Fluorogenic enzymatic assay

Many factors may confound the assessment of the D DI potential of early discovery compounds [93], Limited or no solubility data exist to understand the likelihood that the compound will precipitate out of an in vitro incubation. The compounds have generally not been analyzed from a spectroscopic perspective their characteristics may interfere with a fluorogenic DDI assay. Metabolism data are typically not available. The binding of a compound to plasma proteins or microsomal incubation constituents is not well understood, which may lead to underprediction of its inhibitory potential. The compounds are typically delivered in DMSO, which may cause solvent-related inhibition of the enzymatic assay. Also, since little is known about in vivo concentrations or projected dose, framing the consequences of an early DDI in vitro experiment may be difficult. With these factors in mind, general experimental paradigms have been developed to help minimize their potential impact. 

Fluorogenic substrate molecules that produce fluorescent product molecules by enzymatic reactions, as proposed for horseradish peroxidase [28]. Because fluorophores are continuously replenished and diffuse away from the excitation volume, this assay circumvents the photobleaching problem that hampers all the other assays, and provides extremely long time traces [29,30]... 

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