CN108743590A - There is the micromolecular compound and application thereof of affinity with VISTA - Google Patents

There is the micromolecular compound and application thereof of affinity with VISTA Download PDF

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CN108743590A
CN108743590A CN201810471846.1A CN201810471846A CN108743590A CN 108743590 A CN108743590 A CN 108743590A CN 201810471846 A CN201810471846 A CN 201810471846A CN 108743590 A CN108743590 A CN 108743590A
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substituted
carbon
heteroaryl
alkyl
vista
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柳军
轩春晓
王岚岚
江经纬
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China Pharmaceutical University
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China Pharmaceutical University
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Priority to CN201910309631.4A priority patent/CN109893530A/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A61K31/421—1,3-Oxazoles, e.g. pemoline, trimethadione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4245—Oxadiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Abstract

The invention discloses compounds or its pharmaceutically acceptable salt or ester or solvated compounds for developing VISTA target drugs；And the pharmaceutical composition containing the compound or its pharmaceutically acceptable salt or ester or solvated compounds and pharmaceutically acceptable carrier.The micromolecular compound disclosed by the invention for having affinity with VISTA albumen tests the Suppressive effect in murine melanoma body by compound, it was demonstrated that the compound on tumor filtered out is inhibited, can be used in developing VISTA inhibitor.

Description

There is the micromolecular compound and application thereof of affinity with VISTA
Technical field
The present invention relates to a kind of micromolecular compounds with VISTA with affinity, for developing VISTA target drugs.
Background technology
T cell activation mortifier immune globulin variable region structural domain (V-domain immunoglobulin Suppressor of T-cell activation, VISTA) it is the negative sense immunologic test point that can inhibit T cell immune response. VISTA is also known as PD-1H, C10orf54 and Dies1, is I type transmembrane protein, it is thin to be mainly expressed in hematopoietic cell, granulocyte and T Born of the same parents, natural bind receptor are VSIG8 (V-Set and Immunoglobulin domain containing 8)/VSIG3 (V-set and Ig domain-containing protein 3).VISTA has the dual function of receptor and ligand, swollen It expresses and increases in tumor microenvironment and tumor-infiltrated lymph node, can inhibit response of the T cell to tumour antigen, inhibit T cell differentiation For regulatory T cells (Treg), the generation of interleukin 2 (IL-2) is reduced, there is immunosupress and immunoregulatory function. But VISTA can't have an impact the proliferation of B cell.VISTA and metastatic melanoma, inductivity melanoma and age phase The generation of the inflammation phenotype of pass is related, and with the signal different from death protein 1 and ligand 1 (PD-1/PD-L1) Access.
In addition, VISTA also shows significant adjustment effect in autoimmune disease and organ transplant model, it is such as small Mouse model of systemic lupus erythematosus and acute graft versus host disease model.In the mouse squamous cell tumor model first line of a couplet With VISTA it is related to Cytotoxic T lymphocytes (cytotoxic T lymphocyte-associated antigen-4, CTLA-4) monoclonal antibody effectively can have a rest phase T cell and the T cell transformation exhausted is divided into effector T cell by quiet, clinical Research confirms that VISTA expresses notable raising on Human Gastric carcinoma, oral squamous cell carcinoma and non-small cell lung cancer, and is using The expression of VISTA can obviously rise after her wood treatment prostate cancer.These results of study show that VISTA is immune as negative sense Checkpoint potential treatment effect existing for immunotherapy of tumors field.In addition to this, VISTA is in asthma, arthritis etc. itself It also plays an important role in terms of immunity disease, it was demonstrated that potential values of the VISTA as novel targets in terms of drug development.
Therefore, there is an urgent need for there is the micromolecular compound of affinity with VISTA albumen, for developing VISTA inhibitor.
Invention content
The purpose of the present invention is to provide a kind of small molecule chemical combination in albumen level and VISTA albumen with affinity Object, for developing specific VISTA target spots inhibitor.
To achieve the above object, the present invention adopts the following technical scheme that：
The compound of I-formula of formula III or its pharmaceutically acceptable salt or ester or solvated compounds are for developing VISTA target spots Drug；
R in formula I1And R in I-a of formula5、R6、R7For hydrogen, halogen, 1~6 carbon substituted or non-substituted alkyl (such as methyl, Ethyl), substituted or non-substituted benzyl, substituted or non-substituted heteroaryl methylene (such as pyridine methylene, ptfe pyridine methylene Base), substituted or non-substituted alkyl oxy (such as methoxyl group), cyano (such as acetonitrile), nitro (such as first nitro), the carboxylic of 1~6 carbon Base, ester group (such as carbomethoxy), amino, substituted-amino (such as methylamino), hydroxyl, amide (such as formamide), sulfonamide (such as methylsulfonyl Amine), substituted or non-substituted benzoyl (such as tetrafluoro benzoyl), substituted or non-substituted heteroaryl formoxyl (such as tetrafluoro Pyridine), substituted or non-substituted benzenesulfonyl (such as tetrafluoro benzenesulfonyl), substituted or non-substituted aryl or heteroaryl (such as three Chlorobenzene ring), alkyl sulfoxide base (such as methylsulfonyl), the ArCO (CH of the alkyl sulfuryl of 1~6 carbon or 1~6 carbon2)m-、Ar(CH2) mCO-、ArCOCO-、ArCO(CH2)mCO-、ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2) mOOC- or Ar (CH2) MNHOC-, wherein Ar is substituted or non-substituted aryl or heteroaryl, m=1~6；
R in formula I2、R3For substituted or non-substituted aromatic ring, hydrogen, halogen, 1~6 carbon substituted or non-substituted alkyl (such as Methyl, ethyl), substituted or non-substituted benzyl, substituted or non-substituted heteroaryl methylene (such as pyridine methylene, tetrafluoro pyrrole Pyridine methylene), substituted or non-substituted alkyl oxy (such as methoxyl group), cyano (such as acetonitrile), nitro (such as first nitre of 1~6 carbon Base), carboxyl, ester group (such as carbomethoxy), amino, substituted-amino (such as methylamino), hydroxyl, amide (such as formamide), sulfonamide (such as Methanesulfomide), substituted or non-substituted benzoyl (such as tetrafluoro benzoyl), substituted or non-substituted heteroaryl formoxyl (such as ptfe pyridine), substituted or non-substituted benzenesulfonyl (such as tetrafluoro benzenesulfonyl), substituted or non-substituted aryl or heteroaryl Alkyl sulfoxide base (such as methylsulfonyl), the ArCO (CH of base (such as trichlorine phenyl ring), the alkyl sulfuryl of 1~6 carbon or 1~6 carbon2) m-、Ar(CH2)mCO-、ArCOCO-、ArCO(CH2)mCO-、ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2) mOOC- or Ar(CH2) mNHOC-, wherein Ar is substituted or non-substituted aryl or heteroaryl, m=1~6；
R in formula I4For hydrogen, halogen, the substituted or non-substituted alkyl (such as methyl, ethyl) of 1~6 carbon, substitution or non-take The benzyl in generation, substituted or non-substituted heteroaryl methylene (such as pyridine methylene, ptfe pyridine methylene), 1~6 carbon take Generation or non-substituted alkyl oxy (such as methoxyl group), cyano (such as acetonitrile), nitro (such as first nitro), carboxyl, ester group (such as methyl esters Base), amino, substituted-amino (such as methylamino), hydroxyl, amide (such as formamide), sulfonamide (such as Methanesulfomide), substitution or non-take The benzoyl (such as tetrafluoro benzoyl) in generation, substituted or non-substituted heteroaryl formoxyl (such as ptfe pyridine), substitution or non- Substituted benzenesulfonyl (such as tetrafluoro benzenesulfonyl), substituted or non-substituted aryl or heteroaryl (such as trichlorine phenyl ring), 1~6 Alkyl sulfoxide base (such as methylsulfonyl), the ArCO (CH of the alkyl sulfuryl of carbon or 1~6 carbon2)m-、Ar(CH2)mCO-、ArCOCO-、 ArCO(CH2)mCO-、ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2)mOOC-、Ar(CH2) mNHOC-, wherein Ar is to take Generation or non-substituted aryl or heteroaryl, m=1~6；Or I-a of section type, wherein n=0~6；R5、R6Respectively hydrogen, deuterium, halogen Or methyl, and as n=0, R4、R5It is asynchronously hydrogen；
