posted on 2023-01-05, 19:34authored byAnuj K Yadav, Zhenxiang Zhao, Yourong Weng, Sarah H Gardner, Catharine J Brady, Oliver D Pichardo Peguero, Jefferson Chan
Activity-based sensing (ABS) probes equipped with a NIR
bioluminescence
readout are promising chemical tools to study cancer biomarkers owing
to their high sensitivity and deep tissue compatibility. Despite the
demand, there is a dearth of such probes because NIR substrates (e.g.,
BL660 (a NIR luciferin analog)) are not equipped with an appropriate
attachment site for ABS trigger installation. For instance, our attempts
to mask the carboxylic acid moiety with standard self-immolative benzyl
linkers resulted in significant background signals owing to undesirable
ester hydrolysis. In this study, we overcame this longstanding challenge
by rationally designing a new hydrolysis-resistant ester-based linker
featuring an isopropyl shielding arm. Compared to the parent, the
new design is 140.5-fold and 67.8-fold more resistant toward spontaneous
and esterase-mediated hydrolysis, respectively. Likewise, we observed
minimal cleavage of the ester moiety when incubated with a panel of
enzymes possessing ester-hydrolyzing activity. These impressive in
vitro results were corroborated through a series of key experiments
in live cells. Further, we showcased the utility of this technology
by developing the first NIR bioluminescent probe for nitroreductase
(NTR) activity and applied it to visualize elevated NTR expression
in oxygen deficient lung cancer cells and in a murine model of non-small
cell lung cancer. The ability to monitor the activity of this key
biomarker in a deep tissue context is critical because it is associated
with tumor hypoxia, which in turn is linked to drug resistance and
aggressive cancer phenotypes.