CN109988135B - 一种小分子抑制剂azin19及其在制药中的应用 - Google Patents

一种小分子抑制剂azin19及其在制药中的应用 Download PDF

Info

Publication number
CN109988135B
CN109988135B CN201910272138.XA CN201910272138A CN109988135B CN 109988135 B CN109988135 B CN 109988135B CN 201910272138 A CN201910272138 A CN 201910272138A CN 109988135 B CN109988135 B CN 109988135B
Authority
CN
China
Prior art keywords
azin19
cells
small molecule
molecule inhibitor
polyamine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910272138.XA
Other languages
English (en)
Other versions
CN109988135A (zh
Inventor
金涛
欧洁
吴红艳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Yichang No1 People's Hospital (people's Hospital Of China Three Gorges University)
Original Assignee
Yichang No1 People's Hospital (people's Hospital Of China Three Gorges University)
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yichang No1 People's Hospital (people's Hospital Of China Three Gorges University) filed Critical Yichang No1 People's Hospital (people's Hospital Of China Three Gorges University)
Priority to CN201910272138.XA priority Critical patent/CN109988135B/zh
Publication of CN109988135A publication Critical patent/CN109988135A/zh
Application granted granted Critical
Publication of CN109988135B publication Critical patent/CN109988135B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
    • C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
    • C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
    • C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
    • C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

本发明提供一种小分子抑制剂AZIN19，该小分子抑制剂的结构式为：
Figure DDA0002018760160000011
本发明将所述的小分子抑制剂AZIN19在制备抑制抗酶抑制因子的药物上的应用。结果显示：AZIN19抑制人前列腺癌PC3细胞增殖，降低细胞内ODC蛋白及多胺含量，改变PC3细胞生长周期，同时诱导PC3细胞发生凋亡。