R in formula II1、R2、R3、R4And R in II-a of formula5、R6、R7For hydrogen, the substituted or non-substituted alkane of halogen, 1~6 carbon Base (such as methyl, ethyl), substituted or non-substituted benzyl, substituted or non-substituted heteroaryl methylene (such as pyridine methylene, four Fluorine pyridine methylene), the substituted or non-substituted alkyl oxy (such as methoxyl group) of 1~6 carbon, cyano (such as acetonitrile), nitro (such as First nitro), carboxyl, ester group (such as carbomethoxy), amino, substituted-amino (such as methylamino), hydroxyl, amide (such as formamide), sulphonyl Amine (such as Methanesulfomide), substituted or non-substituted benzoyl (such as tetrafluoro benzoyl), substituted or non-substituted heteroaryl first Acyl group (such as ptfe pyridine), substituted or non-substituted benzenesulfonyl (such as tetrafluoro benzenesulfonyl), substituted or non-substituted aryl or Alkyl sulfoxide base (such as methylsulfonyl), the ArCO of heteroaryl (such as trichlorine phenyl ring), the alkyl sulfuryl of 1~6 carbon or 1~6 carbon (CH2)m-、Ar(CH2)mCO-、ArCOCO-、ArCO(CH2)mCO-、ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2) MOOC- or Ar (CH2) mNHOC-, wherein Ar is substituted or non-substituted aryl or heteroaryl, m=1~6；
R in formula II3For hydrogen, halogen, the substituted or non-substituted alkyl (such as methyl, ethyl) of 1~6 carbon, substitution or non- Substituted benzyl, substituted or non-substituted heteroaryl methylene (such as pyridine methylene, ptfe pyridine methylene), 1~6 carbon Substituted or non-substituted alkyl oxy (such as methoxyl group), cyano (such as acetonitrile), nitro (such as first nitro), carboxyl, ester group (such as methyl esters Base), amino, substituted-amino (such as methylamino), hydroxyl, amide (such as formamide), sulfonamide (such as Methanesulfomide), substitution or non-take The benzoyl (such as tetrafluoro benzoyl) in generation, substituted or non-substituted heteroaryl formoxyl (such as ptfe pyridine), substitution or non- Substituted benzenesulfonyl (such as tetrafluoro benzenesulfonyl), substituted or non-substituted aryl or heteroaryl (such as trichlorine phenyl ring), 1~6 Alkyl sulfoxide base (such as methylsulfonyl), the ArCO (CH of the alkyl sulfuryl of carbon or 1~6 carbon2)m-、Ar(CH2)mCO-、ArCOCO-、 ArCO(CH2)mCO-、ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2)mOOC-、Ar(CH2) mNHOC-, wherein Ar is to take Generation or non-substituted aryl or heteroaryl, m=1~6；Or II-a of formula；
R in formula III1、R2、R3、R4And R5For hydrogen, substituted or non-substituted alkyl (such as methyl, second of halogen, 1~6 carbon Base), substituted or non-substituted benzyl, substituted or non-substituted heteroaryl methylene (such as pyridine methylene, ptfe pyridine methylene Base), substituted or non-substituted alkyl oxy (such as methoxyl group), cyano (such as acetonitrile), nitro (such as first nitro), the carboxylic of 1~6 carbon Base, ester group (such as carbomethoxy), amino, substituted-amino (such as methylamino), hydroxyl, amide (such as formamide), sulfonamide (such as methylsulfonyl Amine), substituted or non-substituted benzoyl (such as tetrafluoro benzoyl), substituted or non-substituted heteroaryl formoxyl (such as tetrafluoro Pyridine), substituted or non-substituted benzenesulfonyl (such as tetrafluoro benzenesulfonyl), substituted or non-substituted aryl or heteroaryl (such as three Chlorobenzene ring), alkyl sulfoxide base (such as methylsulfonyl), the ArCO (CH of the alkyl sulfuryl of 1~6 carbon or 1~6 carbon2)m-、Ar(CH2) mCO-、ArCOCO-、ArCO(CH2)mCO-、ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2) mOOC- or Ar (CH2) MNHOC-, wherein Ar is substituted or non-substituted aryl or heteroaryl, m=1~6.
Preferably, the compound of the I-formula of formula III includes following compounds or its pharmaceutically acceptable salt or ester or molten Immunomodulator compounds：
Preferably, the drug is antitumor drug and/or treats the immunosupress class drug of autoimmune disease.
Preferably, the tumour include fibrosarcoma, mucous membrane sarcoma, embryonal-cell lipoma, chondrosarcoma, osteosarcoma, chordoma, Angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphangioendothelial sarcoma, synovial sarcoma, celiothelioma, Ewing' s tumor, smooth muscle Tumor, rhabdomyosarcoma, colon cancer, cancer of pancreas, syringocarcinoma, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, bronchiolar carcinoma, marrow sample The kidneys such as cancer, clear-cell carcinoma and kidney mother cell cancer, liver cancer, cholangiocarcinoma, choriocarcinoma, spermatogonium cancer, embryonal carcinoma, cervical carcinoma, Orchioncus, lung cancer, Small Cell Lung Cancer, carcinoma of urinary bladder, epithelioma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, The skins such as ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, meningioma, oligodendroglioma, melanoma The nervous system cancers such as cancer, neuroblastoma, retinoblastoma, hematologic cancers, human primary gastrointestinal cancers, cancer of the esophagus and urinary system Associated cancer；The autoimmune disease includes but not limited to lupus erythematosus, asthma, arthritis, AIDS, psoriasis and device Official's graft rejection.
Further, a kind of pharmaceutical composition contains above compound or its pharmaceutically acceptable salt or ester or solvation Close object and pharmaceutically acceptable carrier.
Further, described pharmaceutical composition is tablet, capsule, granule, powder, syrup, oral solution or injection.
The beneficial effects of the invention are as follows：The invention discloses a kind of the micromolecular compound of affinity with VISTA albumen, The Suppressive effect in murine melanoma body is tested by compound, it was demonstrated that the compound on tumor filtered out, which has, to be inhibited to make With, can be used in develop VISTA inhibitor.
Description of the drawings：
Fig. 1 is mouse VISTA expressing quantities after present invention transfection with the variation of expression time；
Wherein, D2, D3, D4, D5 and D6 are respectively the 2nd day, the 3rd day, the 4th day, the 5th day and the 6th day after transfecting；
Fig. 2 is that Western Blot of the present invention identify mouse VISTA albumen distribution situations in elution fraction；
Fig. 3 is that coomassie brilliant blue staining of the present invention identifies mouse VISTA albumen and foreign protein distribution situation in elution fraction；
Fig. 4 is variation of the present inventor VISTA expressing quantities with expression time；
Wherein, D2, D3, D4, D5, D6 and D7 are respectively the 2nd day, the 3rd day, the 4th day, the 5th day, the 6th day and the 7th after transfecting It；
Fig. 5 is that Western Blot of the present invention identify people's VISTA albumen distribution situations in elution fraction；
Fig. 6 is coomassie brilliant blue staining purification Identification descendant VISTA albumen of the present invention and foreign protein distribution situation；
Fig. 7 is MST detection compounds G639-1017 of the present invention and people's VISTA protein affinities；
Fig. 8 is MST detection compounds M351-0056 of the present invention and people's VISTA protein affinities；
Fig. 9 is that mouse VISTA albumen of the present invention inhibits primary T cells IL-2 secretions；
Figure 10, which is candidate compound of the present invention, influences inhibition of the mouse VISTA albumen to primary T cells stirring effect；
Figure 11 is that the present inventor's VISTA albumen inhibits Jurkat cell IL-2 secretions；
Figure 12 is the inhibition that candidate compound of the present invention influences people VISTA albumen to Jurkat cell.
