Luciferase-Specific Coelenterazine Analogues for Optical Contamination-Free Bioassays

Abstract

Spectral overlaps among the multiple optical readouts commonly cause optical contamination in fluorescence and bioluminescence. To tackle this issue, we created five-different lineages of coelenterazine (CTZ) analogues designed to selectively illuminate a specific luciferase with unique luciferase selectivity. In the attempt, we found that CTZ analogues with ethynyl or styryl groups display dramatically biased bioluminescence to specific luciferases and pHs by modifying the functional groups at the C-2 and C-6 positions of the imidazopyradinone backbone of CTZ. The optical contamination-free feature was exemplified with the luciferase-specific CTZ analogues, which illuminated anti-estrogenic and rapamycin activities in a mixture of optical probes. This unique bioluminescence platform has great potential for specific and high throughput imaging of multiple optical readouts in bioassays without optical contamination.

Highly bright and stable NIR-BRET with blue-shifted coelenterazine derivatives for deep-tissue imaging of molecular events in vivo

“…Furthermore, our earlier studies also suggest that the use of a flexible alkyl linker moiety in the CTZ may relieve its steric hindrance, and that could enhance the enzymatic efficiency of RLuc 24, 25. Based on this knowledge, we synthesized 18 novel CTZ derivatives, where the ( p -hydroxy)-phenyl group at the C-6 position was intentionally modified by alkylation (Figure 1B and Supplementary Figure 1).…”

“…We previously demonstrated that modification of nCTZ at the C-6 position allows a large spectral shift towards blue- of BL signal without any influence on the enzymatic recognition by RLuc variants 15, 24. The rigidity of the ethynyl group at the C-6 position of nCTZ decreases the BL intensity by increasing the steric bulkiness.…”

“…The rigidity of the ethynyl group at the C-6 position of nCTZ decreases the BL intensity by increasing the steric bulkiness. The ethynyl group causes steric hindrance by interacting with the key amino acids such as, D120, E144 and H285, located within the active site (Figure 1A) 24-26.…”

Advances in bioluminescence imaging: new probes from old recipes

“…[33**] Simultaneous engineering of enzymes and substrates has also been applied to luciferases that use coelenterazine. [34]…”

Biotechnological Advances in Luciferase Enzymes

“…Other analogs, such as coelenterazine-f, -h, and -e have been shown to increase signal intensity by 4-to 8-fold relative to coelenterazine in RLuc-expressing mammalian cells in vitro, but each has failed to compete with the native coelenterazine in living animal imaging [40]. Despite these setbacks, Nishihara et al have reported that analogs with ethynyl or styryl group substitutions at the C-6 position significantly increased bioluminescent output and signal stability in RLuc8 and RLuc8.6-535 [41,42], which suggest that the development of new synthetic coelenterazine analogs will continue to be a promising route for enhancing RLuc functionality.…”