以番茄斑萎病毒核蛋白为靶点的荧光偏振药物筛选体系的建立及应用
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摘要
目的:基于荧光偏振技术建立靶向番茄斑萎病毒(Tomato spotted wilt virus,TSWV)核蛋白(NP)与核酸相互作用的药物筛选体系,并应用该体系展开药物筛选。方法:将目的基因克隆到pGEX-6p-1表达载体上,并采用大肠杆菌表达系统进行目的蛋白的异源表达。建立靶向TSWV NP与核酸相互作用的荧光偏振药物筛选体系,对体系的结合时间、DMSO耐受、变异性和稳定性进行研究,并展开药物筛选。结果:成功构建重组质粒pGEX-6p-1-NP,在大肠杆菌中表达并分离纯化出高质量的核蛋白。基于荧光偏振技术建立了信噪比为8∶1,Z因子为0. 82的稳定的靶向TSWV NP与核酸相互作用的药物筛选体系,并对化合物库中1 000种化合物展开药物筛选,经过初步筛选获得了1种IC_(50)为4. 15μmol/L的化合物。结论:建立了稳定的荧光偏振筛选体系,适用于靶向NP与核酸相互作用的药物的筛选。筛选到的化合物为番茄斑萎病毒的预防和控制提供参考。
Objective: Based on fluorescence polarization technology, a binding assay targeting the interaction between tomato spotted wilt virus( TSWV) nucleoprotein( NP) and nucleic acid was established and used for drug screening. Methods: The full-length NP DNA amplicon was cloned into the pGEX-6p-1 expression vector,and the construct pGEX-6p-1-NP was transformed into E. coli strain BL( DE3). Recombinant NP protein was purified with a general protocol. A fluorescence polarization assay that is sensitive to inhibitors disrupting TSWV NP/nucleic acid interactions was established. The assay's DMSO tolerance,incubation time,stability and variability were studied and a pilot drug screening was performed. Results: The recombinant plasmid pGEX-6p-1-NP was successfully constructed and the high quality recombinant NP was purified. A 384-well fluorescence polarization assay targeting TSWV NP and nucleic acid interaction was developed and validated,with a signal-tonoise ratio of 8∶ 1 and a Z factor of 0. 82 was obtained,demonstrating the assay is HTS compatible. The assay was used to screen 1 000 compounds in the chemical libraries. After the primary screening,one compound with IC_(50) of 4. 146μmol/L was identified. Conclusion: The fluorescence polarization assay is stable and suitable for the screening inhibitors blocking the interaction between NP and nucleic acids,and the compound provides a reference for the prevention and control of TSWV.
Objective: Based on fluorescence polarization technology, a binding assay targeting the interaction between tomato spotted wilt virus( TSWV) nucleoprotein( NP) and nucleic acid was established and used for drug screening. Methods: The full-length NP DNA amplicon was cloned into the pGEX-6p-1 expression vector,and the construct pGEX-6p-1-NP was transformed into E. coli strain BL( DE3). Recombinant NP protein was purified with a general protocol. A fluorescence polarization assay that is sensitive to inhibitors disrupting TSWV NP/nucleic acid interactions was established. The assay's DMSO tolerance,incubation time,stability and variability were studied and a pilot drug screening was performed. Results: The recombinant plasmid pGEX-6p-1-NP was successfully constructed and the high quality recombinant NP was purified. A 384-well fluorescence polarization assay targeting TSWV NP and nucleic acid interaction was developed and validated,with a signal-tonoise ratio of 8∶ 1 and a Z factor of 0. 82 was obtained,demonstrating the assay is HTS compatible. The assay was used to screen 1 000 compounds in the chemical libraries. After the primary screening,one compound with IC_(50) of 4. 146μmol/L was identified. Conclusion: The fluorescence polarization assay is stable and suitable for the screening inhibitors blocking the interaction between NP and nucleic acids,and the compound provides a reference for the prevention and control of TSWV.
引文
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[16]Munson P J,Rodbard D.An exact correction to the“ChengPrusoff”correction.Journal of Receptor Research,1988,8(1-4):533-546.
[2]Komoda K,Narita M,Yamashita K,et al.The asymmetric trimeric ring structure of the nucleocapsid protein of Tospovirus.Journal of Virology,2017,91(20):e01002-17.
[3]Guo Y,Liu B,Ding Z,et al.Distinct mechanism for the formation of the ribonucleoprotein complex of tomato spotted wilt virus.Journal of Virology.2017,91(23):e00892-17.
[4]Wilson J,Rossi C P,Carboni S,et al.A homogeneous 384-well high-throughput binding assay for a TNF receptor using alphascreen technology.Journal of Biomolecular Screening,2003,8(5):522-532.
[5]Checovich W J,Bolger R E,Burke T,et al.Fluorescence polarization:a new tool for cell and molecular biology.Nature,1995,375(6528):254-256.
[6]Kakehi K,Oda Y,Kinoshita M,et al.Fluorescence polarization:analysis of carbohydrate-protein interactions.Analytical Biochemistry,2001,297(2):111-116.
[7]Moerke N J.Fluorescence polarization(FP)assays for monitoring peptide-protein or nucleic acid-protein binding.Current Protocols in Chemical Biology,2009,1(1):1-15.
[8]Terpetschnig E,Szmacinski H,Lakowicz J R,et al.Longlifetime metal-ligand complexes as probes in biophysics and clinical chemistry.Methods in Enzymology,1997,278(278):295-321.
[9]Burke T J,Loniello K R,Beebe J A,et al.Development and application of fluorescence polarization assays in drug discovery.Combinatorial Chemistry&High Throughput Screening,2003,6(3):183-194.
[10]Jameson D M,Sawyer W H.Fluorescence anisotropy applied to biomolecular interactions.Methods in Enzymology,1995,246(28):283-300.
[11]Hill J J,Royer C A.Fluorescence approaches to study of proteinnucleic acid complexation.Methods in Enzymology,1997,278(97):390-416.
[12]Roehrl M H,Wang J Y,Wagner G,et al.A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization.Biochemistry,2004,28;43(51):16056-16066.
[13]Roehrl M H,Wang J Y,Wagner G,et al.Discovery of smallmolecule inhibitors of the NFAT-calcineurin interaction by competitive high-throughput fluorescence polarization screening.Biochemistry,2004,43(51):16067-16075.
[14]Zhang J H,Chung T D,Oldenburg K R,et al.A simple statistical parameter for use in evaluation and validation of high throughput screening assays.Journal of Biomolecular Screening,1999,4(2):67-73.
[15]Arkin M R,Glicksman M A,Fu H,et al.Inhibition of ProteinProtein Interactions:Non-Cellular Assay Formats//Sittampalam G S,Coussens N P,Nelson H,et al.Assay guidance manual.Bethesda:Eli Lilly&Company and the National Center for Advancing Translational Sciences,2004.
[16]Munson P J,Rodbard D.An exact correction to the“ChengPrusoff”correction.Journal of Receptor Research,1988,8(1-4):533-546.