Specific implementation mode
The present invention is described in further detail below in conjunction with attached drawing, the given examples are served only to explain the present invention, not For limiting the scope of the present invention.
It is prepared by the expression of 1 mouse VISTA-Fc fusion proteins of embodiment
ExpiCHO-STMCells (Gibco, A29127), ExpiFectamineTM CHO Transfection Kit (Gibco, A29129) is purchased from Thermofisher Scientific companies；Mouse VISTA-Fc albumen plasmid is by U.S.'s prestige Si Kangxin medical colleges Lily Wang give in laboratory；The anti-VISTA antibody (AF7005) of sheep is purchased from U.S. R&D Systems；It is continuous Sheep secondary antibody (CW0240S) is century bio tech ltd purchased from health；Enhanced BCA albumen concentration detection kit (P0009) it is purchased from green skies bio tech ltd.
Ⅰ、ExpiCHO-STMThe recovery culture of cell
The ExpiCHO-S that will be frozenTMCell goes out to take out from liquid nitrogen, is dipped in 38-39 DEG C of water-bath and melts, and does not stop to shake always It is dynamic, make only to be left very small amount of ice cube in its fast melt to pipe, cell taken out from water-bath, be jetted through after 75% alcohol in It is opened in super-clean bench, the cell suspension (1mL) in cryopreservation tube, which is transferred to preprepared, contains 29mL Expi CHO expression In the cell shaking flask of culture medium (Gibco, A2910001), cell shaking flask is placed in 37 DEG C, CO2A concentration of 8%, shaking speed is It is cultivated in the incubator of 130rpm.
After recovery three days, when cell density reaches 4 × 106–6×106Cell passage is carried out when a living cells/mL, by cell With density for 0.2 × 106–0.3×106A living cells/mL is inoculated in Expi CHO expression culture mediums, and cell culture volumes are 30mL.Cell is put back to 37 DEG C, CO2A concentration of 8%, shaking table (amplitude 19mm) rotating speed be 130rpm incubator in cultivate.
II, cell transfecting
(use ExpiFectamineTMCHO Transfection Kit (Gibco, A29129))
Cultivate ExpiCHO-STMCell density reaches 4 × 106–6×106A living cells/mL carries out sub-bottle (this to cell When be first day), it is 3 × 10 to make the whole density of cell after sub-bottle6–4×106A living cells/mL puts back to cell raw in incubator It is long to stay overnight.
Check cell density and living cell rate within second day.Cell should reach 7 × 106–10×106A living cells/mL is living Cell rate is in 95%-99%.By cell with the fresh ExpiCHO for being preheated to 37 DEG CTMExpression Medium (Gibco, A2910001 6 × 10) are diluted to6A living cells/mL, guarantee transfection volume are 25ml.Gently turn Tissue Culture Flask is with mixing Cell.Soft turning upside down mixes well ExpiFectamine CHO Reagent.Mouse is diluted with OptiPRO SFM VISTA-Fc albumen plasmid and transfection reagent ExpiFectamine CHO Reagent shake centrifuge tube mixing.One side turn from The ExpiFectamine CHO diluted are added in heart pipe into mouse VISTA-Fc albumen Plasmid DNA dilutions on one side Reagent.ExpiFectamine CHO/DNA compound 5min are incubated at room temperature, complex solution is then slowly added into cell In culture bottle, side edged gently shakes Tissue Culture Flask.Tissue Culture Flask is finally put back to 37 DEG C, CO2A concentration of 8%, shaking table Rotating speed is in the incubator of 130rpm.
After transfecting 20h, 150 μ L ExpiCHO Enhancer and 6ml ExpiCHO Feed are added into Tissue Culture Flask, Then culture bottle is put back to incubator and continues culture 8 days.
Each reagent adds volume when 1 25ml transfection volumes of table
III, Identification of Fusion Protein
(1) sample preparation
The cell suspension 1000rpm room temperatures centrifugation 5min after 100 μ L transfections is taken, supernatant is drawn.According to 5 × Loading Buffer is with beta -mercaptoethanol according to 4：1 is configured to sample-loading buffer (5 × Loading buffer, 20 μ L, 5 μ of beta -mercaptoethanol L), then by sample-loading buffer：Sample volume ratio is 1：4 (25 μ L sample-loading buffers are added in 100 μ L samples) mix, in 100 Thermal denaturation 10min at DEG C.Postcooling centrifugation is completed, is frozen in -20 DEG C.
(2)Western Blot
Judge that the molecular weight of estimating of albumen is 83.25KD according to the plasmid map of VISTA-Fc, determines resolving gel concentration It is 8%.8% separation gel and 5% concentration glue are prepared according to table 2.
The film prepared is fixed in electrophoresis tank, electrophoretic buffer is poured into, is cleaned after each loading hole, by preparation The 2nd day to the 6th day cell conditioned medium sample sequentially adds in hole (per 10 μ L of hole) after transfection.Deposition condition is constant pressure 80V, 40min；120V, 1h.Electrophoresis takes out film after terminating, and puts pvdf membrane, gel, filter paper according to the sequence of " black glue tunica albuginea " Ice chest is put into transferring film slot to ensure low temperature at the centre that sandwich presss from both sides, transferring film.Transferring film condition is fixed current 200mA, Transferring film time 2.5h.
Film is placed in 5% skimmed milk power after transferring film, shaking table slowly closes 1h under room temperature.After closing, The liquid of falling deblocking is added VISTA specific antibodies (the anti-VISTA antibody of sheep) and (presses antibody mother liquor：Confining liquid=1：200 dilutions) 4 DEG C are incubated overnight.Second day, antibody is recycled, film is washed with TBST, 80rpm is cleaned five times, each 10min.Again by film with rabbit-anti silk floss Sheep secondary antibody (presses antibody mother liquor：Confining liquid=1：10000 dilutions) it is incubated 1h；Antibody is recycled, film is washed with TBST, 80rpm is washed 10min is operated five times.Finally film is placed in exposure instrument, is uniformly coated with ECL luminescent solutions (180-5001, day energy) (A liquid：B liquid =1：1) it, is exposed.Fig. 1 is the result shows that successful expression goes out mouse VISTA albumen in cell conditioned medium, and since the 2nd day, with The extension of expression time, the content of VISTA albumen gradually increases in supernatant.
Table 2SDS-PAGE gels are prepared
IV, protein concentration
Culture medium in cell shaking flask after transfection is collected into 50ml centrifuge tubes, 1000rpm low-temperature centrifugation 10min, Supernatant, then 7000rpm low-temperature centrifugation 40min are taken, supernatant is collected, 0.45 μm of filter filtering is used in combination, filtrate is collected to be centrifuged in 50ml Guan Zhong.It selects 10KD super filter tube concentrating cells supernatants, 7000rpm to centrifuge 30min according to molecular weight of albumen, collects in super filter tube Concentrate in pipe.
V, protein purification
It is limited purchased from Nanjing Jin Sirui biotechnologies to purify Protein A affinity chromatography mediums (L00400) the column material used Company.
