New enzymatic assay for the AKR1C enzymes

• Published in: Chemico-biological interactions 2013-02, Vol.202 (1-3), p.204-209
• Main Authors: Beranič, Nataša, Štefane, Bogdan, Brus, Boris, Gobec, Stanislav, Rižner, Tea Lanišnik
• Format: Article
• Language: English
• Subjects: 20-Hydroxysteroid Dehydrogenases - antagonists & inhibitors; 20-Hydroxysteroid Dehydrogenases - metabolism; 3-Hydroxysteroid Dehydrogenases - antagonists & inhibitors; 3-Hydroxysteroid Dehydrogenases - metabolism; acetates; AKR1C isoforms; Alcohol Oxidoreductases - metabolism; Aldehyde Reductase; Aldo-Keto Reductase Family 1 Member C3; Aldo-Keto Reductases; Artificial substrates; Catalysis; Cyclopentane derivatives; Cyclopentanes - pharmacology; Enzymatic assay; Enzyme Assays - methods; Enzyme Inhibitors - pharmacology; enzymes; Humans; Hydroxyprostaglandin Dehydrogenases - antagonists & inhibitors; Hydroxyprostaglandin Dehydrogenases - metabolism; Kinetics; medroxyprogesterone; Medroxyprogesterone Acetate - pharmacology; NAD (coenzyme); NAD - metabolism; NADP (coenzyme); NADP - metabolism; neoplasms; new drugs; oxidation; Oxidation-Reduction; ursodeoxycholic acid; Ursodeoxycholic Acid - pharmacology
• ISSN: 0009-2797; 1872-7786
• DOI: 10.1016/j.cbi.2012.12.003

► The AKR1C1–AKR1C3 enzymes are emerging targets for the development of new drugs. ► The majority of current biochemical assays for the AKR1C enzymes have limitations. ► New AKR1C enzymatic assays with two cyclopentane substrates are introduced. ► These assays are appropriate for the future search for AKR1C inhibitors. The imbalance in expression of the human aldo–keto reductases AKR1C1–AKR1C3 is related to different hormone dependent and independent cancers and some other diseases. The AKR1C1–3 enzymes thus represent emerging targets for the development of new drugs. Currently, various enzymatic assays are used in the search for AKR1C inhibitors, and consequently the results of different research groups are not necessarily comparable. During our recent search for AKR1C inhibitors, we found a cyclopentanol derivative (2-(4-chlorobenzylidene)cyclopentanol, CBCP-ol) and its respective cyclopentanone counterpart (2-(4-chlorobenzylidene)cyclopentanone, CBCP-one) that acted as AKR1C substrates. We determined the kinetic parameters KM, kcat and kcat/KM for oxidation of CBCP-ol and reduction of CBCP-one by AKR1C enzymes in the presence of NAD+/NADP+ and NADH/NADPH, respectively. The catalytic efficiencies for the oxidation of CBCP-ol in the presence of NAD+ or NADP+ were in general higher when compared to the catalytic efficiencies for reduction of CBCP-one in the presence of NADH or NADPH. When NADPH was used, as compared to NADH, the reductions of CBCP-one by AKR1C1, AKR1C2 and AKR1C3 were 14-, 51- and 31-fold more efficient, respectively. When comparing to oxidations of the well-known artificial substrates, 1-acenaphthenol and S-tetralol, we observed similar catalytic efficiencies as for CBCP-ol oxidation with NAD+ and NADP+. The comparison of CBCP-one reduction with NADPH to reductions of physiological substrates revealed in general higher efficiencies, except for reduction of 9-cis-retinaldehyde by AKR1C3. This NADPH-dependent reduction of CBCP-one was then used to re-evaluate inhibitory potencies of the known inhibitors of the target AKR1C3 and the anti-target AKR1C2, medroxyprogesterone acetate and ursodeoxycholic acid, respectively, showing Ki constants similar to the reported values. Our data thus confirm that the new enzymatic assays with two cyclopentane substrates CBP-ol and CBP-one, and especially reduction of CBCP-one with NADPH, are appropriate for the evaluation of AKR1C inhibitors.