bone cancer, pancreatic cancer, skin cancer, oral cancer, head or neck cancer, breast cancer, lung cancer, skin or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal region cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, non-hodgkin's lymphoma, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, childhood cancer, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system tumor, primary CNS lymphoma, tumor angiogenesis, spinal cord axis tumor, brain stem glioma, pituitary adenoma, kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmental carcinoma, hematological malignancy, examples include multiple myeloma, B-cell lymphoma, hodgkin's lymphoma/primary mediastinal B-cell lymphoma, non-hodgkin's lymphoma, acute myeloid lymphoma, chronic myelogenous leukemia, chronic lymphoid leukemia, follicular lymphoma, diffuse large B-cell lymphoma, burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, degenerative large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combination thereof.

Preferably, the compound 5,6,7,8,3',4' -hexamethoxyflavanone treats or prevents tumors by inhibiting LAG3 protein binding to MHC ii proteins.

Preferably, the compound 5,6,7,8,3',4' -hexamethoxyflavanone is added with pharmaceutically acceptable carriers and/or auxiliary materials to be prepared into any one of tablets, sprays, granules, capsules, oral liquid, injection and suspension.

The beneficial effects of the invention are as follows: the invention selects the compound 5,6,7,8,3',4' -hexamethoxyflavanone as the active ingredient of the inhibitor, can effectively inhibit the combination of LAG3 protein and MHC II protein, further treat tumors, and also provides a new idea for developing novel anticancer drugs.

Drawings

FIG. 1 inhibitory Effect of Compounds 5,6,7,8,3',4' -hexamethoxyflavanone on binding of LAG3 protein and MHC II protein

FIG. 2 curves of the binding of Compound 5,6,7,8,3',4' -hexamethoxyflavanone to LAG3 protein

Detailed Description

The present invention will be further described in detail with reference to the following examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The reagent materials used in the examples described below are commercially available unless otherwise specified.

Example one, extraction of Compounds

(1) The roots of Scutellaria baicalensis (Scutellaria baicalensis REHDERIANA DIELS) obtained from Gansu west of Gansu Province were dried in the shade and crushed to obtain 10kg of plant samples. It was immersed 3 times in 60L of methanol at room temperature for 7 days each. All the extracts were concentrated and combined to give 620g of crude extract, which was dissolved in hot distilled water and extracted sequentially with ethyl acetate and n-butanol.

(2) 100G of ethyl acetate extract is separated on macroporous resin, and methanol/water is used for gradient elution according to the volume ratio (v: v) of 30:70, 50:50, 80:20 and 100:0, so that the corresponding four parts of 30%,50%,80% and 100% of extract are obtained.

(3) 50G of the 80% fraction from step (2) was separated on an MCI column, gradient eluted with methanol/water (10:90-100:0, v: v) and 10 fractions (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%) were collected according to TLC detection analysis.

(4) 30G of the 60% fraction obtained in the step (3) is separated by normal phase silica gel, petroleum ether/acetone (3:1-1:3) is used as eluent, and 5 components, 6A-6E, are obtained by TLC detection and analysis.

(5) The fragment 6A (2 g) obtained in step (4) was purified using sephadex (LH-20) (CHCl ₃: meOH, 1:1) as eluent to give compound (40 mg).

Compounds of formula (I) ：¹H NMR(300MHz,CDCl₃δin ppm,Jin Hz):δ_H6.52(s,H-3),7.33(d,J＝1.8Hz,H-2′),6.90(d,J＝8.7Hz,H-5′),7.44(dd,J＝8.7,1.8Hz,H-6′),3.86(s,5-OCH₃),3.88(s,6-OCH₃),4.02(s,7-OCH₃),3.87(s,8-OCH₃),4.01(s,3′-OCH₃),3.99(s,4′-OCH₃);¹³C NMR(75MHz,CDCl₃):δ_C 160.7(C-2),106.5(C-3),177.2(C-4),143.8(C-5),137.7(C-6),151.3(C-7),147.6(C-8),148.7(C-9),114.7(C-10),123.6(C-1′),108.5(C-2′),149.2(C-3′),151.8(C-4′),111.2(C-5′),119.3(C-6′),62.1(5-OCH₃),55.9(6-OCH₃),61.9(7-OCH₃),55.8(8-OCH₃),61.8(3′-OCH₃),61.4(4′-OCH₃).

