The current arsenal of tools and methods for the continuous monitoring and imaging of redox metabolic pathways in the context of intact cells is limited.
Fluorogenic substrates allow for direct measurement of
enzyme activity in situ; however, in contrast to
proteases and exo-
glycosidases, there are no simple guidelines for the design of selective probes for redox metabolic
enzymes. Here, we introduce redox probe 1 and demonstrate its high selectivity in living cells for human
hydroxysteroid dehydrogenases (HSDs) of the
aldo-keto reductase (AKR) superfamily. AKR1C
isoforms perform multiple functions among which the metabolism of potent
steroid hormones is well documented. Moreover, expression of these
enzymes is responsive to cellular stress and pathogenesis, including
cancer. Our probe design is based on redox-sensitive optical switches, which couple a
ketone-alcohol redox event to a profound change in fluorescence. The high selectivity of phenyl
ketone 1 for AKR1C2 over the many endogenous
reductases present in mammalian cells was established by a quantitative comparison of the metabolic rates between null control cells (COS-1) and AKR1C2-transfected cells. Phenyl
ketone 1 is a cell-permeable fluorogenic probe that permits a direct, real-time, and operationally simple readout of AKR1C2
enzyme activity in intact mammalian cells. Furthermore, it was demonstrated that probe 1 enables the quantitative examination of physiological substrate 5alpha-dihydrotestosterone ("dark substrate") in situ by means of a two-substrate competitive assay. Similarly, inhibitor potency of physiological (ursodeoxycholate) and synthetic inhibitors (
flufenamic acid,
ibuprofen, and
naproxen) was also readily evaluated.