(1) sample preparation
Before crossing column, the protein concentrated solution that step IV obtains first is replaced as buffer components with equilibration buffer.Displacement is logical Super filter tube progress is crossed, by protein concentrated solution and equilibration buffer 1:After 1 dilution, 7000rpm low-temperature centrifugation 30min, repeatedly twice.
(2) column is filled
It fully shakes up so that resin is resuspended, draws in 2ml slurries to new chromatographic column, 2ml balances are previously added in chromatographic column Buffer solution.It allows resin natural subsidence, flows out equilibration buffer.It is added in 10ml equilibration buffers to chromatographic column and balances resin, press The flow velocity of about 1ml/min flows out equilibration buffer.
(3) chromatographic purifying
By sample according in the flow velocity loading to chromatographic column of about 1ml/min, efflux is collected, is labeled as Flow Through, i.e. FT.With the equilibration buffer solution resin of 30ml, flow velocity maintains about 2ml/min, and a group is collected as per 2ml Point.With 15ml elution buffer antibody elutions, flow velocity maintains about 1ml/min, and is added immediately 1/10 elution buffer volume Neutralization buffer adjusts pH to 7.4, and a component is collected as per 2ml.
Equilibration buffer (pH 8.0)：NaCl 2.19g, Na2HPO4·12H2O 1.79g, after adding ultrapure water dissolution, use is dense Hydrochloric acid tune pH is 8.0, then is settled to 250ml with ultra-pure water；It is used after 0.22 μm of membrane filtration, room temperature preservation.
Elution buffer (pH 2.5)：Glycine 1.88g is 2.5 with concentrated hydrochloric acid tune pH, then use after adding ultrapure water dissolution Distilled water is settled to 250ml；It is used after 0.22 μm of membrane filtration, room temperature preservation.
Neutralization buffer (pH 8.5)：Tris 30.285g are 8.5 with concentrated hydrochloric acid tune pH, then use after adding ultrapure water dissolution Distilled water is settled to 50ml；It is used after 0.22 μm of membrane filtration, room temperature preservation.
The component being collected into purification process passes through Western Blot and coomassie brilliant blue staining is identified.FT in Fig. 2 Component does not detect destination protein, illustrates that sample is fully incorporated on column material, and after Equilibration buffer wash and elution is slow After fliud flushing elution, VISTA albumen is successfully eluted from column material.Coomassie brilliant blue staining result in Fig. 2 compares figures 3 Show that the purity for being eluted albumen is high, has no the miscellaneous band of other molecular weight.
VI, collecting protein with it is quantitative
(1) collecting protein
It collects and verifies the elution fraction containing destination protein through Western Blot, 10KD super filter tubes is used in combination to concentrate, centrifugation Condition is 7000rpm low-temperature centrifugations 40min.After concentration, then albumen is preserved into system and is replaced as PBS buffer solution, i.e., by concentrate With PBS buffer solution 1:14 mixing, 7000rpm low-temperature centrifugation 40min, repeatedly twice.Finally collect albumen, frozen after packing in- 80℃。
(2) BCA is quantitative
Illustrate configuration protein standard substance according to enhanced BCA albumen concentration detection kit.By working solution A and B with 50: 1 ratio is made into mixed liquor, and the protein standard substance of 20 μ l is added per hole into 96 orifice plates, then the mixed of 200 μ l is added into every hole Liquid is closed, each concentration does 3 secondary orifices, after 37 DEG C are incubated 30min, OD values is measured at 562nm wavelength, are calculated according to standard curve A concentration of 2.3mg/ml of destination protein shows that successful expression prepares mouse VISTA-Fc fusion proteins.
2 competitive ELISA method of embodiment screens the affinity molecule of VISTA albumen
Competitive ELISA method screen VISTA albumen affinity molecule, by simply incubate, wash altogether and development step i.e. Height of the screening object for VISTA protein affinities can be characterized.Horseradish peroxidase is relied primarily on to react with TMB developing solutions Color change occurs and is detected.VISTA albumen is coated on by physisorption on ELISA Plate when experiment, by washing Wash with the nonspecific binding site that be saturated by albumen existing for closing step removal board bottom, add object to be measured and VISTA specific antibody mixtures are eventually adding secondary antibody, TMB colour developings, detect absorbance at 450nm.If detect object not with VISTA protein bindings, detection antibody will be reacted with VISTA completely, this reaction group absorbance value is maximum, i.e. positive control.If Object and VISTA protein bindings are detected, since detection object occupies a part of binding site of VISTA albumen, detects antibody And the combination of VISTA albumen is reduced, and 450nm, which goes out absorbance value, can also reduce.It can be reflected using the power of absorbance value Screen height of the object for VISTA protein affinities.
Albumen is mouse VISTA-Fc fusion proteins prepared by the expression of embodiment 1, and the compound of experiment screening is by Jiangsu Province New medicament screen key lab provides, and experiment screening compound stock solutions are 10mg/ml, screen final concentration of 10 μ g/ml；Primary antibody is The anti-VISTA antibody (AF7005) of sheep, secondary antibody are sheep secondary antibody (CW0240S).
3 experimental design of table
Experiment is divided into 3 groups：Negative control, positive control and experimental group；Negative control is the group for not being incubated primary antibody, at this time Board bottom VISTA protein binding sites are not occupied, which is 0%；In positive controls, primary antibody and two are added Anti-, the binding site of board bottom VISTA should be fully saturated by primary antibody, which is 100%；The change being added in experimental group If closing object can should decline with VISTA protein bindings, the absorbance value of the group, Percentage bound is in 0%-100%.With coating buffer solution Mouse VISTA-Fc fusion proteins after purification are diluted to 0.09818 μ g/ml, the protein solution of 100 μ l of addition per hole, 4 degree Coating is overnight.Coating protein liquid is discarded within second day, PBST is washed 3 times, and 200 μ l confining liquids are added per hole, are discarded after room temperature closing 2h Confining liquid, PBST are washed 5 times.By 100 μ g/ml samples working solutions of preparation and primary antibody 1:(sample is dense eventually after mixing for addition after 9 mixing Degree is 10 μ g/ml, the final concentration of 0.5 μ g/ml of primary antibody), per 100 μ l of hole, reaction solution is discarded after reacting at room temperature 2h, PBST is washed 5 times. Secondary antibody anti-sheep-HRP (1 is added:10000), per 100 μ l of hole, PBST is washed 5 times after being incubated at room temperature 1h.100 μ l TMB are added Developing solution, dark place are incubated 9min, add and read absorbance value after 50 μ l reaction terminating liquids at 450nm.
The Percentage bound of mouse VISTA-Fc fusion proteins and screening compounds is calculated according to absorbance value.
Percentage bound %=(1- (OD-ODNegative control)/(ODPositive control-ODNegative control)) × 100%
The results show that screening compounds 44 of the present invention, wherein the preferable compound data of activity is as shown in table 4.
4 compound structure of table and concrete activity data
It is coated with buffer solution (50mM carbonate buffer solutions)：Na2CO30.375g, NaHCO30.7325g is dissolved in ultra-pure water In, it is settled to 250ml, 9.6,4 DEG C of preservations of PH.
Confining liquid/antibody diluent (PBS solution of 2.5%BSA)：BSA 0.1g, PBS 10ml, now with the current, mistake It is used after 0.22 μm of filter, 4 DEG C of preservations.
Reaction terminating liquid (2N H2SO4)：18N concentrated sulfuric acid 4ml ultra-pure waters are taken to be diluted to 36ml, room temperature preservation.
TMB (P0209) developing solution is purchased from green skies biological reagent company.
