CN106074476A - 帕莫酸在制备BLyS拮抗剂的用途 - Google Patents
帕莫酸在制备BLyS拮抗剂的用途 Download PDFInfo
- Publication number
- CN106074476A CN106074476A CN201610411081.3A CN201610411081A CN106074476A CN 106074476 A CN106074476 A CN 106074476A CN 201610411081 A CN201610411081 A CN 201610411081A CN 106074476 A CN106074476 A CN 106074476A
- Authority
- CN
- China
- Prior art keywords
- blys
- pamoic acid
- binding
- taci
- bcma
- 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.)
- Pending
Links
- 102000016605 B-Cell Activating Factor Human genes 0.000 title claims abstract description 71
- 108010028006 B-Cell Activating Factor Proteins 0.000 title claims abstract description 71
- 239000005557 antagonist Substances 0.000 title claims abstract description 10
- SHWNNYZBHZIQQV-UHFFFAOYSA-J EDTA monocalcium diisodium salt Chemical compound [Na+].[Na+].[Ca+2].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O SHWNNYZBHZIQQV-UHFFFAOYSA-J 0.000 title 1
- 229950009379 palmoxiric acid Drugs 0.000 title 1
- WLJNZVDCPSBLRP-UHFFFAOYSA-N pamoic acid Chemical compound C1=CC=C2C(CC=3C4=CC=CC=C4C=C(C=3O)C(=O)O)=C(O)C(C(O)=O)=CC2=C1 WLJNZVDCPSBLRP-UHFFFAOYSA-N 0.000 claims abstract description 29
- 238000002360 preparation method Methods 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 abstract description 15
- 238000002474 experimental method Methods 0.000 abstract description 7
- 230000002596 correlated effect Effects 0.000 abstract description 4
- 230000027455 binding Effects 0.000 description 26
- 150000001875 compounds Chemical class 0.000 description 18
- 230000003993 interaction Effects 0.000 description 15
- 150000003384 small molecules Chemical class 0.000 description 14
- 102000006942 B-Cell Maturation Antigen Human genes 0.000 description 13
- 108010008014 B-Cell Maturation Antigen Proteins 0.000 description 13
- 101000795167 Homo sapiens Tumor necrosis factor receptor superfamily member 13B Proteins 0.000 description 13
- 102100029675 Tumor necrosis factor receptor superfamily member 13B Human genes 0.000 description 13
- BGFTWECWAICPDG-UHFFFAOYSA-N 2-[bis(4-chlorophenyl)methyl]-4-n-[3-[bis(4-chlorophenyl)methyl]-4-(dimethylamino)phenyl]-1-n,1-n-dimethylbenzene-1,4-diamine Chemical compound C1=C(C(C=2C=CC(Cl)=CC=2)C=2C=CC(Cl)=CC=2)C(N(C)C)=CC=C1NC(C=1)=CC=C(N(C)C)C=1C(C=1C=CC(Cl)=CC=1)C1=CC=C(Cl)C=C1 BGFTWECWAICPDG-UHFFFAOYSA-N 0.000 description 11
- 238000002965 ELISA Methods 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 210000003719 b-lymphocyte Anatomy 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 102100036922 Tumor necrosis factor ligand superfamily member 13B Human genes 0.000 description 5
- 101710181056 Tumor necrosis factor ligand superfamily member 13B Proteins 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 102000004196 processed proteins & peptides Human genes 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- -1 small molecule compounds Chemical class 0.000 description 5
- 208000023275 Autoimmune disease Diseases 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000003032 molecular docking Methods 0.000 description 4
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 4
- 238000003041 virtual screening Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000005711 Benzoic acid Substances 0.000 description 3
