An ER-Targeting Near-Infrared Fluorogenic Probe for In Situ hCES2A Functional Imaging and Inhibitor Discovery

Abstract Targeted inhibition of intestinal human carboxylesterase 2A (hCES2A) represents a promising strategy to mitigate irinotecan-induced intestinal toxicity (IIT). Herein, a rational molecular engineering strategy was adopted for a high-performance near-infrared (NIR) fluorogenic probe of hCES2A. First, HDCI was identified as an ideal fluorophore scaffold (λex/λem = 510 nm/720 nm), exhibiting a large Stokes shift (210 nm), high metabolic stability, and excellent endoplasmic reticulum (ER)-targeting capability. Further investigations established PA-HDCI as a robust ER-localized probe for monitoring hCES2A activity across diverse biological systems. The PA-HDCI-based high-throughput screening assay identified A4 as an extremely potent covalent hCES2A inhibitor (IC50 = 0.052 nM). In vivo studies showed that A4 exhibited favorable safety profiles and robust therapeutic efficacy to ameliorate IIT in tumor-bearing mice. Collectively, this work devises a novel NIR fluorogenic probe for in situ monitoring of hCES2A in living systems, and establishes a high-throughput platform for discovering hCES2A inhibitors to combat IIT.