Probing Biochemical Mechanisms of Action of Muscarinic M3 Receptor Antagonists with Label-Free Whole Cell Assays

    Authors

    H. Y. Deng; C. M. Wang; M. Su;Y. Fang

    Comments

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    Abbreviated Journal Title

    Anal. Chem.

    Keywords

    TARGET RESIDENCE TIME; OPTICAL BIOSENSOR; DRUG DISCOVERY; GROWTH-FACTOR; IN-VIVO; BINDING; ACETYLCHOLINE; SUBTYPE; OPINION; HT-29; Chemistry, Analytical

    Abstract

    Binding kinetics of drugs is increasingly recognized to be important for their in vivo efficacy and safety profiles. However, little is known about the effect of drug binding kinetics on receptor signaling in native cells. Here we used label-free whole cell dynamic mass redistribution (DMR) assays under persistent and duration-controlled stimulation conditions to investigate the influence of the binding kinetics of four antagonists on the signaling of endogenous muscarinic M3 receptor in native HT-29 cells. Results showed that DMR assays under different conditions differentiated the biochemical mechanisms of action of distinct M3 antagonists. When co-stimulated with acetylcholine, tiotropium, a relatively slow binding antagonist, was found to selectively block the late signaling of the receptor, suggesting that acetylcholine attains its binding equilibrium faster than tiotropium does, thereby still being able to initiate its rapid response until the antagonist draws up and fully blocks the signaling. Furthermore, DMR assays under microfluidics allowed estimation of the residence times of these antagonists acting at the receptor in native cells, which were found to be the determining factor for the blockage efficiency of M3 receptor signaling under duration-controlled conditions. This study demonstrates that DMR assays can be used to elucidate the functional consequence of kinetics-driven antagonist occupancy in native cells.

    Journal Title

    Analytical Chemistry

    Volume

    84

    Issue/Number

    19

    Publication Date

    1-1-2012

    Document Type

    Article

    Language

    English

    First Page

    8232

    Last Page

    8239

    WOS Identifier

    WOS:000309493200023

    ISSN

    0003-2700

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