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    Molecular Basis for the High Affinity Binding and Stabilization of Firefly Luciferase by PTC124

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    1. Auld et al_PNAS_combined100108 copy.pdf (4.721Mb)
    Issue Date
    2010-03-16
    Author
    Auld, Douglas S.
    Lovell, Scott
    Thorne, Natasha
    Lea, Wendy A.
    Maloney, David J.
    Shen, Min
    Rai, Ganesha
    Battaile, Kevin P.
    Thomas, Craig J.
    Simeonov, Anton
    Hanzlik, Robert P.
    Inglese, James
    Publisher
    National Academy of Sciences
    Type
    Article
    Article Version
    Scholarly/refereed, author accepted manuscript
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    Abstract
    Firefly luciferase (FLuc), an ATP-dependent bioluminescent reporter enzyme, is broadly used in chemical biology and drug discovery assays. PTC124 (Ataluren; (3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid) discovered in an FLuc-based assay targeting nonsense codon suppression, is an unusually potent FLuc-inhibitor. Paradoxically, PTC124 and related analogs increase cellular FLuc activity levels by posttranslational stabilization. In this study, we show that FLuc inhibition and stabilization is the result of an inhibitory product formed during the FLuc-catalyzed reaction between its natural substrate, ATP, and PTC124. A 2.0 Å cocrystal structure revealed the inhibitor to be the acyl-AMP mixed-anhydride adduct PTC124-AMP, which was subsequently synthesized and shown to be a high-affinity multisubstrate adduct inhibitor (MAI; KD = 120 pM) of FLuc. Biochemical assays, liquid chromatography/mass spectrometry, and near-attack conformer modeling demonstrate that formation of this novel MAI is absolutely dependent upon the precise positioning and reactivity of a key meta-carboxylate of PTC124 within the FLuc active site. We also demonstrate that the inhibitory activity of PTC124-AMP is relieved by free coenzyme A, a component present at high concentrations in luciferase detection reagents used for cell-based assays. This explains why PTC124 can appear to increase, instead of inhibit, FLuc activity in cell-based reporter gene assays. To our knowledge, this is an unusual example in which the “off-target” effect of a small molecule is mediated by an MAI mechanism.
    URI
    http://hdl.handle.net/1808/8633
    DOI
    https://doi.org/10.1073/pnas.0909141107
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    • Distinguished Professors Scholarly Works [918]
    • Medicinal Chemistry Scholarly Works [244]
    Citation
    Douglas S. Auld, Scott Lovell, Natasha Thorne, Wendy A. Lea, David J. Maloney, Min Shen, Ganesha Rai, Kevin Battaile, Craig J. Thomas, Anton Simeonov, Robert P. Hanzlik, and James Inglese, "Molecular Basis for the High Affinity Binding and Stabilization of Firefly Luciferase by PTC124." Proc. Nat. Acad. Sci. USA 2010, 107, 4878-83. PMID 20194791 http://dx.doi.org/10.1073/pnas.0909141107

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    Contact KU ScholarWorks
    785-864-8983
    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    785-864-8983

    KU Libraries
    1425 Jayhawk Blvd
    Lawrence, KS 66045
    Image Credits
     

     

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