Fluorescent Probes for Rapid Screening of Potential Drug–Drug Interactions at the CYP3A4 Level

Abstract

Steroid derivatives bearing fluorescent groups such as anthracene, dansyl, deazaflavin, and pyrene attached to C6 were synthesized. These compounds are unique inhibitors of cytochrome P450 3A4 (CYP3A4) and display similar IC(50) 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 photophysical interaction of the fluorophore with the heme. The addition of drug candidates with binding constants 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 determined by fluorescence measurements.

Cytochromes P450 in Nanodiscs

“…Detection of binding events can be accomplished either by monitoring an LSPR optical signal, as described above, or by direct observation of binding or release of fluorescent substrate or substrate analog [16, 24-25]. …”

Current Approaches for Investigating and Predicting Cytochrome P450 3A4-Ligand Interactions

“…Several fluorescent inhibitors specific for CYP3A4 have been synthesized by attaching a dansyl, deazaflavin or pyrene group to the C6 atom of testosterone [49]. Fluorescence of steroid derivatives is quenched upon reaction with the heme but can be restored when the activesite-bound fluorophore is displaced by another compound, which makes possible fluorimetric determination of relative affinities of various compounds and monitoring of drug-drug interactions.…”

Substrate binding to cytochromes P450

“…In these studies, fluorescence lifetime measurements (mainly that of tryptophan residues) are used together with FRET experiments to study conformational dynamics of P450s. Among the studied fluorescent P450 3A4 ligands are 2- p -toluidinylnaphthalene-6-sulfonic acid (TNS) (which fluoresces only in a highly hydrophobic environment [86, 88]), a synthetic deazaflavin-substituted testosterone analog [89, 90], and Nile Red [87, 91]. An alternate approach in studying substrate binding via fluorescence spectroscopy is the modification of the P450 of interest by a thiol-reactive fluorescent probe, which has been applied to P450 107A1 [92] and P450 3A4 [93] in order to probe substrate-induced conformational changes.…”