- 235000010233 benzoic acid Nutrition 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 230000006957 competitive inhibition Effects 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 235000020183 skimmed milk Nutrition 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 102100029690 Tumor necrosis factor receptor superfamily member 13C Human genes 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000011960 computer-aided design Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000000329 molecular dynamics simulation Methods 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 108700015048 receptor decoy activity proteins Proteins 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000007536 B-Cell Activation Factor Receptor Human genes 0.000 description 1
- 108010046304 B-Cell Activation Factor Receptor Proteins 0.000 description 1
- 108010003455 BLyS receptor Proteins 0.000 description 1
- 101100177114 Caenorhabditis elegans his-69 gene Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229940124602 FDA-approved drug Drugs 0.000 description 1
- 102100025594 Guided entry of tail-anchored proteins factor CAMLG Human genes 0.000 description 1
- 101000932902 Homo sapiens Guided entry of tail-anchored proteins factor CAMLG Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 101710178302 Tumor necrosis factor receptor superfamily member 13B Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960003270 belimumab Drugs 0.000 description 1
- 229940022836 benlysta Drugs 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000000205 computational method Methods 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000002898 library design Methods 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 230000029174 mature B cell apoptotic process Effects 0.000 description 1
- 210000003519 mature b lymphocyte Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000012900 molecular simulation Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000012268 protein inhibitor Substances 0.000 description 1
- 229940121649 protein inhibitor Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000000547 structure data Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- 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
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/194—Carboxylic acids, e.g. valproic acid having two or more carboxyl groups, e.g. succinic, maleic or phthalic acid
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了帕莫酸在制备BLyS拮抗剂的用途。实验表明：帕莫酸能够抑制BLyS与TACI‑Fc的结合。抑制作用与帕莫酸浓度的浓度呈正相关。并证明帕莫酸的抑制作用是特异和有效的。实验表明：帕莫酸能够抑制BLyS与BCMA‑Fc的结合。抑制作用与帕莫酸浓度的浓度呈正相关。并证明帕莫酸的抑制作用是特异和有效的。
Description
技术领域
本发明属于生物医药领域，涉及帕莫酸在制备BLyS拮抗剂的用途。
背景技术
B淋巴细胞刺激因子(B lymphocyte stimulator,BLyS)，又称为B细胞激活因子(Bcell activating factor belonging to the TNF family,BAFF)，是TNF家族中的一员。由单核细胞和巨噬细胞持续合成、分泌。BLyS能够结合B细胞表面的三种受体，B细胞成熟抗原(B cell mature antigen,BCMA)，跨膜激活物和CAML结合物(Transmembrane activatorand CAML-interactor,TACI)以及BAFF受体3(BR3)。受体结合BAFF后，减少成熟B细胞的凋亡。如果敲除BAFF，小鼠体内的成熟B细胞完全缺失。由于BLyS在B淋巴细胞活化、增殖中发挥重要作用，而B淋巴细胞产生的抗体所介导的体液免疫在众多已发现的自身免疫性疾病当中处于中心地位。目前认为BLyS在体内的过量表达与某些自身免疫性疾病如系统性红斑狼疮(SLE)、类风湿性关节炎(RA)等病程的发生、发展密切相关。因此，以阻断BLyS生物学功能为策略，探讨BLyS抑制拮抗剂治疗系统性红斑狼疮和类风湿性关节炎等自身免疫性疾病和B细胞肿瘤疾病的研究正在国外如火如荼的进行中。目前，针对BLyS的抑制剂的研究主要集中在研制具有中和BLyS作用的BLyS诱饵受体(decoy receptor)、抗BLyS抗体和拮抗肽[1-3]。2011年3月美国FDA批准由人类基因科学公司(Human Genome Sciences)和葛兰士-史克(GlaxoSmithKline)公司联合研发的抗BLyS抗体BENLYSTA(Belimumab)治疗系统性红斑狼疮。这是50年来，FDA首次批准治疗该类疾病的药物。但是蛋白抑制剂作为治疗药物具有一定的局限性，例如来源较少和分离纯化难度大以及药物稳定性较差，另外还存在口服的生物利用度较低，主要是因为体内各种酶对多肽类药物的降解性高，从而会导致这一类药物的作用半衰期大大缩短。因此，开发小分子化合物作为BLyS抑制剂的研究具有重要的现实意义。
目前，尚未有帕莫酸在制备BLyS拮抗剂的用途的报道。
1.Jian Sun*(孙剑),Zhou Lin,Jiannan Feng,Yan Li,Beifen Shen.(2008)BAFF-targeting therapy,a promising strategy for treating autoimmunediseases.Eur J Pharmacol 597:1–5.
