Rational Engineering of Isoform-Specific hSULT1E1 Fluorogenic Substrates for Functional Analysis and Inhibitor Screening

Human estrogen sulfotransferase (hSULT1E1), an important conjugative enzyme, plays crucial roles in both estrogen homeostasis and xenobiotic metabolism. Herein, a rational substrate engineering strategy was adopted to construct highly specific fluorogenic substrates for hSULT1E1. In the 1 ^st round of structure-based virtual screening, 4-hydroxyl-1,8-naphthalimide (4-HN) was identified as a suitable scaffold for constructing hSULT1E1 substrates. Subsequently, structural modifications on the north part of 4-HN generated a panel of derivatives as substrate candidates, in which HN-299 was identified as a highly selective fluorogenic substrate for hSULT1E1. In the 3 ^rd round of structural optimization, a “molecular growth” strategy on the south part of HN-299 was used to develop a highly selective and reactive substrate (HN-375). Under physiological conditions, HN-375 could be readily sulfated by hSULT1E1 to generate a single fluorescent product, which emitted bright green signals at around 510 nm and was fully identified as HN-375 4-O-sulfate (HNS). Further investigations indicated that HN-375 exhibited excellent isoform-specificity, rapid-response, ultrahigh sensitivity, and high signal-to-noise ratio, and as such was subsequently used for sensing SULT1E1 activity in hepatocellular carcinoma specimens and live organs. With HN-375 in hand, a practical fluorescence-based assay was established for high-throughput screening and characterization of hSULT1E1 inhibitors, as such two potent hSULT1E1 inhibitors were identified from in-house compound libraries. Collectively, this study showcases a groundbreaking strategy for engineering highly specific and sensitive fluorogenic substrates for target conjugative enzyme(s), while HN-375 emerges as a practical tool for sensing SULT1E1 activity in a biological context and for the high-throughput screening of inhibitors.