Description

一种小分子抑制剂AZIN19及其在制药中的应用
技术领域
本发明提供一种抑制抗酶抑制因子的小分子抑制剂，同时将该小分子抑制剂用于制备治疗肿瘤疾病的药物上的应用。
背景技术
多胺是细胞生长和发育的基本调节剂，对多胺代谢的精密调控为正常生命活动所必需。研究发现，多胺代谢失调与多种疾病，包括癌症，炎症，动脉粥样硬化，中风，肾衰竭和糖尿病等的发生与发展密切相关。细胞的快速分裂高度依赖于细胞内的多胺含量，由此肿瘤细胞中的总多胺含量也显著性高于正常细胞。研究发现，细胞内多胺水平升高能促进肿瘤生长和侵袭转移，而降低多胺含量则能抑制肿瘤细胞增殖，由此调控多胺代谢途径已经成为肿瘤防治的重要手段。
在正常生理条件下，细胞内多胺含量受复杂的合成、分解代谢及转运机制所调控，由于多胺合成代谢限速酶ODC在控制细胞内多胺含量中发挥最重要的作用，因而对该酶调控的研究也最为深入。抗酶(antizyme,AZ)是细胞内天然存在的ODC抑制因子，它通过双重效应影响细胞内的多胺水平：(1)与ODC结合形成杂二聚体并加快其降解，由此封闭ODC活性和降低细胞内多胺合成；(2)与细胞膜上的多胺转运载体结合，由此抑制细胞外环境中的多胺向细胞内转移。细胞内的AZ活性同时又受另一种因子，抗酶抑制因子(antizymeinhibitor,AZIN)的调节，AZIN以更高的亲和力与AZ形成杂二聚体，由此竞争性释放被AZ捕获的ODC并恢复其活性。此外，细胞内多胺含量是影响AZ活性的另一重要因素，高多胺浓度能通过诱发一种特殊的移码翻译机制而合成功能性AZ蛋白分子。因此，细胞内存在一种以AZ-AZIN为核心的ODC调控网络，是理想的多胺调控分子靶点。
细胞水平上的研究发现，以二氟甲基鸟氨酸(difluoromethylormithine,DFMO)为代表的ODC抑制剂能通过封闭ODC活性而耗竭细胞内多胺，由此抑制肿瘤细胞增殖并诱发肿瘤细胞凋亡。但将这些直接靶向ODC的抑制剂作为抗肿瘤药物用于临床却疗效不佳，究其原因，一是这些药物需要高剂量使用，由此带来病人难以承受的副作用，二是当细胞内的多胺合成受到抑制后，细胞外环境中的多胺能通过细胞膜上的多胺转运载体(polyaminetransport)进入细胞而代偿细胞内由合成抑制引起的多胺含量下降。由此，探索新的能抑制ODC活性的方法和途径，成为该领域的研究热点，具有重要的临床意义和应用前景。
发明内容：
本发明的目的，是以该调控网络中的AZ和AZIN为分子靶点，运用计算机辅助药物设计技术设计和筛选一种AZIN抑制剂，干扰AZ-AZIN的相互作用，降低细胞内ODC和多胺的水平，可以用于制备治疗肿瘤的药物。
该小分子抑制剂AZIN19的具体结构式为：
Figure BDA0002018760140000021
所述的小分子抑制剂AZIN19在制备抑制抗酶抑制因子的药物上的应用。
所述的小分子抑制剂AZIN19在制备抑制人前列腺癌的药物上的应用。
所述的制备抑制人前列腺癌的药物上的应用，具体是在制备抑制人前列腺癌PC3细胞生长繁殖的药物上的应用。
附图说明
图1筛选AZIN小分子抑制剂的药效团模型。
图2免疫印迹法检测PC3细胞内ODC、AZ-1、AZIN-1蛋白含量的变化。
图3HPLC检测PC3细胞内多胺的含量，**：p<0.01，*：p<0.05。
图4MTT法检测小分子药物AZIN19对PC3细胞生长抑制作用。
图5流式细胞术法检测小分子药物AZIN19对PC3细胞生长周期的影响。
图6流式细胞术检测小分子药物AZIN19诱导PC3细胞凋亡。
具体实施方式：
一种小分子抑制剂AZIN19，的具体结构式为：
Figure BDA0002018760140000022
药效团筛选结果
以AZIN的晶体结构(3BTN.pdb)为初始结构，利用口袋探测模块来寻找受体中可能的结合部位，共找到了5个可能的结合位点。结合AZ和AZIN结合界面的突变数据，选择了包含结合界面残基最多的口袋用于进一步筛选小分子抑制剂。以确定的活性位点的关键氨基酸残基特征为基础，利用药效团方法构建结合口袋的药效团模型(图1)。利用构建的药效团模型搜索筛选SPECS化合物数据库，最终购买获得AZIN19化合物。