It is prepared by the expression of 3 plancenta hominis outer segment VISTA-His fusion proteins of embodiment
Mouse anti human VISTA antibody (ab201565) is purchased from Abcam companies；Mouse secondary antibody (A0216) is purchased from green skies life Object Science and Technology Ltd.；People's VISTA-His albumen plasmid is synthesized by Nanjing Genscript Biotechnology Co., Ltd.'s customization.
Ⅰ、ExpiCHO-STMThe recovery culture of cell (with embodiment 1)
II, cell transfecting (with embodiment 1)
III, Identification of Fusion Protein
(1) sample preparation (with embodiment 1)
(2)Western Blot
Judge that the molecular weight of estimating of albumen is 22.75KD according to the plasmid map of people VISTA-His, according to various concentration The optimum separative capacity of separation gel selects 10% resolving gel concentration.Remaining is the same as embodiment 1.It takes after transfecting the 2nd day to the 7th day Cell conditioned medium be prepared into Western Blot loading samples, Fig. 4 is the result shows that successful expression goes out mouse VISTA- in cell conditioned medium His albumen, and with the extension of expression time, the content of VISTA albumen gradually increases in supernatant.
IV, protein concentration (with embodiment 1)
V, protein purification
Ni Sepharose6Fast Flow affinity chromatography mediums (17-5318-01) the column materials used are purified to give birth to purchased from GE Order scientific company.
(1) sample preparation
Before crossing column, first with 1 × nickel column balance buffering liquid by the volume displaced at buffer components of protein concentrated solution.Displacement is logical Super filter tube progress is crossed, by protein concentrated solution and equilibration buffer 1:After 1 dilution, 7000rpm low-temperature centrifugation 30min, repeatedly twice.
(2) dress column (with embodiment 1)
(3) chromatographic purifying
Sample collects efflux according in the flow velocity loading to chromatographic column of about 1ml/min, is labeled as Flow Through, That is FT.It is washed respectively with the imidazole buffer (preparation method is shown in Table 5) of 20mM, 40mM, 60mM, 80mM, 100mM, 250mM, 500mM Resin is washed, flow velocity maintains about 1ml/min, each imidazole gradient to rinse 20ml buffer solution volumes, and a component is collected as per 2ml. 20ml 1 × nickel column wash buffer column materials are used after elution, and finally column material is immersed in 20% ethanol solution, 4 DEG C of guarantors It deposits.The component being collected into purification process is passed through WesternBlot and is identified.Fig. 5 is the result shows that people's VISTA-His albumen exists 250mM imidazole concentrations are eluted.
The preparation of 5 various concentration imidazole gradient solution of table
5 × nickel column balance buffering liquid：Na2HPO4·12H2O 17.9g, NaCl 73.125g, add constant volume after ultrapure water dissolution To 500ml, room temperature preservation.
1 × nickel column balance buffering liquid：5 × nickel column balance buffering liquid 100ml, ultra-pure water 400ml, when use, first add ultra-pure water 300ml is 7.4 with concentrated hydrochloric acid tune PH, then be settled to 500ml with ultra-pure water；It is used after 0.22 μm of membrane filtration, room temperature preservation.
500mM imidazole buffers：1 × nickel column balance buffering liquid 250ml, imidazoles 8.51g, after adding ultrapure water dissolution, use is dense Hydrochloric acid tune pH is 7.4, then is settled to 250ml with ultra-pure water；It is used after 0.22 μm of membrane filtration, room temperature preservation.
VI, collecting protein with it is quantitative
(1) collecting protein
It collects through the authenticated elution fractions containing destination protein of Western Blot, 3KD super filter tubes is used in combination to concentrate, centrifugation Condition is 7000rpm low-temperature centrifugations 40min.After concentration, then albumen is preserved into system and is replaced as PBS buffer solution, i.e., by concentrate With PBS buffer solution 1:14 mixing, 7000rpm low-temperature centrifugation 40min, repeatedly twice.Albumen is finally collected, and samples and carries out examining horse It dispenses and is frozen in -80 DEG C after this brilliant blue dyeing identification purity.The results are shown in Figure 6 for the coomassie brilliant blue staining of albumen after purification, Show that the purity of protein prepared is high, without other molecular weight foreign proteins.
(2) BCA quantitative (with embodiment 1)
A concentration of 1.6765mg/ml of destination protein is calculated according to standard curve, shows that successful expression prepares plancenta hominis outer segment VISTA-His fusion proteins.
4 competitive ELISA of embodiment is tested and the affinity molecule of MST experiment screenings identification VISTA albumen
ELISA method by simply incubating, washing and development step can characterize screening object for VISTA albumen parent altogether With the height of power.The preferable molecule of activity after ELISA experiment screenings, the later stage continue through the MST experimental verifications molecule with The affinity of VISTA albumen.MST (micro thermophoresis is dynamic, Microscale Thermophoresis) experiment refers to molecule microcosmic Temperature gradient field in directed movement, by the cooling of the infrared excitation at capillary syring center and periphery, in capillary syring The albumen of required measurement and small molecule mixed solution are filled into capillary by the temperature gradient difference for generating accurate microcosmos area Among suction pipe, the power of albumen and small molecule binding ability is then can detect that by monitoring the distribution of fluorescent molecular signal. It keeps the albumen concentration of fluorescent marker constant, increases the concentration of testing molecule, by the conjugate molecule pair for detecting various concentration The influence of the distribution of fluorescent molecular thermophoresis dynamic equilibrium state, and then obtain the K of the two interactiondValue.
I, ELISA screening experiments
Albumen is plancenta hominis outer segment VISTA-His fusion proteins prepared by the expression of embodiment 3, and the compound of experiment screening is by river Su Sheng new medicament screens key lab provides, and experiment screening compound stock solutions are 10mg/ml, and screening concentration is 10 μ g/ml；Primary antibody For mouse anti human VISTA antibody (ab201565), it is purchased from Abcam companies；Secondary antibody is mouse secondary antibody (A0216), is purchased from the green skies Bio tech ltd.
6 experimental design of table
Experiment is divided into 3 groups：Negative control, positive control and experimental group；Negative control is the group for not being incubated primary antibody, at this time Board bottom VISTA protein binding sites are not occupied, which is 0%；In positive controls, primary antibody and two are added Anti-, the binding site of board bottom VISTA should be fully saturated by primary antibody, which is 100%；The change being added in experimental group If closing object can should decline with VISTA protein bindings, the absorbance value of the group, Percentage bound is in 0%-100%.With coating buffer solution Mouse extracellular fragment fusion protein after purification is diluted to 0.8959 μ g/ml, the protein solution of 100 μ l, 4 degree of coatings are added per hole Overnight.Coating protein liquid is discarded within second day, PBST is washed 3 times, and 200 μ l confining liquids are added per hole, closing is discarded after room temperature closing 2h Liquid, PBST are washed 5 times.By 100 μ g/ml samples working solutions of preparation and primary antibody 1:(sample is final concentration of after mixing for addition after 9 mixing 10 μ g/ml, the final concentration of 0.5 μ g/ml of primary antibody), per 100 μ l of hole, reaction solution is discarded after reacting at room temperature 2h, PBST is washed 5 times.It is added Secondary antibody anti-mouse-HRP (1:10000), per 100 μ l of hole, PBST is washed 5 times after being incubated at room temperature 1h.100 μ l TMB colour developings are added Liquid, dark place are incubated 9min, add and read absorbance value after 50 μ l reaction terminating liquids at 450nm.
The Percentage bound of people VISTA-Fc fusion proteins and screening compounds is calculated according to absorbance value.
Percentage bound %=(1- (OD-ODNegative control)/(ODPositive control-ODNegative control)) × 100%
The results show that screening compounds 48 of the present invention, wherein the preferable compound data of activity is as shown in table 7.
