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.
Highlights
The efficacy of a drug relies upon interaction with a relevant therapeutic target protein at the physiological site of activity
HEK-huKMO cell lysate was analysed for kynurenine 3-monooxygenase (KMO) enzymatic activity by measuring the amount of KYN converted to 3HK detected using liquid chromatography-mass spectrometry (LC-MS/MS) following incubation with the compounds screened in the TAPS assay using a method we described previously [17]
These results show that the TAPS assay protocol can be utilised to identify cell permeable compounds with specific binding affinity for KMO
Summary
The efficacy of a drug relies upon interaction with a relevant therapeutic target protein at the physiological site of activity Direct detection of this interaction in vitro remains a challenge, if isolated protein cannot be obtained or if a suitable assay method cannot be designed. TAPS selects, in a single assay method, compounds with sufficient cellular permeability, and excludes, via necessary controls, compounds which lack specificity or demonstrate high cytotoxicity. This method is generally applicable for any intracellular target which can be expressed as a fluorescent fusion protein and its applicability to challenging membrane proteins is demonstrated here
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