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Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.

Zhang M, Peh J, Hergenrother PJ, Cunningham BT - J. Am. Chem. Soc. (2014)

Bottom Line: Here we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with subpicometer wavelength accuracy.Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets, pairs that have binding affinities or inhibition constants ranging from subnanomolar to low micromolar.Finally, a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II is performed, in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, ‡Department of Chemistry, §Department of Bioengineering, and ⊥Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

ABSTRACT
High-throughput screening has enabled the identification of small molecule modulators of important drug targets via well-established colorimetric or fluorimetric activity assays. However, existing methods to identify small molecule binders of nonenzymatic protein targets lack either the simplicity (e.g., require labeling one of the binding partners with a reporter) or throughput inherent in enzymatic assays widely used for HTS. Thus, there is intense interest in the development of high-throughput technologies for label-free detection of protein-small molecule interactions. Here we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with subpicometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets, pairs that have binding affinities or inhibition constants ranging from subnanomolar to low micromolar. Finally, a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II is performed, in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

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Specific detection of HSA-warfarin interaction.(a) Mixture ofsmall molecules used in the test. Warfarin is highlighted in red.(b) LWV shift due to the binding of warfarin to HSA in the compoundmixture. The dotted line indicates the addition of the compound mixtureto both the active and reference wells. Values shown are the meanof at least three independent measurements; error bars represent standarderror of mean.
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fig3: Specific detection of HSA-warfarin interaction.(a) Mixture ofsmall molecules used in the test. Warfarin is highlighted in red.(b) LWV shift due to the binding of warfarin to HSA in the compoundmixture. The dotted line indicates the addition of the compound mixtureto both the active and reference wells. Values shown are the meanof at least three independent measurements; error bars represent standarderror of mean.

Mentions: To confirm thatthe ECL biosensor instrument is sufficiently robustfor screening protein–small molecule interactions, detectionof warfarin binding to immobilized human serum albumin (HSA) (KD = 1.2 μM)18 was tested. When the active and reference sensors were exposed toa cocktail of 5 nonbinding compounds (Figure 3a), no binding signal was observed in the absence of warfarin, whilethe addition of warfarin in the cocktail resulted in a LWV shift of14 pm (Figure 3b) due to its binding to immobilizedHSA. Importantly, it was determined that the observed shift of 14pm correlates well with the concentration of warfarin available tobind with HSA, based on further dose-response measurements (Figure S5). This set of data demonstrates thespecificity of the ECL biosensor for detection of protein–smallmolecule binding interactions.


Detection of protein-small molecule binding using a self-referencing external cavity laser biosensor.

Zhang M, Peh J, Hergenrother PJ, Cunningham BT - J. Am. Chem. Soc. (2014)

Specific detection of HSA-warfarin interaction.(a) Mixture ofsmall molecules used in the test. Warfarin is highlighted in red.(b) LWV shift due to the binding of warfarin to HSA in the compoundmixture. The dotted line indicates the addition of the compound mixtureto both the active and reference wells. Values shown are the meanof at least three independent measurements; error bars represent standarderror of mean.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4333586&req=5

fig3: Specific detection of HSA-warfarin interaction.(a) Mixture ofsmall molecules used in the test. Warfarin is highlighted in red.(b) LWV shift due to the binding of warfarin to HSA in the compoundmixture. The dotted line indicates the addition of the compound mixtureto both the active and reference wells. Values shown are the meanof at least three independent measurements; error bars represent standarderror of mean.
Mentions: To confirm thatthe ECL biosensor instrument is sufficiently robustfor screening protein–small molecule interactions, detectionof warfarin binding to immobilized human serum albumin (HSA) (KD = 1.2 μM)18 was tested. When the active and reference sensors were exposed toa cocktail of 5 nonbinding compounds (Figure 3a), no binding signal was observed in the absence of warfarin, whilethe addition of warfarin in the cocktail resulted in a LWV shift of14 pm (Figure 3b) due to its binding to immobilizedHSA. Importantly, it was determined that the observed shift of 14pm correlates well with the concentration of warfarin available tobind with HSA, based on further dose-response measurements (Figure S5). This set of data demonstrates thespecificity of the ECL biosensor for detection of protein–smallmolecule binding interactions.

Bottom Line: Here we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with subpicometer wavelength accuracy.Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets, pairs that have binding affinities or inhibition constants ranging from subnanomolar to low micromolar.Finally, a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II is performed, in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, ‡Department of Chemistry, §Department of Bioengineering, and ⊥Department of Electrical and Computer Engineering, University of Illinois at Urbana-Champaign , Urbana, Illinois 61801, United States.

ABSTRACT
High-throughput screening has enabled the identification of small molecule modulators of important drug targets via well-established colorimetric or fluorimetric activity assays. However, existing methods to identify small molecule binders of nonenzymatic protein targets lack either the simplicity (e.g., require labeling one of the binding partners with a reporter) or throughput inherent in enzymatic assays widely used for HTS. Thus, there is intense interest in the development of high-throughput technologies for label-free detection of protein-small molecule interactions. Here we describe a novel self-referencing external cavity laser (ECL) biosensor approach that achieves high resolution and high sensitivity, while eliminating thermal noise with subpicometer wavelength accuracy. Using the self-referencing ECL biosensor, we demonstrate detection of binding between small molecules and a variety of immobilized protein targets, pairs that have binding affinities or inhibition constants ranging from subnanomolar to low micromolar. Finally, a "needle-in-the-haystack" screen for inhibitors against carbonic anhydrase isozyme II is performed, in which known inhibitors are clearly differentiated from inactive molecules within a compound library.

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