New tools for molecular imaging of redox metabolism: development of a fluorogenic probe for 3 alpha-hydroxysteroid dehydrogenases.
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.
Department of Chemistry, Columbia University, 3000 Broadway, New York, New York 10027, USA.
Dominic J
DJ
Yee
Vojtech
V
Balsanek
Dalibor
D
Sames
D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific) / Q000737:chemistry / Q000378:metabolism*; D000438:Alcohols / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D006736:Horses; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007659:Ketones / Q000138:chemical synthesis* / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction
D000438:Alcohols; D005456:Fluorescent Dyes; D007527:Isoenzymes; D007659:Ketones; D009249:NADP; D045002:3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)