7 compound structure of table and concrete activity data
II, MST screening experiments
MST tests the protein fluorescence labelling kit Monolith Protein Labeling Kit used MicroScale Termophoresis Grade RED-NHS (MO-L001) and loading capillary Monolith NT.115Series Capillaries MicroScale Termophoresis Grade Standard Treated(MO- K002 NanoTemper companies) are purchased from.
The compound of experiment screening is to screen active molecule through ELISA.By diluted chemical compound at a system when test The concentration gradient of row mixes upper machine testing after incubating altogether with the albumen of fluorescent marker.DMSO in the reaction system of every hole when experimental implementation Final concentration of 0.5%.
(1) protein dissolution system exchanges
Add the buffer salt in 3ml distilled waters dissolving label buffer solution.Column A is placed in 1.5ml EP pipes, 3000rpm centrifugations 1min removes excessive liquid in column A, and the label buffer solution of 300 μ l is added, again 3000rpm centrifugations 1min, 3 times repeatedly.It will Column A is added in the protein solution of 100 10 μM of μ l, column A is put into new EP pipes, and 3000rpm centrifuges 2min to get exchange at 4 DEG C The albumen of buffer solution.
(2) label of albumen
The DMSO dissolved solid dyestuffs of 50 μ L, mixing system so that dyestuff fully dissolves is added.Buffer solution is marked in 95 μ l 5 μ l dyestuffs of middle addition, then with protein solution 1:1 volume mixture, room temperature, which is protected from light, is incubated 30min.
(3) purifying of albumen
The storing liquid in column B is outwelled, with 8ml MST buffer solution balance columns B.The label reaction solution of 200 μ L volumes is added extremely The centre positions column B, allow reaction solution to be completely immersed in column B, add the MST buffer solutions of 300 μ L to column B, discard efflux.It waits for The MST buffer solutions of 300 μ L are added the flushing liquor of 600 μ L volumes, collect the liquid eluted up to glimmering completely into column B Signal albumen.
(4) compound test
With MST buffer solutions by diluted chemical compound at a series of concentration gradient, containing for DMSO in each concentration gradient is controlled Amount is 1%, and compound volume is 10 μ l in each concentration gradient.10 μ l labelled proteins are added to each component again, with compound Room temperature, which is protected from light, after mixing is incubated 30min.After incubation, a small amount of compound protein mixed liquor is drawn with capillary Upper machine testing.As a result as follows：
The compound G639-1017 and protein bound Kd of people VISTA are 11.7 ± 1.49 μM (as shown in Figure 7)；
The compound M351-0056 and protein bound Kd of people VISTA are 12.6 ± 3.84 μM (as shown in Figure 8)；
The protein bound MST fitting effects of compound M351-0110 and people VISTA are bad, and Kd values are insincere.
The experimental results showed that compound G639-1017 and M351-0056 and people's VISTA protein binding power are stronger, compound M351-0110 has in conjunction with trend with people's VISTA albumen but binding ability is not strong.
MST buffer solutions：Tris-HCl 3.94g, NaCl 4.383g, MgCl20.4761g, 0.05% polysorbas20,250 μ L is dissolved in ultra-pure water, is settled to 500ml, PH 7.4；It is used after 0.22 μm of membrane filtration, room temperature preservation.
The foundation of 5 mouse primary t cell activation model of embodiment is used for authenticating compound cell activity
Mouse primary T cell can be activated by CD-3 antibody inductions, the phenomenon that cell Proliferation occur and generate cell factor. VISTA can inhibit the proliferation of T cell with the secretion of cell factor as the inhibition signal of T cell activation.In cell model In building process, while T cell CD-3 antibody and VISTA albumen are given, by the secretion for analyzing cell factor in cell conditioned medium Situation can symbolize the case where T cell is activated.
C57BL/6 male mice 8-12 week old is purchased from Beijing Vital River Experimental Animals Technology Co., Ltd.；Mouse IL- 2ELISA detection kits (431004) are purchased from Biolegand companies.
I, the acquisition of mouse spleen lymph suspension
Disconnected neck puts to death mouse, and spleen is taken to be placed in sieve, and screen bed base is in 6 orifice plates, and the tissue extraction containing 4ml is slow in hole Fliud flushing is ground with syringe piston.The cell suspension for collecting filtration, adds the tissue extraction buffer solution for cleaning ware bottom of 1ml (for grinding Grind the board bottom of the ware plate of tissue).1500rpm centrifuge cells 6min.Cell, meter are hanged with the fresh tissue extraction buffer solution punchings of 1ml Number.
II, cell sorting
It is 1 × 10 to adjust cell density8A/ml, control sample volume is in 0.25ml-2ml.With the volume of 50 μ l/ml point Not Jia Ru rat blood serum and separation magnetic bead, be incubated at room temperature 10min after mixing.Vortex magnetic bead RapidSpheres, with 75 μ l/ml's Volume is added in above-mentioned sample.2.5min is incubated at room temperature after mixing.The adjustment of cell sorting buffer solution is added and is incubated sample after magnetic bead Volume is to 2.5ml.Sample cell is put into magnetic pole and is incubated 5min.The cell that sorting is completed is poured into new centrifuge tube, 1500rpm centrifuge cell 6min hang cell with the complete 1640 culture medium punching of 3ml, count spare.
III, t cell activation is tested
First day：Coating protein
It is coated with the mixed liquor of CD-3 and CD-3 and VISTA albumen respectively in 96 orifice plates.Blank group is not coated with any albumen, It substitutes and PBS is added.Excitement group coating CD-3, is not coated with VISTA albumen, CD-3 is diluted to 2.5 μ g/ml using PBS；Inhibition group It is coated with CD-3 and VISTA albumen, keeps CD-3 antibody concentrations constant, is 2.5 μ g/ml, but give three kinds of different VISTA albumen Concentration, respectively 2.5 μ g/ml, 5 μ g/ml, 10 μ g/ml, albumen are diluted with PBS, and it is molten that 100 μ l albumen are added per hole when coating Liquid, 4 DEG C of coatings are overnight.
Table 8T cell-stimulatings test group setting
Annotation："+" indicates that coated albumen system contains the albumen；"-" indicates that coated albumen system does not contain the egg In vain.
Second day：
The coating protein solution in hole is sucked, is cleaned 2 times with PBS, per 200 μ l of hole.It is added per hole thin after 100 μ l sortings Born of the same parents, per 100000, hole cell.Every pore volume is supplied to 200 μ l with fresh complete 1640 culture medium, and 96 orifice plates are put to 37 In DEG C cell incubator, cell conditioned medium is collected after 48h, detects the secretory volume of interleukin-22.The results are shown in Figure 9, after CD-3 excitements T cell has secreted a large amount of IL-2, and VISTA albumen inhibits the secretion of T cell IL-2 in inhibition group.
IV, Compound cellular activity experiment
First day：
It is coated with the mixed liquor of CD-3 and CD-3 and VISTA albumen respectively in 96 orifice plates.Blank group is not coated with any albumen, It substitutes and PBS is added.CD-3 is diluted to 2.5 μ g/ml by exciting group using PBS, and inhibition group and compound experimental group CD-3 are a concentration of 2.5 a concentration of 10 μ g/ml of μ g/ml, VISTA albumen.Albumen is diluted with PBS, is added 100 μ l protein solutions when coating per hole, and 4 DEG C coating overnight.
