Bifunctional molecules evade cytochrome P₄₅₀ metabolism by forming protective complexes with FK506-binding protein

Despite their large size and complexity, the macrolide natural products rapamycin and FK506 have excellent pharmacological characteristics. We hypothesize that these unexpected properties may arise from protective, high affinity interactions with the cellular FK506-binding protein, FKBP. In this model, the drug–FKBP complex might sequester the small molecule and limit its degradation by restricting access to metabolic enzymes. In support of this idea, we found that adding FKBP blocks binding of FK506 to the common cytochrome P₄₅₀ enzyme CYP3A4 in vitro. To further test this idea, we have systematically modified a small collection of otherwise unrelated compounds, such that they acquire affinity for FKBP. Strikingly, we found that many of these synthetic derivatives, but not the unmodified parent compounds, are also protected from CYP3A4-mediated metabolism. Depending on the properties of the linker, the bifunctional molecules exhibited up to a 3.5-fold weaker binding to CYP3A4, and this protective effect was observed in the presence of either purified FKBP or FKBP-expressing cells. Together, these results suggest that the surprising pharmacology of rapamycin and FK506 might arise, in part, from binding to their abundant, intracellular target, FKBP. Furthermore, these findings provide a framework by which other small molecules might be systematically modified to impart this protective effect.