Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis

Wilson, K., Webster, S. P., Iredale, J. P., Zheng, X., Homer, N. Z., Pham, N. T., Auer, M. and Mole, D. J. (2018) Detecting drug-target binding in cells using fluorescence-activated cell sorting coupled with mass spectrometry analysis. Methods and Applications in Fluorescence, 6(1), 015002. (doi: 10.1088/2050-6120/aa8c60) (PMID:28901950)

Abstract

The assessment of drug-target engagement for determining the efficacy of a compound inside cells remains challenging, particularly for difficult target proteins. Existing techniques are more suited to soluble protein targets. Difficult target proteins include those with challenging in vitro solubility, stability or purification properties that preclude target isolation. Here, we report a novel technique that measures intracellular compound-target complex formation, as well as cellular permeability, specificity and cytotoxicity-the toxicity-affinity-permeability-selectivity (TAPS) technique. The TAPS assay is exemplified here using human kynurenine 3-monooxygenase (KMO), a challenging intracellular membrane protein target of significant current interest. TAPS confirmed target binding of known KMO inhibitors inside cells. We conclude that the TAPS assay can be used to facilitate intracellular hit validation on most, if not all intracellular drug targets.

• Item Type: Articles
• Additional Information: This work was supported by and MRC studentship to KW. DJM held a Health Foundation/Academy of Medical Sciences Clinician Scientist Fellowship during this work and now gratefully acknowledges the support of the UK Medical Research Council through a Senior Clinical Fellowship.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Wilson, Dr Kris
• Authors: Wilson, K., Webster, S. P., Iredale, J. P., Zheng, X., Homer, N. Z., Pham, N. T., Auer, M., and Mole, D. J.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity
• Journal Name: Methods and Applications in Fluorescence
• Publisher: IOP Publishing
• ISSN: 2050-6120
• ISSN (Online): 2050-6120
• Published Online: 18 December 2017
• Copyright Holders: Copyright © 2017 IOP Publishing Ltd
• First Published: First published in Methods and Applications in Fluorescence 6(1): 015002
• Publisher Policy: Reproduced under a Creative Commons License