Fluorescent probes for rapid screening of potential drug-drug interactions at the CYP3A4 level

Steroid derivs. bearing fluorescent groups such as anthracene, dansyl, deazaflavin, and pyrene attached to C6 were synthesized.  For example, reacting testosterone deriv. I with deazaflavinyl alc. II gave ester III.  These compds. are unique inhibitors of cytochrome P 450 3A4 (CYP3A4) and display similar IC50 values in the microM range for the CYP3A4 substrates midazolam, testosterone, and nifedipine.  On binding to CYP3A4, the fluorescence of the dansyl, deazaflavin, and pyrene probes is quenched by photophys. interaction of the fluorophore with the heme.  The addn. of drug candidates with binding consts. in the nM-microM range causes displacement of the probes from the active site, and hence leads to restoration of fluorescence.  Accordingly, relative affinities of drug candidates to CYP3A4 can be easily and accurately detd. by fluorescence measurements.