小分子抑制剂AZIN19对PC3细胞内ODC、AZ、AZIN和多胺含量的影响
1.免疫印迹法检测PC3细胞内ODC、AZ-1和AZIN-1蛋白含量的变化
收集药物处理的细胞和对照组细胞，并用RIPA细胞裂解液裂解半个小时后，经离心机以12000rmp离心后收集蛋白，之后经BCA法定量蛋白。随后以每组等量的蛋白经电泳分离后，电转移至PVDF膜，以5％脱脂牛奶(20mmol/L Tris-HCl,150mmol/LNaCl,0.05％Tween-20,pH7.4)封闭一小时后，与抗ODC、AZIN-1和AZ-1单克隆抗体孵育过夜，后与多克隆抗体室温孵育一小时。通过ECL上机检测蛋白表达量。结果如图2所示，与对照细胞相比较，ODC蛋白含量显著降低，提示我们AZIN19能有效干扰AZ和AZIN蛋白之间的相互作用，加速ODC的降解，从而降低细胞中ODC蛋白的含量。
2.HPLC检测AZIN19对人前列腺癌PC3细胞中多胺含量的影响
收集药物处理的细胞和对照组细胞，用RAPA细胞裂解液裂解获取蛋白之后，通过苯甲酰化反应后抽提多胺，并通过Waters 2695型高效液相色谱仪、2489UV/Vis型阵列检测器和MYC色谱柱(150mmx4.6mm，5μm)，以乙腈-水(40：60)、流速1.0mL/min、254nm和室温(柱温)的条件下检测多胺含量。结果显示，与对照细胞相比，AZIN19处理后细胞中腐胺、精胺及精脒含量减少，表明AZIN19干扰细胞多胺代谢，如图3所示。
小分子抑制剂AZIN19抗肿瘤活性研究
AZIN19有效抑制人前列腺癌PC3细胞的生长繁殖
取对数生长期的PC3细胞以4×103个/孔的浓度接种96孔细胞培养板，培养24h后，分别向细胞孔内加入含有小分子药物AZIN19的1640培养基，使AZIN19的终浓度分别为0μM、12.5μM、25μM、50μM、75μM和100μM(每组设4个复孔)。同时设有不加药对照组和空白组。分别在继续培养24h，48h和72h后，去除培养板中细胞培养基，加入终浓度为0.2g/L的MTT试剂，37℃孵育4h后加DMSO 150μL,震荡混匀后测量570nm处的吸光度值。细胞增殖抑制率＝(A对照孔-A实验孔)/A对照孔×100％。
MTT法检测SLD9059对A549细胞的抑制作用，结果显示所有浓度的AZIN19号均能显著抑制PC3细胞的生长(P<0.001)，且对PC3的生长随着药物的浓度增加抑制作用增加，并随着时间的增长抑制作用增加(P<0.001)(图4)。
小分子抑制剂AZIN19抗肿瘤作用机制研究
1.AZIN19改变人前列腺癌PC3细胞生长周期
取对数生长期的PC3细胞以1.3×105个/孔的浓度接种于6孔细胞培养板，培养24h后，向细胞孔内加入含有小分子药物AZIN19的1640培养基，使AZIN19的终浓度为42μM，同时设有不加药对照组，两组均处理48h。按照细胞周期检测试剂盒说明书处理细胞并经流式细胞仪检测细胞周期。结果表明：AZIN19能引起PC3细胞G0/G1期阻滞(图5)，由64.2％增加到74.5％(P<0.05)。
2.AZIN19诱导人前列腺癌PC3细胞发生凋亡
取对数生长期的PC3细胞以1.3×105个/孔的浓度接种于6孔细胞培养板，培养24h后，向细胞孔内加入含有小分子药物AZIN19的1640培养基，使AZIN19的终浓度为42μM，同时设有不加药对照组，两组均处理48h。按照细胞凋亡试剂盒说明书处理细胞，并经流式细胞仪检测细胞凋亡情况。结果如图6：与对照细胞相比，早期凋亡细胞(AnnexinV+/PI-)比率从1.15％增加到26.3％，晚期凋亡细胞(AnnexinV+/PI+)比率从1.87％增加到69.5％。提示我们AZIN19有效诱导PC3细胞发生凋亡。