9 Compound cellular activity experiment group of table is arranged
Second day：
The coating protein solution in hole is sucked, is cleaned 2 times with PBS, per 200 μ l of hole.The cell of 100 μ l is first added per hole, 100000, every hole cell.100 μ l, 20 μM of untested compound are added per hole for compound test set.Control group is equal with blank group Complete 1640 culture mediums of 100 μ l are added.96 orifice plates are put to 37 DEG C of cell incubators, cell conditioned medium is collected after 48h, detection is white The secretory volume of interleukin 2.The results are shown in Figure 10, and candidate compound changes the inhibition that VISTA secretes T cell IL-2, Compound 6809-0223,6809-0226, M355-0156 inhibit the effect of VISTA, promote the secretion of IL-2；Compound G176-0096 promotes VISTA inhibiting effect, reduces the secretion of T cell IL-2.
Complete 1640 culture medium：RPMI 1640 basal medium 178ml, fetal calf serum 20ml, Pen .- Strep are double Anti- 2ml.
Tissue extraction buffer solution：PBS 49ml, fetal calf serum 1ml, after mixing, 4 degree of preservations.
Cell sorting buffer solution：PBS 48.5ml, fetal calf serum 1ml, 100mM EDTA 0.5ml, after mixing, 4 degree of guarantors It deposits.
The foundation of embodiment 6JurkatT cell excitement models is used for authenticating compound cell activity
The one kind and lectin of PMA (Phorbol-12-myristate-13-acetate, PMA) as phorbol exters (Phytohemagglutinin, PHA) can combine exciting Jurkat T cells, stimulate the proliferation of T cell and point of interleukin-22 It secretes.While cell-stimulating, if there are the inhibition signals that VISTA albumen generates, agonistic effects to weaken in system.In cell In the building process of model, while agonist and VISTA albumen are given, by the secretion feelings for analyzing cell factor in cell conditioned medium Condition judges the case where T cell is activated.
Jurkat cell is purchased from Cell Bank of Chinese Academy of Sciences；Human IL-2's ELISA detection kit (431804) is purchased from Biolegand companies.
I, Jurkat cell activation experiment
First day：
VISTA protein solutions are coated in 96 orifice plates.VISTA albumen is diluted to 5 μ g/ml, 10 μ g/ respectively using PBS ml.100 μ l protein solutions are added per hole, 4 DEG C of coatings are overnight.
Second day：
The coating protein solution in hole is sucked, is cleaned 2 times with PBS, per 200 μ l of hole.It is thin that 100 μ l Jurkat are added per hole Born of the same parents, per 20000, hole cell.The mixed liquor that 100 μ l PMA and PHA are added per hole, controls the final concentration of 1ng/ml of PMA, The final concentration of 6 μ g/ml of PHA.96 orifice plates are put to 37 DEG C of cell incubators, cell conditioned medium is collected after 48h and detects interleukin-22 Secretory volume.As a result as shown in figure 11, the people VISTA albumen of 5 μ g/ml and 10 μ g/ml can inhibit PMA-PHA to induce The secretion of Jurkat T cells IL-2.
Table 10T cell-stimulatings test group setting
II, compound activity test experience
First day：
VISTA protein solutions are coated in 96 orifice plates.VISTA albumen is diluted to 5 μ g/ml using PBS.It is added per hole 100 μ l protein solutions, 4 DEG C of coatings are overnight.
Second day：
The coating protein solution in hole is sucked, is cleaned 2 times with PBS, per 200 μ l of hole.100 μ l Jurkat are first added per hole Cell, per 20000, hole cell.The mixed liquor and 50 μ l a concentration of 40 of the PMA and PHA of 50 μ l is added in compound test set μM untested compound.The mixed liquor of 100 μ l PMA and PHA is added in control group.The complete medium of 100 μ l is added in blank group. The final concentration of 1ng/ml, the final concentration of 6 μ g/ml of PHA of PMA are controlled between group.96 orifice plates are put to 37 DEG C of cell incubators, The secretory volume of cell conditioned medium detection interleukin-22 is collected after 48h.As a result as shown in figure 12, compound M351-0056, M351-0110 The Jurkat cell IL-2 that PMA-PHA inductions can be reduced at 50 μM and 25 μM secretes and has dose dependent, showing The inhibiting effect that object M351-0056, M351-0110 promote VISTA albumen is closed, is the agonist of VISTA albumen；And compound Agonistic effects only faint at 50 μM G639-1017.
11 Compound cellular activity experiment group of table is arranged
7 compound of embodiment is to the Suppressive effect in murine melanoma body
I, cell culture
The culture in 1640 complete mediums (containing 10%FBS) of B16-F10 cells, waits for that cell is in exponential phase, raw When long degrees of fusion is 80%~90%, pancreatin had digestive transfer culture.
II, mouse melanoma B16-F10 plants tumor experiment
(1) mice group
The mouse for taking growth conditions almost the same totally 48, is divided into 8 groups, including control group, 6809-0223,6809- 0226, C176-0096, M355-0156, G639-1017, M351-0056, M351-0110 group；Using PBS as blank control, with 6809-0223,6809-0226, C176-0096, M355-0156, G639-1017, M351-0056, M351-0110 are drug, Dosage is 20mg/kg, gastric infusion.
(2) 0 cell of mouse intradermal vaccination melanoma B16-F1
1. anesthetized mice：Mixing is overturned by being managed equipped with the EP of 0 cell of melanoma B16-F1 6 times, draws cell culture fluid To 0.4ml；
2. to the both sides groin intracutaneous injection B16F10 cells of every mouse, per 100 μ l of side.
(3) mouse medication
It is denoted as D0 days on the day of planting tumor, starts within D3 days to be administered, gastric infusion, 20mg/kg/day.Start within D9 days, uses isoflurane It by mouse anesthesia, weighs, with vernier caliper measurement the right and left tumor size.Wait for that control group gross tumor volume is about 2500mm3When, Stop administration, put to death mouse, terminates zoopery.
Each group mouse tumor volume comparison result is as shown in table 12, shows the compound 6809- that present invention screening obtains 0223,6809-0226, C176-0096, M355-0156, G639-1017, M351-0056, M351-0110 have suppression to tumour It makes and uses.
12 each group mouse tumor volume of table
Note：All experimental results use Mean ± SD to indicate.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all the present invention spirit and Within principle, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.

