New Tools for Molecular Imaging of Redox Metabolism:  Development of a Fluorogenic Probe for 3α-Hydroxysteroid Dehydrogenases

Abstract

A new fluorogenic substrate was developed for 3alpha-hydroxysteroid dehydrogenases (3alpha-HSD), including the human enzymes implicated in important physiological functions (androgen deactivation, neurosteroid activation). While ketone 5 is nonfluorescent, the corresponding alcohol exhibits high fluorescence with emission maximum at 510 nm, thus constituting a redox optical switch. This study began with a chemical concept of a ketone-alcohol optical switch which guided the synthesis of a focused array of compounds. Subsequently, seven compounds were selected (1-7) on the basis of their optical and chemical (stability) properties and were submitted to a screen against a panel of dehydrogenase enzymes. Probe 5 was found to be highly selective for bacterial, rat, and human 3alpha-HSD enzymes. The kinetic parameters were obtained for human 3alpha-HSD enzyme (type 2 isozyme, AKR 1C3; Km = 2.5 muM, kcat = 8.2 min-1). Remarkably, comparison to 5alpha-dihydrotestosterone (5alpha-DHT, Km = 26 muM, kcat = 0.25 min-1, Figure 4), a likely physiological substrate in prostate, revealed that synthetic probe 5 is in fact a far better substrate for this enzyme. Structure 5 represents an exciting lead for the development of a redox imaging probe.

Fluorogenic click reaction

“…In contrast, addition of electron donating groups at the 4-, 6-, or 7-positions or electron withdrawing groups at the 3-position can shift the fluorescence band to longer wavelengths. [11][12][13] Based on this principle, Zhou and Fahrni prepared the non-fluorescent 1, where a 7-alkynyl group was introduced to quench the fluorescence of the coumarin core. After CuAAC reaction with the azide 2, the fluorescence could be restored by the formation of the triazole compound 3 (Scheme 1a).…”

Fluorogenic metabolic probes for direct activity readout of redox enzymes: Selective measurement of human AKR1C2 in living cells

“…Fluorogenic substrates 1 and 3 were prepared and stored as 5 mM stock solutions in acetonitrile or DMSO at 0°C. In vitro kinetics were determined as described (9). In short, 1-ml reaction mixtures containing 100 mM potassium phosphate buffer (pH 6), 0.250 mM NADPH (Roche), and 0.2K M to 5K M fluorogenic ketone 1 or 3 in acetonitrile (up to 4% vol͞vol) were initiated by 2 l of diluted AKR1C2.…”

“…We recently described fluorogenic substrate methylketone 3 (9). This probe was developed in two major stages: first, by designing optical switches, which are organic compounds that translate a redox chemical transformation to a profound change in the emission profile, and second, by screening these compounds against a panel of purified dehydrogenases.…”

“…In this instance, a synthetic molecule, bearing distant resemblance to a physiological substrate, would have to function as a competitive substrate. Thus, these approaches depend on significant substrate plasticity of the enzymes of interest (9)(10)(11). Furthermore, with activity comes the issue of selectivity, and as with all nonpeptidic ligand-protein interactions, there are no straightforward design guidelines.…”

“…We recently described a novel fluorogenic substrate for human hydroxysteroid dehydrogenases (HSDs) of the aldo-keto reductase (AKR) superfamily (9). It has been proposed that these enzymes function as prereceptor switches by activating or deactivating steroid hormones through redox chemistry (12).…”