Developing Colorimetric and Luminescence-Based High-Throughput Screening Platforms for Monitoring the GTPase Activity of Ferrous Iron Transport Protein B (FeoB)

被引:8
作者
Veloria, John [1 ]
Shin, Minhye [2 ]
Devkota, Ashwini K. [1 ]
Payne, Shelley M. [2 ]
Cho, Eun Jeong [1 ]
Dalby, Kevin N. [1 ,3 ]
机构
[1] Univ Texas Austin, Coll Pharm, Targeted Therapeut Drug Discovery & Dev Program, 107 W Dean Keaton St,Stop C0850,BME 6-202E, Austin, TX 78712 USA
[2] Univ Texas Austin, Coll Nat Sci, Dept Mol Biosci, Austin, TX 78712 USA
[3] Univ Texas Austin, Coll Pharm, Div Chem Biol & Med Chem, 107 W Dean Keeton St,Stop C0850, Austin, TX 78712 USA
关键词
NFeoB; GTPase; malachite green; inhibitor; luminescence; high-throughput screening; ASSAY; INHIBITOR;
D O I
10.1177/2472555219844572
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Iron is an essential requirement for the survival and virulence for bacteria. The bacterial ferrous iron transporter protein B (FeoB) functions as a major iron transporter in prokaryotes and has an N-terminal domain (NFeoB) with homology to eukaryotic G-proteins. Its GTPase activity is required for ferrous iron uptake, making it a potential target for antivirulence therapies. Here, two assay strategies relying on different spectroscopic readouts are described to monitor NFeoB GTPase activity. The first one is the colorimetric-based platform that utilizes a malachite green reagent to monitor phosphate production from GTP hydrolysis. The absorbance change directly relates to the GTPase activity of NFeoB. The assay was further improved by the addition of Tween-20 and miniaturized in a 384-well plate format with a 10 mu L assay volume. The second format is a luminescence-based platform, measuring the GTP depletion by using a modified GTPase-Glo assay from Promega. In this platform, the luminescence signal correlates to the amount of GTP remaining, allowing for the direct calculation of GTP hydrolysis by NFeoB. The colorimetric platform was tested in a high-throughput manner against a custom-assembled library of a similar to 2000 small molecules and was found to be simple, cost-effective, and robust. Additionally, the luminescence-based platform demonstrated its capability as an orthogonal assay to monitor GTPase activity, providing a valid and convenient method to filter false hits. These two assay platforms are proven to offset the limitations of each platform while enhancing overall quality and success rates.
引用
收藏
页码:597 / 605
页数:9
相关论文
共 19 条
  • [1] Potassium-activated GTPase Reaction in the G Protein-coupled Ferrous Iron Transporter B
    Ash, Miriam-Rose
    Guilfoyle, Amy
    Clarke, Ronald J.
    Guss, Mitchell
    Maher, Megan J.
    Jormakka, Mika
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2010, 285 (19) : 14594 - 14602
  • [2] A MALACHITE GREEN PROCEDURE FOR ORTHO-PHOSPHATE DETERMINATION AND ITS USE IN ALKALINE PHOSPHATASE-BASED ENZYME-IMMUNOASSAY
    BAYKOV, AA
    EVTUSHENKO, OA
    AVAEVA, SM
    [J]. ANALYTICAL BIOCHEMISTRY, 1988, 171 (02) : 266 - 270
  • [3] A High-Throughput Screen of the GTPase Activity of Escherichia coli EngA to Find an Inhibitor of Bacterial Ribosome Biogenesis
    Bharat, Amrita
    Blanchard, Jan E.
    Brown, Eric D.
    [J]. JOURNAL OF BIOMOLECULAR SCREENING, 2013, 18 (07) : 830 - 836
  • [4] A robotics-based automated assay for inorganic and organic phosphates
    Cogan, EB
    Birrell, GB
    Griffith, OH
    [J]. ANALYTICAL BIOCHEMISTRY, 1999, 271 (01) : 29 - 35
  • [5] Structural basis of GDP release and gating in G protein coupled Fe2+ transport
    Guilfoyle, Amy
    Maher, Megan J.
    Rapp, Mikaela
    Clarke, Ronald
    Harrop, Stephen
    Jormakka, Mika
    [J]. EMBO JOURNAL, 2009, 28 (17) : 2677 - 2685
  • [6] Is the bacterial ferrous iron transporter FeoB a living fossil?
    Hantke, K
    [J]. TRENDS IN MICROBIOLOGY, 2003, 11 (05) : 192 - 195
  • [7] The [35S]GTPγS binding assay:: approaches and applications in pharmacology
    Harrison, C
    Traynor, JR
    [J]. LIFE SCIENCES, 2003, 74 (04) : 489 - 508
  • [8] MALACHITE GREEN MICROMETHOD FOR DETERMINATION OF INORGANIC-PHOSPHATE
    HOHENWALLNER, W
    WIMMER, E
    [J]. CLINICA CHIMICA ACTA, 1973, 45 (02) : 169 - 175
  • [9] A Pan-GTPase Inhibitor as a Molecular Probe
    Hong, Lin
    Guo, Yuna
    BasuRay, Soumik
    Agola, Jacob O.
    Romero, Elsa
    Simpson, Denise S.
    Schroeder, Chad E.
    Simons, Peter
    Waller, Anna
    Garcia, Matthew
    Carter, Mark
    Ursu, Oleg
    Gouveia, Kristine
    Golden, Jennifer E.
    Aube, Jeffrey
    Wandinger-Ness, Angela
    Sklar, Larry A.
    [J]. PLOS ONE, 2015, 10 (08):
  • [10] Structural fold, conservation and Fe(II) binding of the intracellular domain of prokaryote FeoB
    Hung, Kuo-Wei
    Chang, Yi-Wei
    Eng, Edward T.
    Chen, Jai-Hui
    Chen, Yi-Chung
    Sun, Yuh-Ju
    Hsiao, Chwan-Deng
    Dong, Gang
    Spasov, Krasimir A.
    Unger, Vinzenz M.
    Huang, Tai-huang
    [J]. JOURNAL OF STRUCTURAL BIOLOGY, 2010, 170 (03) : 501 - 512