Claims (6)

1. the compound of I-formula of formula III or its pharmaceutically acceptable salt or ester or solvated compounds are for developing VISTA target spot medicines Object；
R in formula I1And R in I-a of formula5、R6、R7For hydrogen, the substituted or non-substituted alkyl, substituted or non-substituted of halogen, 1~6 carbon Benzyl, substituted or non-substituted heteroaryl methylene, the substituted or non-substituted alkyl oxy of 1~6 carbon, cyano, nitro, It is carboxyl, ester group, amino, substituted-amino, hydroxyl, amide, sulfonamide, substituted or non-substituted benzoyl, substituted or non-substituted Heteroaryl formoxyl, substituted or non-substituted benzenesulfonyl, substituted or non-substituted aryl or heteroaryl, 1~6 carbon alkane The alkyl sulfoxide base of base sulfuryl or 1~6 carbon, ArCO (CH2)m-、Ar(CH2)mCO-、ArCOCO-、ArCO(CH2)mCO-、 ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2) mOOC- or Ar (CH2) mNHOC-, wherein Ar is substituted or non-substituted Aryl or heteroaryl, m=1~6；
R in formula I2、R3For substituted or non-substituted aromatic ring, hydrogen, halogen, the substituted or non-substituted alkyl of 1~6 carbon, substitution or Substituted or non-substituted alkyl oxy, the cyanogen of non-substituted benzyl, substituted or non-substituted heteroaryl methylene, 1~6 carbon Base, nitro, carboxyl, ester group, amino, substituted-amino, hydroxyl, amide, sulfonamide, substituted or non-substituted benzoyl, substitution Or non-substituted heteroaryl formoxyl, substituted or non-substituted benzenesulfonyl, substituted or non-substituted aryl or heteroaryl, 1~6 The alkyl sulfoxide base of the alkyl sulfuryl of a carbon or 1~6 carbon, ArCO (CH2)m-、Ar(CH2)mCO-、ArCOCO-、ArCO(CH2) mCO-、ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2) mOOC- or Ar (CH2) mNHOC-, wherein Ar is substitution or non- Substituted aryl or heteroaryl, m=1~6；
R in formula I4For hydrogen, halogen, the substituted or non-substituted alkyl of 1~6 carbon, substituted or non-substituted benzyl, substitution or non- Substituted heteroaryl methylene, the substituted or non-substituted alkyl oxy of 1~6 carbon, cyano, nitro, carboxyl, ester group, amino, Substituted-amino, hydroxyl, amide, sulfonamide, substituted or non-substituted benzoyl, substituted or non-substituted heteroaryl formoxyl, Substituted or non-substituted benzenesulfonyl, substituted or non-substituted aryl or heteroaryl, the alkyl sulfuryl of 1~6 carbon or 1~6 Alkyl sulfoxide base, the ArCO (CH of carbon2)m-、Ar(CH2)mCO-、ArCOCO-、ArCO(CH2)mCO-、ArCONH(CH2)m-、 ArCOO(CH2)m-、Ar(CH2)mOOC-、Ar(CH2) mNHOC-, wherein Ar is substituted or non-substituted aryl or heteroaryl, m =1~6；Or I-a of section type, wherein n=0~6；R5、R6Respectively hydrogen, deuterium, halogen or methyl, and as n=0, R4、R5No It is hydrogen simultaneously；
R in formula II1、R2、R3、R4And R in II-a of formula5、R6、R7For hydrogen, halogen, 1~6 carbon substituted or non-substituted alkyl, take Generation or non-substituted benzyl, substituted or non-substituted heteroaryl methylene, 1~6 carbon substituted or non-substituted alkyl oxy, Cyano, carboxyl, ester group, amino, substituted-amino, hydroxyl, amide, sulfonamide, substituted or non-substituted benzoyl, takes nitro Generation or non-substituted heteroaryl formoxyl, substituted or non-substituted benzenesulfonyl, substituted or non-substituted aryl or heteroaryl, 1 The alkyl sulfuryl of~6 carbon or alkyl sulfoxide base, the ArCO (CH of 1~6 carbon2)m-、Ar(CH2)mCO-、ArCOCO-、ArCO (CH2)mCO-、ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2) mOOC- or Ar (CH2) mNHOC-, wherein Ar is substitution Or non-substituted aryl or heteroaryl, m=1~6；
R in formula II3For hydrogen, halogen, the substituted or non-substituted alkyl of 1~6 carbon, substituted or non-substituted benzyl, substitution or non- Substituted heteroaryl methylene, the substituted or non-substituted alkyl oxy of 1~6 carbon, cyano, nitro, carboxyl, ester group, amino, Substituted-amino, hydroxyl, amide, sulfonamide, substituted or non-substituted benzoyl, substituted or non-substituted heteroaryl formoxyl, Substituted or non-substituted benzenesulfonyl, substituted or non-substituted aryl or heteroaryl, the alkyl sulfuryl of 1~6 carbon or 1~6 Alkyl sulfoxide base, the ArCO (CH of carbon2)m-、Ar(CH2)mCO-、ArCOCO-、ArCO(CH2)mCO-、ArCONH(CH2)m-、 ArCOO(CH2)m-、Ar(CH2)mOOC-、Ar(CH2) mNHOC-, wherein Ar is substituted or non-substituted aryl or heteroaryl, m =1~6；Or II-a of formula；
R in formula III1、R2、R3、R4And R5For hydrogen, the substituted or non-substituted alkyl, substituted or non-substituted of halogen, 1~6 carbon Substituted or non-substituted alkyl oxy, cyano, nitro, the carboxylic of benzyl, substituted or non-substituted heteroaryl methylene, 1~6 carbon It is base, ester group, amino, substituted-amino, hydroxyl, amide, sulfonamide, substituted or non-substituted benzoyl, substituted or non-substituted The alkyl of heteroaryl formoxyl, substituted or non-substituted benzenesulfonyl, substituted or non-substituted aryl or heteroaryl, 1~6 carbon The alkyl sulfoxide base of sulfuryl or 1~6 carbon, ArCO (CH2)m-、Ar(CH2)mCO-、ArCOCO-、ArCO(CH2)mCO-、 ArCONH(CH2)m-、ArCOO(CH2)m-、Ar(CH2) mOOC- or Ar (CH2) mNHOC-, wherein Ar is substituted or non-substituted Aryl or heteroaryl, m=1~6.
2. the compound of I-formula of formula III according to claim 1 or its pharmaceutically acceptable salt or ester or solvated compounds Purposes for developing VISTA target drugs, which is characterized in that the compound includes following compounds or it can pharmaceutically connect The salt or ester or solvated compounds received：
3. the compound of I-formula of formula III according to claim 1 or 2 or its pharmaceutically acceptable salt or ester or solvation Close the purposes that object is used to develop VISTA target drugs, which is characterized in that the drug is antitumor drug and/or treats itself The immunosupress class drug of immunity disease.
4. the compound of I-formula of formula III according to claim 3 or its pharmaceutically acceptable salt or ester or solvated compounds Purposes for developing VISTA target drugs, which is characterized in that the tumour includes fibrosarcoma, mucous membrane sarcoma, fatty meat Tumor, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangioendothelial sarcoma, lymphangioendothelial sarcoma, synovial membrane meat Tumor, celiothelioma, Ewing' s tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, syringocarcinoma, carcinoma of sebaceous glands, mamillary Cancer, papillary adenocarcinoma, bronchiolar carcinoma, cephaloma, clear-cell carcinoma, kidney mother cell cancer, liver cancer, cholangiocarcinoma, choriocarcinoma, essence are former thin Born of the same parents' cancer, embryonal carcinoma, cervical carcinoma, orchioncus, lung cancer, Small Cell Lung Cancer, carcinoma of urinary bladder, epithelioma, glioma, astrocytoma, Medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, meningioma, oligodendroglia are thin Born of the same parents' tumor, melanoma, neuroblastoma, retinoblastoma, hematologic cancers, human primary gastrointestinal cancers, cancer of the esophagus and urinary system cancer Disease；The autoimmune disease includes lupus erythematosus, asthma, arthritis, AIDS, psoriasis and organ-graft refection.
5. a kind of pharmaceutical composition, which is characterized in that the compound containing I-formula of formula III as claimed in claim 1 or 2 or its medicine Acceptable salt or ester or solvated compounds and pharmaceutically acceptable carrier on.
6. pharmaceutical composition according to claim 5, which is characterized in that described pharmaceutical composition is tablet, capsule, particle Agent, powder, syrup, oral solution or injection.
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CN109893530A (en) * 2018-05-17 2019-06-18 中国药科大学 The medical usage and its pharmaceutical composition of VISTA small molecule agonist
CN112250637A (en) * 2020-10-30 2021-01-22 中山大学 Application of virtually screened compound in preparation of anti-tumor drug and drug thereof

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US9631018B2 (en) * 2010-03-26 2017-04-25 The Trustees Of Dartmouth College Vista regulatory T cell mediator protein, vista binding agents and use thereof
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CN108743590A (en) * 2018-05-17 2018-11-06 中国药科大学 There is the micromolecular compound and application thereof of affinity with VISTA

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* Cited by examiner, † Cited by third party
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CN109893530A (en) * 2018-05-17 2019-06-18 中国药科大学 The medical usage and its pharmaceutical composition of VISTA small molecule agonist
CN112250637A (en) * 2020-10-30 2021-01-22 中山大学 Application of virtually screened compound in preparation of anti-tumor drug and drug thereof

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