Functional Imaging of CYP3A4 at Multiple Dimensions Using an AI-Driven High Performance Fluorogenic Substrate.

Area of Science:

• Biochemistry
• Pharmacology
• Chemical Biology

Background:

• Cytochrome P450 3A4 (CYP3A4) is crucial for drug metabolism and drug-drug interactions (DDIs).
• Developing orally active, specific fluorogenic substrates for in vivo enzyme imaging remains a significant challenge.

Purpose of the Study:

• To create a highly specific, orally active fluorogenic substrate for sensing and imaging CYP3A4 activity in biological systems.
• To utilize an artificial intelligence (AI)-driven strategy for designing novel enzyme substrates.

Main Methods:

• An AI-driven approach fused drug-like fragments with a CYP3A4-preferred fluorophore.
• Three candidate substrates were synthesized and evaluated for specificity, sensitivity, safety, and bioavailability.
• The lead candidate, NFa, was used for in situ imaging, inhibitor screening, and DDI assessment.

Main Results:

• NFa demonstrated excellent CYP3A4 isoform-specificity, high sensitivity, and good spatial resolution.
• NFa exhibited favorable safety profiles and acceptable oral bioavailability for in vivo applications.
• NFa successfully enabled functional in situ imaging, endoplasmic reticulum (ER) colocalization, and high-resolution imaging of CYP3A4.
• NFa facilitated high-throughput screening of CYP3A4 inhibitors, leading to the discovery of a novel inhibitor (D13).

Conclusions:

• An AI-powered strategy successfully developed NFa, the first orally available fluorogenic substrate for sensing and imaging CYP3A4.
• NFa serves as a valuable tool for fundamental research on CYP3A4 and accelerates the drug discovery process, including DDI assessment.