弓形虫毒力效应因子ROP18竞争性抑制剂的筛选及亲和活性分析
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摘要
目的采用药物靶标虚拟筛选技术筛选靶向弓形虫毒性因子ROP18的小分子竞争性抑制剂,并对中靶小分子进行活性分析。方法通过MOE软件的Structure Prepare完成ROP18三维晶体结构的结构准备;以ATP结合口袋作为筛选起点,分别参考EHT药效团注释及构建方法,以及TypeⅠ激酶抑制剂药效团特征构建靶向天然配体口袋的药效团;通过Pharmacophore Search导入包含202 919个化合物优势构象的SPECS数据库,通过EHT药效团注释方法进行小分子相似性筛选;筛选的小分子经分子对接和打分函数排序后,对排名前100的分子构象进行SAR分析。结果构建的药效团模型具有4个典型的TypeⅠ激酶抑制剂特征,保证了筛选的多样性和互作特异性;在SPECS数据库中共计通过了1 314个符合该模型的小分子,最终选取了25种活性较高的小分子。这些小分子具有2种母环核心Scaffold骨架聚类,分别命名为ScaffoldⅠ类及ScaffoldⅡ类。另有13种Scaffold各异的化合物类型。结论成功构建了靶向ROP18的ATP-结合口袋的药效团模型,筛选得到25种代表性小分子,形成2种Scaffold聚类及13种多样性骨架结构,为开发新型抗弓形虫病药物先导物奠定了基础。
Objective To screen the competitive inhibitors targeted to ROP18 and analyze the activity of them. Method The preparation of ROP18 structure was conducted by a "Structure Prepare" function of Molecular Operating Environment(MOE) software. The ATP-binding pocket was selected to be the starting point for virtual screening. Construction of pharmacophore model was referred to methods of EHT half-quantitative measurement and construction, as well as characteristics of TypeⅠkinase inhibitors. The pharmacophore model of ROP18 was imported into the SPECs database which contains 202 919 compounds dominant conformation, then the small compounds similarity screening was performed through EHT parmacophore measurement. Hit compounds which pass the screening were selected by functions of London dG and GBVI/WSA scoring, and the top 100 hits were analyzed by Structure Activity Relationship(SAR) analysis. Results The final ROP18 parmacophore consisted four typical characteristics of Type Ⅰ kinase inhibitors, such as 3 hydrogen bond acceptors/donors, 2 ring aromatic features occupied in hydrophodic core, 1 cation group feature targeted to the terminus of ATP. This features ensured the diversity and specific recognition of the virtual screening. 1 314 hit compounds analogous to the ROP18 parmacophore were passed through the SPECS database, the screen identified 25 drug-like inhibitors which had the highest affinity activity score and belong to 2 chemical core scaffolds which were named as Scaffold Ⅰ and Scaffold Ⅱ. Additionally, the screen also identified 13 inhibitors with distinct scaffold structure. The docking models and SAR analysis demonstrated that these hits coule engage in multiple hydrogen bonds, salt bridges halogen bonds, and hydrophobic interactions with ROP18, and para-position halo substituents on the benzene ring may enhance their affinity scoring. Conclusion A conservative pharmacophore targeted ROP18 ATP-binding pocket was successfully constructed, and a total of 25 representative inhibitors were screened among SPECS database which belong to 2 scaffold clusters and 13 diverse chemical structure. Our study has laid a research foundation for the development of new anti-toxoplasmosis drug leads.
Objective To screen the competitive inhibitors targeted to ROP18 and analyze the activity of them. Method The preparation of ROP18 structure was conducted by a "Structure Prepare" function of Molecular Operating Environment(MOE) software. The ATP-binding pocket was selected to be the starting point for virtual screening. Construction of pharmacophore model was referred to methods of EHT half-quantitative measurement and construction, as well as characteristics of TypeⅠkinase inhibitors. The pharmacophore model of ROP18 was imported into the SPECs database which contains 202 919 compounds dominant conformation, then the small compounds similarity screening was performed through EHT parmacophore measurement. Hit compounds which pass the screening were selected by functions of London dG and GBVI/WSA scoring, and the top 100 hits were analyzed by Structure Activity Relationship(SAR) analysis. Results The final ROP18 parmacophore consisted four typical characteristics of Type Ⅰ kinase inhibitors, such as 3 hydrogen bond acceptors/donors, 2 ring aromatic features occupied in hydrophodic core, 1 cation group feature targeted to the terminus of ATP. This features ensured the diversity and specific recognition of the virtual screening. 1 314 hit compounds analogous to the ROP18 parmacophore were passed through the SPECS database, the screen identified 25 drug-like inhibitors which had the highest affinity activity score and belong to 2 chemical core scaffolds which were named as Scaffold Ⅰ and Scaffold Ⅱ. Additionally, the screen also identified 13 inhibitors with distinct scaffold structure. The docking models and SAR analysis demonstrated that these hits coule engage in multiple hydrogen bonds, salt bridges halogen bonds, and hydrophobic interactions with ROP18, and para-position halo substituents on the benzene ring may enhance their affinity scoring. Conclusion A conservative pharmacophore targeted ROP18 ATP-binding pocket was successfully constructed, and a total of 25 representative inhibitors were screened among SPECS database which belong to 2 scaffold clusters and 13 diverse chemical structure. Our study has laid a research foundation for the development of new anti-toxoplasmosis drug leads.
