Coelenterazine Analogs for Bioassays and Molecular Imaging

Abstract

Coelenterazine (CTZ) is a common substrate of marine luciferases upon emission of bioluminescence (BL) in living organisms. Because CTZ works as a "luminophore" in the process of BL emission, the chemical modification has been centered for improving the optical properties of BL. In this review, we showcase recent advances in CTZ designs with unique functionalities. We first elucidate the light-emitting mechanisms of CTZ, and then focus on how the rational modification of CTZ analogs developed in recent years are connected to the development of unique functionalities even without luciferases, which include color tunability covering the visible region, specificity to various proteins (e.g., luciferase, albumin, and virus protein), and activatability to ions or reactive oxygen species (ROS) and anticancer drugs. This review provides new insights into the broad utilities of CTZ analogs with designed functionalities in bioassays and molecular imaging.

Keywords: bioassay; bioluminescence; coelenterazine; imaging; luciferase.

Figure 1

(A) The imidazopyrazinone structure of CTZ and its oxidative reaction to generate luminescence, giving coelenteramide (CTMD). (B) The intermediate forms of CTZ in the process of the oxidative reaction with molecular oxygen. (C) The detailed mechanism of chemiluminescence emission of CTZ through its oxidative reaction.

Figure 2

A proposed catalytic mechanism of CTZ-powered Renilla-type bioluminescence. This figure was modified from a reference [19]. Inset a shows the binding mode between Renilla-type luciferase (AncFT) and CTMD (CEI) based on the PDB structure, 7QXQ. This tertiary structure shows involvement of D160 besides the catalytic pentad, N51, D118, W119, E142, and H283.

Figure 3

Overview of the CTZ analogs with typical modification at the imidazopyrazinone backbone. The C-2, C-5, C-6, and C-8 positions of the backbone were modified for better optical properties. Group A represents the historically old and structurally simple chemical CTZ analogs; Group B shows CTZ analogs with a specific elbow at the C-8 position; Group C categorizes CTZ analogs with a π-electron extension at the C-6 position; Group D represents CTZ analogs with a bridge connecting the C-5 and C-6 positions; and Groups E and F are designed for FMZ analogs.

Figure 4

(A) BL spectra of CTZ analogs, C3 and C6, according to marine luciferases. The right panel showcases the spectral signatures of representative C-series CTZ analogs that represent each marine luciferase. (B) Binding of selected CTZ analogs with marine luciferases, explaining the spectral signatures.

Figure 5

(A) BL spectra of C- and S-series CTZ analogs according to marine luciferases. (B) Diverse BL spectra of bottle-series CTZ analogs with RLuc8.6-535. (C) Dye-conjugated CTZ analogs. The red colored frames represent the fluorescent dyes. (D) The relative TBET spectra of Cy5-CTZ with ALuc30, where the peak heights were normalized to the peak height contributed by the CTZ part (474 nm) in the green region. The legend indicates the relative ratios of the luciferase (left) to the substrate (right). The numbers on the peaks denote the maximal wavelengths (λ_max) of the spectra.

Figure 6

(A) Relative luciferase specificity of representative CTZ analogs, 6et-OH-CTZ, 6pi-OH-2H-CTZ, and 6pi-OH-CTZ. The bottom panel shows the corresponding chemical structures of the substrates. The imidazopyrazinone backbone was highlighted in green and the characteristic functional groups were marked in red. (B) Relative luciferase specificity of K-series CTZ analogs according to marine luciferases. The overall BL intensities were depicted in relative values compared to that of the substrate K6 with RLuc86SG. (C) Comparison of the luciferase specificity of M2 with those of nCTZ and CTZh. (D) Relative luminescence intensities of S-series CTZ analogs in response to various serum albumins. Abbreviations: HSA, human serum albumin; BSA, bovine serum albumin; OVA, ovalbumin.

Figure 7

(A) BL-emitting mechanism of a caged CTZ analog A5 for determining nitroreductase activity. (B) BL-emitting mechanism of a caged CTZ analog, Piv-FMZ-OH and Ad-FMZ-OH, derived from FMZ-OH (Hydrofurimazine; HFz) for determining esterase activity. (C) Design of an analog named Potassiorin that is derived from the diphenylterazine (DTZ) structure. The C-2 position of DTZ is modified with a K⁺-binding crown ether ring to derive Potassiorin. (D) Modification of the C-3 position of the DTZ backbone for improving the BBB permeability and minimizing BBB efflux.

Figure 8

Chemical structures of CTZ analogs working as ROS scavengers and anticancer drugs. Native CTZ (left end in the upper panel) is modified to generate ROS scavengers (MCLA) and cancer drugs (Br-Cla-1 and FBr-Cla). The corresponding reactions produce oxidated compounds in the bottom panel.