The structural identification proves that the compound is 5,6,7,8,3',4' -hexamethoxyflavanone, the structural formula is shown as formula (I), and the molecular formula is shown as the specification: c ₂₁H₂₂O₈, molecular weight: 402.13, the characters are as follows: yellow solid, with CAS number 67549-69-3.

It should be noted that the extraction method described in the first embodiment is only one extraction method of the compound 5,6,7,8,3',4' -hexamethoxyflavanone, and the compound 5,6,7,8,3',4' -hexamethoxyflavanone may be obtained by other routes, such as purchase, synthesis, etc.

Example two, inhibition Activity of Compounds 5,6,7,8,3',4' -hexamethoxyflavanone on protein binding experiments

1. Experimental grouping

Positive control: 4 mu LTag-LAG 3,4 mu LTag2-MHC II, 5 mu L anti-Tag1-Tb ³⁺ and 5 mu L anti-Tag2-XL665, and 2 mu L dilutions

Negative control: 4 mu LTag-MHC II, 5 mu L anti-Tag1-Tb ³⁺ and 5 mu L anti-Tag2-XL665, and 6 mu L dilution

Blank control: mu.L of anti-Tag1-Tb ³⁺ and 10. Mu.L of diluent, and 5. Mu.L of detection buffer.

2. Experimental reagent and source

LAG3/MHC ii kits were all purchased from Cisbio.

Tag1-LAG3, tag2-MHC II, anti-Tag1-Tb ³⁺, anti-Tag2-XL665 and 2. Mu.L dilutions were all from the kit.

3. Experimental method

(1) The test compound 5,6,7,8,3',4' -hexamethoxyflavanone was dissolved using DMSO, and the test compound 5,6,7,8,3',4' -hexamethoxyflavanone was diluted using the dilution liquid prepared in the kit.

(2) Mu.L of the compound 5,6,7,8,3',4' -hexamethoxyflavanone mother liquor, 4 mu LTag-LAG 3 protein and 4 mu LMHC II protein were added to 96-well plates.

(3) Incubation was carried out at room temperature for 15 minutes, and then 5. Mu.L of anti-Tag1-Tb3 ⁺ and 5. Mu.L of anti-Tag2-XL665 (or a premix of 10. Mu.L of anti-Tag1-Tb3 ⁺ and 5. Mu.L of anti-Tag2-XL 665) were added and mixed well, at which time the final volume in each well was 20. Mu.L, keeping DMSO below 0.5% during the assay.

(4) Incubation was performed at room temperature for 1 hour to overnight, and specific emission signals at 665nm were detected using a homogeneous time resolved fluorescence (Homogeneous Time-resolved Fluorescence) detector.

Calculation formula of inhibition ratio of 5,6,7,8,3',4' -hexamethoxyflavanone to LAG3 protein and MHC ii binding:

Inhibition ratio = (positive control group 665nm fluorescent signal-experimental group 665nm fluorescent signal)/(positive control group 665nm fluorescent signal-blank group 665nm fluorescent signal)

4. Experimental results

TABLE 1 binding force of 5,6,7,8,3',4' -hexamethoxyflavanone with LAG3

The compound 5,6,7,8,3',4' -hexamethoxyflavanone has strong inhibition activity on the combination of LAG3 protein and MHC II protein, and the IC ₅₀ value is 1.54 mu M.

In conclusion, the compound 5,6,7,8,3',4' -hexamethoxyflavanone of the invention can be used as LAG3 protein inhibitor with IC ₅₀ value of 1.54 mu M for treating tumor,

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims

1. The use of a compound 5,6,7,8,3',4' -hexamethoxyflavone as LAG3 protein inhibitor for non-diagnostic and non-therapeutic purposes, said compound 5,6,7,8,3',4' -hexamethoxyflavone being capable of inhibiting LAG3 protein binding to MHC ii protein.