引文
[1] Montoya JG, Liesenfeld O. Toxoplasmosis[J]. Lancet, 2004, (363): 1965-76.
[2] Zhou P, Chen Z, Li HL, et al. Toxoplasma gondii infection in humans in China[J]. Parasit Vectors, 2011, 4(1): 1-9.
[3] Jensen KD, Camejo A, Melo MB, et al. Toxoplasma gondii superinfection andvirulence during secondary infection correlate with the exact ROP5/ROP18 allelic combination[J]. MBio, 2015, 6(2): e02280.
[4] Basavaraju A. Toxoplasmosis in HIV infection: An overview [J]. Trop Parasitol, 2016, 6(2):129-35.
[5] Saeij JP, BoyleJP, Coller S, et al. Polymorphic secreted kinases are key virulence factors in toxoplasmosis[J].Science, 2006, 314(5806): 1780-83.
[6] Behnke MS, Khan A, Lauron, EJ, et al. Rhoptry proteins ROP5 and ROP18are major murine virulence factors in genetically divergent SouthAmerican strains of Toxoplasma gondii[J]. PLoS Genet, 2015, 11(8): e1005434.
[7] Etheridge RD, Alaganan A, Tang K, et al. The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice[J]. Cell Host Microbe, 2014, 15(5): 537-50.
[8] Zhang W, Li L, Xia N, et al. Analysis of the virulence determination mechanisms in a local Toxoplasma strain (T.gHB1) isolated from central China[J]. Parasitol Res, 2016, 115(10): 3807-15.
[9] Fentress SJ, Behnke MS, Dunay IR, et al. Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted Kinase promotes macrophage survival and virulence[J]. Cell Host Microbe, 2010, 8(6): 484-95.
[10] Steinfeldt T, Konen-Waisman S, Tong L, et al. Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii[J]. PLoS Biol, 2010, 8(12): e1000576.
[11] Hermanns T, Müller UB, Konen-Waisman S, et al. The Toxoplasma gondii rhoptry protein ROP18 is an Irga6-specific kinase and regulated by the dense granule protein GRA7[J].Cell Microbiol, 2016, 18(2): 244-59.
[12] Yamamoto M, Takeda K. Inhibition of ATF6β-dependent host adaptive immune response by a Toxoplasma virulence factor ROP18[J]. Virulence, 2012, 3(1): 77-80.
[13] Yamamoto M, Ma JS, Mueller C, et al. ATF6β is a host cellular target of the Toxoplasma gondii virulence factor ROP18[J]. J Exp Med, 2011, 208(7): 1533-46.
[14] Virreira Winter S, Niedelman W, Jensen KD, et al. Determinants of GBP recruitment to Toxoplasma gondii vacuoles and the parasitic factors that control it[J]. PLoS One, 2011, 6(9): e24434.
[15] Xia J, Kong L, Zhou LJ, et al. Genome-wide bimolecular fluorescence complementation-based proteomic analysis of Toxoplasma gondii ROP18's human interactome shows its key role in regulation of cell immunity and apoptosis[J]. Front Immunol, 2018(9): 61.
[16] Yang Z, Hou Y, Hao T, et al. A human proteome array approach to identifying key host proteins targeted by Toxoplasma kinase ROP18[J].Mol Cell Proteomics, 2017, 16(3): 469-84.
[17] Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors[J]. Nat Rev Cancer, 2009, 2(1): 28-39.
[18] 尹昆, 寇景轩, 王利磊, 等. 靶向弓形虫毒力决定因子Rop18激酶抑制剂的虚拟筛选[J]. 中国病原生物学杂志, 2015, 10(6): 525-30.
[19] Lim D, Gold DA, Julien L, et al. Structure of the Toxoplasma gondii ROP18 kinase domain reveals a second ligand binding pocket required for acute virulence[J]. J Biol Chem, 2013, 288(48): 34968-80.
[20] Jensen KD, Wang Y, Wojno ED, et al. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation[J]. Cell Host Microbe, 2011, 9(6): 472-83.
[21] 赵桂华, 郝万东, 尹昆. 弓形虫棒状体分泌毒性因子-假激酶的研究进展[J]. 中国人兽共患病学报, 2014, 30(8): 855-9.
