NanoLuc-Driven Multiplex Imaging Platforms Incorporating Novel G-series Coelenterazine Variants

Bioluminescence (BL) is widely used as an optical readout in bioassays and molecular imaging applications. In this study, we present a systematic approach to develop an advanced multiplex imaging system using novel coelenterazine (CTZ) analogs, termed the "G-series." We synthesized a library of 20 CTZ analogs with diverse functional groups at the C-2 and C-8 positions of the imidazopyrazinone backbone and classified them into five categories. These analogs were characterized with respect to luciferase specificity, BL stability, emission spectra, kinetics, and computationally predicted binding modes. Most of the G-series CTZ analogs were found to be water-soluble and exhibited excellent specificity for NanoLuc from Oplophorus luciferase, while G1 alone showed high specificity for ALuc16 derived from a group of copepod luciferases, together with unique emission colors including blue, green, and orange. The applicability of the G-series CTZ analogs for multiplex imaging was further evaluated using single-chain BL probes and by in vivo imaging of xenografts in living mice. The results showed that substrate G6 facilitates specific BL signals (red emission) from the single-chain probe in response to estrogen antagonists. The mice xenografted with subcutaneous (s.c.) tumors expressing ALuc16 and NanoLuc were imaged with excellent signal-to-background ratios using G1 and G20, respectively. This study demonstrates that the G-series CTZ analogs constitute a versatile and tunable molecular imaging platform with superior optical properties for both in vitro and in vivo applications.