2.Yacong Zhao,Xiafei Hao,Jiannan Feng,Beifen Shen,Jing Wei*(魏静),Jian Sun*(孙剑)(2015),The comparison of BLyS-binding peptides from phagedisplay library andcomputer-aided design on BLyS–TACI interaction.IntImmunopharmacol,24:219–223.
3.Xiafei Hao,Yanfeng Zhu,Chang Zheng,Xuegang Fu,Jiannan Feng,BeifenShen,Jing Wei*(魏静),Jian Sun*(孙剑).A comparison of biological activity of Blymphocyte stimulator(BLyS)antagonist peptibodies and the elucidation ofpossible BLyS binding sites.Protein&Peptide Letters,2016,23,17-23
4.Xuegang Fu,Liyan Xuan,Yuzhe Wang,Jing Wei*(魏静),Jian Sun*(孙剑).Molecular mechanism of the affinity interactions between BAFF and itspeptides by molecular simulations.Protein&Peptide Letters,2015,22,992-999.
发明内容
本发明的目的是克服现有技术的不足，提供帕莫酸在制备BLyS拮抗剂药物的用途。
本发明的技术方案概述如下：
帕莫酸在制备BLyS拮抗剂的用途。
本发明的优点：
1.对于BLyS结合TACI具有显著抑制作用
竞争ELISA实验结果表明：帕莫酸浓度在1mg/mL时能够抑制2.71±2.51％BLyS与TACI-Fc的结合；在3mg/mL时能够抑制12.36±6.38％BLyS与TACI-Fc的结合；在5mg/mL时能够抑制79.68±2.68％BLyS与TACI-Fc的结合。抑制作用与帕莫酸浓度的浓度呈正相关。
非结合BAFF的小分子化合物(苯甲酸)，即阴性对照，对BLyS与TACI的相互作用没有明显抑制作用、且没有剂量依赖关系。证明帕莫酸的抑制作用是特异和有效的。
2.对于BLyS结合BCMA具有显著抑制作用
竞争ELISA实验结果表明：帕莫酸浓度在1mg/mL时能够抑制5.59±2.01％BLyS与BCMA-Fc的结合；在3mg/mL时能够抑制8.91±4.36％BLyS与BCMA-Fc的结合；在5mg/mL时能够抑制83.72±0.89％BLyS与BCMA-Fc的结合。抑制作用与帕莫酸浓度的浓度呈正相关。
阴性对照化合物苯甲酸对BLyS与BCMA的相互作用没有明显抑制作用、且没有剂量依赖关系。证明帕莫酸的抑制作用是特异和有效的。
附图说明
图1为BLyS结合受体的关键残基，其中+表示关键残基。
图2为BLyS结合口袋的分子表面和关键残基，D1表示保守性结合区域，D2表示特异性结合区域。
图3为虚拟筛选流程图。
图4为帕莫酸结构式。
图5为帕莫酸与BLyS的相互作用的模式。
图6为帕莫酸抑制TACI对BLyS的结合作用。**代表p<0.01。
图7为帕莫酸抑制BCMA对BLyS的结合作用。**代表p<0.01。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。众多研究已经详细阐述BLyS与受体相互作用的可能模式及关键氨基酸。我们在前期工作中通过分子动力学模拟，基于BLyS和其 受体BCMA的晶体结构，通过计算机模拟BLyS与其三个天然受体的相互作用模式，探究其参与保守性结合作用和亲和力选择性的关键残基。在此基础上，利用“锁钥原理”，针对关键结合区域进行商业化化学小分子数据库进行分子虚拟筛选，预测得到一系列具有潜在抑制作用的小分子化合物，获得其实体并进行体外活性测定发现，二元酸类化合物帕莫酸具有一定的抑制BLyS结合TACI和BCMA的作用。为开发有临床应用前景的新药先导药物奠定了实验研究基础。
下面结合具体实施例对本发明作进一步的说明。本发明的实施例是为了使本领域的技术人员能够实施，但并不对本发明作任何限制。其中的常用试剂的公开了是为了使本领域的技术人员更好的实施本发明，但并不对本发明作任何限制。
BLyS蛋白：孙剑,黎燕,冯建男,孙瑛勋,胡美茹,沈倍奋,.人可溶性B淋巴细胞刺激因子基因的克隆及在大肠杆菌中的表达[J].细胞与分子免疫学杂志,2006,(2)SEQ IDNO.8所示。
TACI-Fc蛋白：冀丽军,白乌仁图雅,柴琳,孙剑,.TACI-Fc融合蛋白的基因构建、原核表达及生物活性鉴定[J].生物技术,2013,(3).SEQ ID NO.9所示
BCMA-Fc蛋白：孙剑,冯建男,林周，黎燕,沈倍奋.计算机辅助设计可溶性BLyS受体,eBCMA-Fc融合蛋白及其在大肠杆菌中的表达.中国生物化学与分子生物学报,2008,24(2)SEQ ID NO.9所示。
Fc蛋白：吴真.BCMA-Fc作为潜在药物的研究[D].天津大学,2012SEQ ID NO.10所示。
