Abstract 4146: Brain permeable bioluminescent substrates for NanoLuc based reporters

Abstract Non-invasive imaging methods for tracking gene expression, cell growth and migration and other biological events in vivo are indispensable tools for biomedical studies. Bioluminescence imaging (BLI) can achieve sensitive detection with low background in vivo, which is desirable for body imaging of small animals. Yet, BLI in the central nervous system (CNS) is challenging because many luciferase substrates show limited permeability of the blood-brain-barrier (BBB). To address this issue, we developed a family of new brain-permeant NanoLuc (NLuc) substrates, cephalofurimazine (CFz). CFz family paired with Antares, a fusion of CyOFP and NLuc that emits orange light instead of blue, produces an order of magnitude more signal from the brain than the standard combination of D-luciferin with firefly luciferase and matches the peak output of AkaLumine with AkaLuc but without the need of ATP. One of the substrates in the family, CFz-9, not only produces outstanding signal as other CFz substrates when paired with Antares, but also is able to be formulated into lyophilized cake that can be reconstituted in aqueous buffers for i.p. injections. Toxicity studies demonstrate that CFz-9 and its reconstituted solutions are non-toxic to mice and thus multiple injections are allowed. Citation Format: Chao Gao, Yichi Su, Yan Wu, Connor Fitzgerald, Tetsuo H. Uyeda, Hui Wang, Thomas A. Kirkland, Michael Z. Lin, Wenhui Zhou. Brain permeable bioluminescent substrates for NanoLuc based reporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4146.