Development of Nonsteroidal Anti-Inflammatory Drug Analogs and Steroid Carboxylates Selective for Human Aldo-Keto Reductase Isoforms: Potential Antineoplastic Agents That Work Independently of Cyclooxygenase Isozymes

Abstract

Human aldo-keto reductases (AKRs) regulate nuclear receptors by controlling ligand availability. Enzymes implicated in regulating ligand occupancy and trans-activation of the nuclear receptors belong to the AKR1C family (AKR1C1-AKR1C3). Nuclear receptors regulated by AKR1C members include the steroid hormone receptors (androgen, estrogen, and progesterone receptors) and the orphan peroxisome proliferator-activated receptor (PPAR␥). In human myeloid leukemia (HL-60) cells, ligand access to PPAR␥ is regulated by AKR1C3, which diverts PGD 2 metabolism away from J-series prostanoids (Desmond et al., 2003). Inhibition of AKR1C3 by indomethacin, a nonsteroidal anti-inflammatory drug (NSAID), caused PPAR␥-mediated terminal differentiation of the HL-60 cells. To discriminate between antineoplastic effects of NSAIDs that are mediated by either AKR1C or cyclooxygenase (COX) isozymes, selective inhibitors are required. We report a structural series of N-phenylanthranilic acid derivatives and steroid carboxylates that selectively inhibit recombinant AKR1C isoforms but do not inhibit recombinant COX-1 or COX-2. The inhibition constants, IC 50 , K I values, and inhibition patterns were determined for the NSAID analogs and steroid carboxylates against AKR1C and COX isozymes. Lead compounds, 4-chloro-N-phenylanthranilic acid and 4-benzoyl-benzoic acid for the N-phenylanthranilic acid analogs and most steroid carboxylates, exhibited IC 50 values that had greater than 500-fold selectivity for AKR1C isozymes compared with COX-1 and COX-2. Crystallographic and molecular modeling studies showed that the carboxylic acid of the inhibitor ligand was tethered by the catalytic Tyr55-OH 2 ϩ and explained why A-ring substituted N-phenylanthranilates inhibited only AKR1C enzymes. These compounds can be used to dissect the role of the AKR1C isozymes in neoplastic diseases and may have cancer chemopreventive roles independent of COX inhibition.

An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3α-HSD, type 5 17β-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone independent malignancies

“…We previously described N-phenylanthranilic acid analogues that inhibit the AKR1C subfamily of enzymes, but do not inhibit COX activities [19]. However, the close sequence identity (>86%) between the human AKR1C isoforms presented a challenge for the development of isoform selective inhibitors.…”

“…A selective inhibitor of AKR1C3 would be an important tool for understanding its role in cancer development and other disorders. We have described analogues of the N-phenylanthranilic acid family of NSAIDs that inhibit the AKR1C enzymes but do not affect the cyclooxygenase (COX) isozymes [19]. However, none of these compounds were selective for AKR1C3 over the other AKR1C isoforms.…”

“…Homogenous recombinant AKR1C1, AKR1C2, and AKR1C3 were purified according to published methods [5;23] and stored at −80 °C in 20 mM potassium phosphate buffer, pH 7.0, containing 30% glycerol, 1 mM EDTA, and 1 mM 2-mercaptoethanol. Enzyme activity was standardized using 1-acenaphthenol as substrate as described previously [19], specific activities for the enzymes were 1.8, 2.4, and 1.4 μmol of 1-acenaphthenol (1 mM) oxidized/ min/mg for AKR1C1, AKR1C2, and AKR1C3, respectively.…”

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

“…Regulation of the prereceptor levels of steroid hormones and prostaglandins by AKR1C enzymes may contribute to the development of human cancer (Byrns et al, 2008;Bauman et al, 2005). AKR1C3 is particularly relevant for the following reasons: 1) its activity in testosterone production in prostate enhances 5α-DHT formation and increases androgen receptor activity.…”

“…To date, only one knockout model of the enzyme of this family, namely 20α-HSD, has been reported, and this led to a decrease in the survival of pups and an increase in the duration of the estrous cycles and pregnancy (Ishida et al, 2007;Piekorz et al, 2005). Development of specific inhibitors and fluorogenic substrates that can be used to interrogate the role of individual AKR1C isoforms, as well as serve as potential drug candidates is under way (Byrns et al, 2008;Bauman et al, 2005;Yee et al, 2006). Approaches based both on similarities in activity (such as e.g.…”