包被液：称量出33.6g碳酸氢钠和63.6g无水碳酸钠,将其放入烧杯中，向内加入600ml蒸馏水，搅拌至溶质全部溶解，再加入蒸馏水中定容到1L。将其放在4℃下保存。
PBST的配制：取250μl吐温-20加入500ml的1×PBS中，混合均匀至完全融合。在室温下保存。
5％脱脂奶粉的配制：称取脱脂奶粉1g，搅拌下溶解于10ml 0.01M PBS中，加PBS定容至20ml，于4℃保存
TMB显色液：取4.95ml底物缓冲液，0.05ml 1％TMB储液，临用前加入5μl 30％过氧化氢。
1％TMB储液：称取1g TMB，搅拌下充分溶解于80ml DMSO中，加DMSO定容至100ml，保存于4℃。
底物缓冲液(pH 5.0)：取24.3ml 0.1M柠檬酸，25.7ml 0.2M磷酸氢二钠，加入约40ml水，调pH至5.0，再加水定容至100ml，保存于室温。TMB工作液(现配现用)。
实施例1：用计算机模拟筛选技术，筛选出BLyS相关小分子化合物。
蛋白结构信息获取：从Protein Date Bank蛋白质数据库(http://www.rcsb.org/pdb)中下载BLyS蛋白结构(PDB ID：1OQD和1OQE)。采用分子动力学等计算方法，从分子水平上探究BLyS与其天然受体(BCMA，TACI，BR3)的相互作用机制。根据每个氨基酸残基对结合能贡献大小，确定其关键残基。图1和图2分别显示了BLyS靶点天然受体的关键作用残基，以及BLyS靶点的结合口袋和关键残基的相关信息。
小分子数据库的获得：小分子数据库含有近万个结构不同的化合物，其主要来源于两个途径：天然产物数据库或商用小分子数据库中的小分子，课题组合成得到的系列化合物。商用小分子数据库主要指百灵威(J&K)、阿法埃莎(Alfa-Aesar)、西格玛奥德里奇(Sigma-Aldrich)三家试剂公司网站得到。对三维小分子结构进行加氢、加电荷处理后，将文件保存成pdbqt文件格式，进行进一步的分子对接研究。
虚拟筛选：借助AutoDock 4.2软件，将数据库中所有小分子与BLyS活性中心区域进行筛选对接计算。分子对接过程中，将三维小分子结构数据逐一放在BLyS的活性位点处，通过不断优化小分子模型的位置、构象、分子内部可旋转键的二面角，寻找化合物与BLyS作用的最佳构象，并预测其结合模式。分析对接结果，根据给出的小分子预测结合分值，考察化合物与BLyS结合中心的匹配度，以及与关键氨基酸相互关系情况，筛选出潜在具有BLyS亲和力的小分子候选化合物。分值越大，代表化合物与BLyS的亲和力越高，即化合物可能有较好的亲和力。选择综合评价最佳的化合物，获得化合物实体，并进行ELISA实验。图3中描述了虚拟筛选的流程图。
实施例2：计算机分析小分子拮抗剂和BLyS结合模式。
利用计算机分子对接方法，结合Chimera软件探讨了帕莫酸(结构式参见图4)与BLyS分子机制研究，分析其与BLyS相互识别的关键氨基酸残基以及关键相互作用(图5)。发现帕莫酸的一个苯环插入到BLyS的保守性疏水口袋，与His69，Leu70和Ile92形成疏水相互作用，并与Arg124和Arg90残基侧链形成π-cation作用，共同加强帕莫酸与BLyS的结合作用；两个羧基与BLyS的关键残基Arg124和Arg90形成稳定的盐桥作用；另外一个苄位的羰基与Arg124形成氢键相互作用。
实施例3：竞争ELISA分析化合物对于BLyS结合TACI/BCMA蛋白的抑制作用。
如果化合物帕莫酸可以与TACI/BCMA竞争结合BLyS活性口袋，则加入的化合物帕莫酸会抑制BLyS结合TACI/BCMA；相反，如果化合物不结合BLyS，也就不存在与TACI/BCMA竞争。则加入化合物帕莫酸对BLyS结合TACI/BCMA没有影响。因此，通过竞争抑制ELISA实验，来测试小分子帕莫酸阻断BLyS与受体TACI和BCMA结合的作用。
首先，按常规ELISA方法做BLyS与TACI-Fc/BCMA-Fc结合的ELISA。确定竞争抑制ELISA实验中使用TACI-Fc/BCMA-Fc的浓度。最终确定浓度是10μg/mL。竞争抑制ELISA的具体操作步骤如下：
a)包被：20g/mL的BLyS与包被液按1:1稀释，制备10μg/mL的BLyS液。将稀释的BLyS液按50微升/孔包被96孔板。4℃过夜。
b)洗板后，用5％的脱脂奶粉室温封闭2小时。
c)洗板，再将10g/mL的TACI-Fc或BCMA-Fc与不同体积的帕莫酸水溶液混合，制备小分子化合物帕莫酸终浓度梯度为0、1、3和5mg/mL的混合溶液，每孔加入50微升。37℃，温育1小时。未加小分子溶液的混合溶液作为空白对照组。苯甲酸为阴性对照化合物。
d)洗板，加入HRP标记羊抗人2抗(按说明书稀释)。
e)洗板后加入TMB显色剂显色。
f)终止反应，测450纳米的OD值。化合物竞争BLyS与TACI-Fc/BCMS-Fc的相互作用的抑制效果用下述公式计算：抑制率％＝[(空白对照组OD450-实验组OD450)/空白对照组OD450]*100％。(抑制率见图6和图7) 。
Claims (1)
