Human Aldo-keto reductases (AKR) of the 1A, 1B, 1C and 1D subfamilies are involved in the pre-receptor regulation of nuclear (steroid hormone and orphan) receptors by regulating the local concentrations of their lipophilic ligands. AKR1C3 is one of the most interesting isoforms. It was cloned from human prostate and the recombinant protein was found to function as a 3-, 17- and 20 ketosteroid reductase with a preference for the conversion of Δ4-androstene-3,17- dione to testosterone implicating this enzyme in the local production of active androgens within the prostate. Using a validated isoform-specific real-time RT-PCR procedure the AKR1C3 transcript was shown to be more abundant in primary cultures of epithelial cells than stromal cells, and its expression in stromal cells increased with benign and malignant disease. Using a validated isoform-specific monoclonal Ab, AKR1C3 protein expression was also detected in prostate epithelial cells by immunoblot analysis. Immunohistochemical staining of prostate tissue showed that AKR1C3 was expressed in adenocarcinoma and surprisingly high expression was observed in the endothelial cells. These cells are a rich source of prostaglandin G/H synthase 2 (COX-2) and vasoactive prostaglandins (PG) and thus the ability of recombinant AKR1C enzymes to act as PGF synthases was compared. AKR1C3 had the highest catalytic efficiency (kcat/Km) for the 11-ketoreduction of PGD2 to yield 9α,11β-PGF2 raising the prospect that AKR1C3 may govern ligand access to peroxisome proliferator activated receptor (PPARγ). Activation of PPARγ is often a pro-apoptotic signal and/or leads to terminal differentiation, while 9α,11β- PGF2 is a pro-proliferative signal. AKR1C3 is potently inhibited by non-steroidal anti- inflammatory drugs suggesting that the cancer chemopreventive properties of these agents may be mediated either by inhibition of AKR1C3 or COX. To discriminate between these effects we developed potent AKR1C inhibitors based on N-phenylanthranilic acids that do not inhibit COX-1 or COX-2. These compounds can now be used to determine the role of AKR1C3 in producing two proliferative signals in the prostate namely testosterone and 9α,11β-PGF2. Structure-activity relationships Thirty-five crystal structures of AKR1C3·NADP + ·inhibitor complexes exist in the PDB. Inspection of these structures shows that if the inhibitor contains a carboxylic acid, it can often form hydrogen bonds with the catalytic tetrad members Tyr55 and His117. Other portions of the inhibitor can occupy one of several subpockets (SP), e.g. SP1 Ser118, Asn167, Phe306, Phe311, and Tyr319 (e.g. occupied by the B-ring of N- cphenylaminobenzoates). The SP2 sub-pocket refers to Ser129, W227, and F311 (e.g. occupied by the side-chain of PGs), and the SP3 sub-pocket which contains Y24, E192, S217, S221, Q222, Y305, and F306. While the presence of these sub-pockets can be rationalized to determine the binding mode and can be used as the basis of docking studies,