An optimized CYP3A4-activatable fluorogenic sensor for <i>in situ</i> functional imaging and multi-dimensional inhibitor assessment

Cytochrome P450 3A4 (CYP3A4), one of the most important drug-metabolizing enzymes, plays a pivotal role in the oxidative metabolism of a wide range of non-polar xenobiotics and endogenous substances. Deciphering the dynamic changes in CYP3A4 activity under specific physiological or pathological conditions, as well as assessing the modulatory effects of therapeutic agents on CYP3A4, requires highly-efficient and reliable tools for sensing CYP3A4 activity within complex biological matrices. Herein, an integrated strategy was adopted for developing an optimized CYP3A4-activatable fluorogenic sensor that enables <i>in situ</i> detection of CYP3A4 activity in living systems without the interference of P-glycoprotein (P-gp), <i>via</i> integrating computer-aided substrate design, drug-likeness filtering, and biochemical assays. Following screening a range of 1,8-naphthalimide derivatives, <i>N</i>-cyclopropylmethyl-1,8-naphthalimide (NCN) was identified as an optimized fluorogenic substrate for CYP3A4, demonstrating exceptional isoform-specificity, single metabolite formation, ultrahigh sensitivity, high binding-affinity, improved cell-membrane permeability, and favorable bio-safety profiles. Notably, both NCN and its fluorogenic metabolite (HNCN) were identified as non-substrates of P-gp, which greatly facilitated <i>in situ</i> functional imaging of CYP3A4 activities in living systems, such as live cells and organs. It was also found that NCN was an orally bioavailable agent, which significantly facilitated the precise assessment of CYP3A4 inhibitors across multi-dimensional biological systems, including <i>in vitro</i>, <i>ex vivo</i>, and <i>in vivo</i>. Collectively, this work showcases an integrated strategy for the rational engineering of isoform-specific and orally bioavailable CYP3A4-activatable fluorogenic substrates for CYP3A4, with NCN emerging as a practical and reliable CYP3A4-activatable tool for <i>in situ</i> imaging and inhibitor assessment.