1.帕莫酸在制备BLyS拮抗剂的用途。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610411081.3A CN106074476A (zh) | 2016-06-08 | 2016-06-08 | 帕莫酸在制备BLyS拮抗剂的用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610411081.3A CN106074476A (zh) | 2016-06-08 | 2016-06-08 | 帕莫酸在制备BLyS拮抗剂的用途 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106074476A true CN106074476A (zh) | 2016-11-09 |
Family
ID=57845456
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610411081.3A Pending CN106074476A (zh) | 2016-06-08 | 2016-06-08 | 帕莫酸在制备BLyS拮抗剂的用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106074476A (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1449375A (zh) * | 2000-06-23 | 2003-10-15 | 希格马托制药工业公司 | 双羟萘酸或其一种衍生物或其一种类似物用于制备治疗以淀粉状蛋白聚集物沉积为特征的疾病的药物的用途 |
| US7858663B1 (en) * | 2007-10-31 | 2010-12-28 | Pisgah Laboratories, Inc. | Physical and chemical properties of thyroid hormone organic acid addition salts |
-
2016
- 2016-06-08 CN CN201610411081.3A patent/CN106074476A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1449375A (zh) * | 2000-06-23 | 2003-10-15 | 希格马托制药工业公司 | 双羟萘酸或其一种衍生物或其一种类似物用于制备治疗以淀粉状蛋白聚集物沉积为特征的疾病的药物的用途 |
| US7858663B1 (en) * | 2007-10-31 | 2010-12-28 | Pisgah Laboratories, Inc. | Physical and chemical properties of thyroid hormone organic acid addition salts |
Non-Patent Citations (2)
| Title |
|---|
| COURTNEY HODGSON ET AL.: "The action of retinoic acid on psoriatic skin", 《CLINICAL AND EXPERIMENTAL DERMATOLOGY》 * |
| PINGWEI ZHAO ET AL.: "Targeting of the Orphan Receptor GPR35 by Pamoic Acid: A Potent Activator of Extracellular Signal-Regulated Kinase and b-Arrestin2 with Antinociceptive Activity", 《MOL PHARMACOL》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11674967B2 (en) | Method of identifying potential inhibitors of APO TNFα trimers | |
| Lu et al. | Harnessing allostery: a novel approach to drug discovery | |
| Lumangtad et al. | The signal peptide as a new target for drug design | |
| Ortiz Zacarías et al. | Design and characterization of an intracellular covalent ligand for CC chemokine receptor 2 | |
| Silvian et al. | Small molecule inhibition of the TNF family cytokine CD40 ligand through a subunit fracture mechanism | |
| Lawrie et al. | Engineering of a small protein scaffold to recognize sulfotyrosine with high specificity | |