Claims (3)

1.小分子抑制剂AZIN19在制备抑制抗酶抑制因子的药物上的应用，其特征在于，所述的小分子抑制剂的结构式为：
Figure FDA0003830673770000011
2.权利要求1所述的小分子抑制剂AZIN19在制备抑制人前列腺癌的药物上的应用，其特征在于，所述的小分子抑制剂的结构式为：
Figure FDA0003830673770000012
3.权利要求2所述的应用，其特征在于，具体在制备抑制人前列腺癌PC3细胞生长繁殖的药物上的应用。
CN201910272138.XA 2019-04-04 2019-04-04 一种小分子抑制剂azin19及其在制药中的应用 Active CN109988135B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910272138.XA CN109988135B (zh) 2019-04-04 2019-04-04 一种小分子抑制剂azin19及其在制药中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910272138.XA CN109988135B (zh) 2019-04-04 2019-04-04 一种小分子抑制剂azin19及其在制药中的应用

Publications (2)

Publication Number Publication Date
CN109988135A CN109988135A (zh) 2019-07-09
CN109988135B true CN109988135B (zh) 2022-10-28

Family

ID=67132408

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910272138.XA Active CN109988135B (zh) 2019-04-04 2019-04-04 一种小分子抑制剂azin19及其在制药中的应用

Country Status (1)

Country Link
CN (1) CN109988135B (zh)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112574224A (zh) * 2019-09-30 2021-03-30 上海迪诺医药科技有限公司 Kras g12c抑制剂及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060160762A1 (en) * 2004-12-13 2006-07-20 Children's Medical Center Corporation Methods for the treatment, diagnosis, and prognosis of cancer

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112574224A (zh) * 2019-09-30 2021-03-30 上海迪诺医药科技有限公司 Kras g12c抑制剂及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
"AZIN-1 小分子抑制剂抗非小细胞肺癌活性研究";王顺超等;《中国药理学通报》;20200407;第36卷;第551-556页 *

Also Published As

Publication number Publication date
CN109988135A (zh) 2019-07-09

Similar Documents

Publication Publication Date Title
Chiaradonna et al. From cancer metabolism to new biomarkers and drug targets
Reis et al. Discovery of new chemical entities for old targets: insights on the lead optimization of chromone-based monoamine oxidase B (MAO-B) inhibitors
Wan et al. A new target for an old drug: identifying mitoxantrone as a nanomolar inhibitor of PIM1 kinase via kinome-wide selectivity modeling
Khanfar et al. Elaborate ligand-based modeling coupled with multiple linear regression and k nearest neighbor QSAR analyses unveiled new nanomolar mTOR inhibitors
Wang et al. Discovering new mTOR inhibitors for cancer treatment through virtual screening methods and in vitro assays
CN110718268B (zh) 虚拟筛选在制备蛋白激酶抑制剂的应用和药物先导化合物
Kozhikkadan Davis et al. Taxifolin as dual inhibitor of Mtb DNA gyrase and isoleucyl-tRNA synthetase: In silico molecular docking, dynamics simulation and in vitro assays
Zhang et al. Ligand-and structure-based identification of novel CDK9 inhibitors for the potential treatment of leukemia
CN109988086B (zh) 一种小分子抑制剂azin30及其在制药中的应用
Triveri et al. Protein allostery and ligand design: Computational design meets experiments to discover novel chemical probes
CN109988135B (zh) 一种小分子抑制剂azin19及其在制药中的应用
Xu et al. Matrine injection inhibits pancreatic cancer growth via modulating carbonic anhydrases-a network pharmacology-based study with in vitro validation
CN109942503B (zh) 一种小分子抑制剂azin18及其在制药中的应用
Pang et al. Iron-sulphur cluster biogenesis factor LYRM4 is a novel prognostic biomarker associated with immune infiltrates in hepatocellular carcinoma
CN109942467B (zh) 一种小分子抑制剂azin32及其在制药中的应用
Chen et al. Discovery of novel dual VEGFR2 and Src inhibitors using a multistep virtual screening approach
Rambabu et al. Virtual screening of National Cancer Institute database for claudin‐4 inhibitors: Synthesis, biological evaluation, and molecular dynamics studies
He et al. In silico approaches using pharmacophore model combined with molecular docking for discovery of novel ULK1 inhibitors
Kumari et al. Recent advances in metabolomics of triple negative breast cancer
Ma et al. Vandetanib drives growth arrest and promotes sensitivity to imatinib in chronic myeloid leukemia by targeting ephrin type‐B receptor 4
Kumar et al. Identification of novel inhibitors of human Chk1 using pharmacophore-based virtual screening and their evaluation as potential anti-cancer agents
Li et al. Inverse screening of Simvastatin kinase targets from glioblastoma druggable kinome
Waenphimai et al. Multiple actions of NMS-P715, the monopolar spindle 1 (MPS1) mitotic checkpoint inhibitor in liver fluke-associated cholangiocarcinoma cells
Hu et al. OTUB2 promotes proliferation and migration of hepatocellular carcinoma cells by PJA1 deubiquitylation
Kandasamy et al. Structural exploration of common pharmacophore based berberine derivatives as novel histone deacetylase inhibitor targeting HDACs enzymes

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant