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Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase
Published on: December 19, 2011
New enzymatic assay for the AKR1C enzymes
Nataša Beranič1, Bogdan Stefane, Boris Brus
1Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Abstract:
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.
Insights
New assays using cyclopentane substrates (CBCP-ol and CBCP-one) effectively evaluate aldo-keto reductase (AKR1C) inhibitors. The reduction of CBCP-one with NADPH shows particular promise for drug discovery targeting AKR1C enzymes in cancer and disease.
Area of Science:
- Biochemistry
- Enzymology
- Drug Discovery
Background:
- Imbalances in human aldo-keto reductases (AKR1C1-AKR1C3) are linked to hormone-dependent and independent cancers and other diseases.
- AKR1C enzymes are emerging targets for novel therapeutic drug development.
- Current enzymatic assays for AKR1C inhibitors lack comparability across research groups.
Purpose of the Study:
- To develop and validate new enzymatic assays for evaluating AKR1C inhibitors.
- To characterize novel cyclopentane derivatives, CBCP-ol and CBCP-one, as substrates for AKR1C enzymes.
- To compare the catalytic efficiencies of these new substrates with existing artificial and physiological substrates.
Main Methods:
- Determined kinetic parameters (KM, kcat, kcat/KM) for AKR1C-mediated oxidation of CBCP-ol and reduction of CBCP-one using NAD+/NADP+ and NADH/NADPH.
- Compared catalytic efficiencies of CBCP-ol/CBCP-one with established substrates like 1-acenaphthenol, S-tetralol, and 9-cis-retinaldehyde.
- Re-evaluated known AKR1C3 and AKR1C2 inhibitors (medroxyprogesterone acetate, ursodeoxycholic acid) using the new NADPH-dependent CBCP-one reduction assay.
Main Results:
- CBCP-ol oxidation showed higher catalytic efficiencies with NAD+/NADP+ compared to CBCP-one reduction with NADH/NADPH.
- NADPH significantly enhanced CBCP-one reduction by AKR1C1, AKR1C2, and AKR1C3 (14-, 51-, and 31-fold, respectively) compared to NADH.
- The new assays, particularly CBCP-one reduction with NADPH, yielded Ki constants for known inhibitors consistent with reported values, validating the assay's utility.
Conclusions:
- The novel cyclopentane substrates, CBCP-ol and CBCP-one, are suitable for AKR1C enzyme studies.
- The NADPH-dependent reduction of CBCP-one provides a robust and comparable assay for evaluating AKR1C inhibitors.
- These findings support the development of more reliable drug discovery pipelines for AKR1C-related diseases.