| Wang et al. | A novel peptide exerts potent immunosuppression by blocking the two-site interaction of NFAT with calcineurin | |
| Pegoli et al. | Conjugation of short peptides to dibenzodiazepinone-type muscarinic acetylcholine receptor ligands determines M2R selectivity | |
| Preti et al. | Design, synthesis, and biological evaluation of novel 2-((2-(4-(substituted) phenylpiperazin-1-yl) ethyl) amino)-5′-N-ethylcarboxamidoadenosines as potent and selective agonists of the A2A adenosine receptor | |
| US8536306B2 (en) | Human A2A adenosine receptor crystals and uses thereof | |
| US7662581B1 (en) | Eg5 co-crystals | |
| Qi et al. | Novel osteogenic peptide from bovine bone collagen hydrolysate: Targeted screening, molecular mechanism, and stability analysis | |
| Ziebell et al. | Peptides that mimic glycosaminoglycans: high-affinity ligands for a hyaluronan binding domain | |
| CN106074488B (zh) | 苯甲氧羰基-L-谷氨酸在制备BLyS拮抗剂的用途 | |
| CN103965295B (zh) | BLyS拮抗肽、含TA-Fc融合蛋白基因的质粒及TA-Fc融合蛋白 | |
| Schwehm et al. | Design and elaboration of a tractable tricyclic scaffold to synthesize druglike inhibitors of dipeptidyl peptidase-4 (DPP-4), antagonists of the C–C Chemokine Receptor Type 5 (CCR5), and highly potent and selective phosphoinositol-3 Kinase δ (PI3Kδ) inhibitors | |
| CN106074476A (zh) | 帕莫酸在制备BLyS拮抗剂的用途 | |
| CN105919989A (zh) | (2R,3R)-二苯甲酰酒石酸在制备BLyS拮抗剂的用途 | |
| Persico et al. | GTP is an allosteric modulator of the interaction between the guanylate-binding protein 1 and the prosurvival kinase PIM1 | |
| CN103936833A (zh) | BLyS拮抗肽、含TC-Fc融合蛋白基因的质粒及TC-Fc融合蛋白 | |
| CN104193806B (zh) | BLyS拮抗肽SS12、含该拮抗肽的融合蛋白SS12‑Fc及基因 | |
| Heng et al. | Design, synthesis, and biological evaluation of JNK1 peptide inhibitors for alleviating insulin resistance in type 2 diabetes | |
| He et al. | Design, synthesis and biological evaluation of potent and selective S1PR1 agonists for the treatment of ulcerative colitis | |
| Roper et al. | “Affimer” synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation | |
| Tanaka et al. | Template-assisted and self-activating clicked peptide as a synthetic mimic of the SH2 domain |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20161109 |
|
| WD01 | Invention patent application deemed withdrawn after publication |