[22] Simpson C, Jones NG, Hull-Ryde EA, et al. Identification of small molecule inhibitors that block the Toxoplasma gondii rhoptry kinase ROP18[J]. ACS Infect Dis, 2016, 2(3): 194-206.
[2] Zhou P, Chen Z, Li HL, et al. Toxoplasma gondii infection in humans in China[J]. Parasit Vectors, 2011, 4(1): 1-9.
[3] Jensen KD, Camejo A, Melo MB, et al. Toxoplasma gondii superinfection andvirulence during secondary infection correlate with the exact ROP5/ROP18 allelic combination[J]. MBio, 2015, 6(2): e02280.
[4] Basavaraju A. Toxoplasmosis in HIV infection: An overview [J]. Trop Parasitol, 2016, 6(2):129-35.
[5] Saeij JP, BoyleJP, Coller S, et al. Polymorphic secreted kinases are key virulence factors in toxoplasmosis[J].Science, 2006, 314(5806): 1780-83.
[6] Behnke MS, Khan A, Lauron, EJ, et al. Rhoptry proteins ROP5 and ROP18are major murine virulence factors in genetically divergent SouthAmerican strains of Toxoplasma gondii[J]. PLoS Genet, 2015, 11(8): e1005434.
[7] Etheridge RD, Alaganan A, Tang K, et al. The Toxoplasma pseudokinase ROP5 forms complexes with ROP18 and ROP17 kinases that synergize to control acute virulence in mice[J]. Cell Host Microbe, 2014, 15(5): 537-50.
[8] Zhang W, Li L, Xia N, et al. Analysis of the virulence determination mechanisms in a local Toxoplasma strain (T.gHB1) isolated from central China[J]. Parasitol Res, 2016, 115(10): 3807-15.
[9] Fentress SJ, Behnke MS, Dunay IR, et al. Phosphorylation of immunity-related GTPases by a Toxoplasma gondii-secreted Kinase promotes macrophage survival and virulence[J]. Cell Host Microbe, 2010, 8(6): 484-95.
[10] Steinfeldt T, Konen-Waisman S, Tong L, et al. Phosphorylation of mouse immunity-related GTPase (IRG) resistance proteins is an evasion strategy for virulent Toxoplasma gondii[J]. PLoS Biol, 2010, 8(12): e1000576.
[11] Hermanns T, Müller UB, Konen-Waisman S, et al. The Toxoplasma gondii rhoptry protein ROP18 is an Irga6-specific kinase and regulated by the dense granule protein GRA7[J].Cell Microbiol, 2016, 18(2): 244-59.
[12] Yamamoto M, Takeda K. Inhibition of ATF6β-dependent host adaptive immune response by a Toxoplasma virulence factor ROP18[J]. Virulence, 2012, 3(1): 77-80.
[13] Yamamoto M, Ma JS, Mueller C, et al. ATF6β is a host cellular target of the Toxoplasma gondii virulence factor ROP18[J]. J Exp Med, 2011, 208(7): 1533-46.
[14] Virreira Winter S, Niedelman W, Jensen KD, et al. Determinants of GBP recruitment to Toxoplasma gondii vacuoles and the parasitic factors that control it[J]. PLoS One, 2011, 6(9): e24434.
[15] Xia J, Kong L, Zhou LJ, et al. Genome-wide bimolecular fluorescence complementation-based proteomic analysis of Toxoplasma gondii ROP18's human interactome shows its key role in regulation of cell immunity and apoptosis[J]. Front Immunol, 2018(9): 61.
[16] Yang Z, Hou Y, Hao T, et al. A human proteome array approach to identifying key host proteins targeted by Toxoplasma kinase ROP18[J].Mol Cell Proteomics, 2017, 16(3): 469-84.
[17] Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase inhibitors[J]. Nat Rev Cancer, 2009, 2(1): 28-39.
[18] 尹昆, 寇景轩, 王利磊, 等. 靶向弓形虫毒力决定因子Rop18激酶抑制剂的虚拟筛选[J]. 中国病原生物学杂志, 2015, 10(6): 525-30.
[19] Lim D, Gold DA, Julien L, et al. Structure of the Toxoplasma gondii ROP18 kinase domain reveals a second ligand binding pocket required for acute virulence[J]. J Biol Chem, 2013, 288(48): 34968-80.
[20] Jensen KD, Wang Y, Wojno ED, et al. Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation[J]. Cell Host Microbe, 2011, 9(6): 472-83.
[21] 赵桂华, 郝万东, 尹昆. 弓形虫棒状体分泌毒性因子-假激酶的研究进展[J]. 中国人兽共患病学报, 2014, 30(8): 855-9.
[22] Simpson C, Jones NG, Hull-Ryde EA, et al. Identification of small molecule inhibitors that block the Toxoplasma gondii rhoptry kinase ROP18[J]. ACS Infect Dis, 2016, 2(3): 194-206.