Selective inhibitors of aldo-keto reductases AKR1C1 and AKR1C3 discovered by virtual screening of a fragment library.

Human aldo-keto reductases 1C1-1C4 (AKR1C1-AKR1C4) function in vivo as 3-keto-, 17-keto-, and 20-ketosteroid reductases and regulate the activity of androgens, estrogens, and progesterone and the occupancy and transactivation of their corresponding receptors. Aberrant expression and action of AKR1C enzymes can lead to different pathophysiological conditions. AKR1C enzymes thus represent important targets for development of new drugs. We performed a virtual high-throughput screen of a fragment library that was followed by biochemical evaluation on AKR1C1-AKR1C4 enzymes. Twenty-four structurally diverse compounds were discovered with low µM K(i) values for AKR1C1, AKR1C3, or both. Two structural series included the salicylates and the N-phenylanthranilic acids, and additionally a series of inhibitors with completely novel scaffolds was discovered. Two of the best selective AKR1C3 inhibitors had K(i) values of 0.1 and 2.7 µM, exceeding expected activity for fragments. The compounds identified represent an excellent starting point for further hit-to-lead development.

Expression of human aldo-keto reductase 1C2 in cell lines of peritoneal endometriosis: potential implications in metabolism of progesterone and dydrogesterone and inhibition by progestins.

The human aldo-keto reductase AKR1C2 converts 5α-dihydrotestosterone to the less active 3α-androstanediol and has a minor 20-ketosteroid reductase activity that metabolises progesterone to 20α-hydroxyprogesterone. AKR1C2 is expressed in different peripheral tissues, but its role in uterine diseases like endometriosis has not been studied in detail. Some progestins used for treatment of endometriosis inhibit AKR1C1 and AKR1C3, with unknown effects on AKR1C2. In this study we investigated expression of AKR1C2 in the model cell lines of peritoneal endometriosis, and examined the ability of recombinant AKR1C2 to metabolise progesterone and progestin dydrogesterone, as well as its potential inhibition by progestins. AKR1C2 is expressed in epithelial and stromal endometriotic cell lines at the mRNA level. The recombinant enzyme catalyses reduction of progesterone to 20α-hydroxyprogesterone with a 10-fold lower catalytic efficiency than the major 20-ketosteroid reductase, AKR1C1. AKR1C2 also metabolises progestin dydrogesterone to its 20α-dihydrodydrogesterone, with 8.6-fold higher catalytic efficiency than 5α-dihydrotestosterone. Among the progestins that are currently used for treatment of endometriosis, dydrogesterone, medroxyprogesterone acetate and 20α-dihydrodydrogesterone act as AKR1C2 inhibitors with low µM K(i) values in vitro. Their potential in vivo effects should be further studied.

Selectivity and potency of the retroprogesterone dydrogesterone in vitro.

Dydrogesterone is widely used for menstrual disorders, endometriosis, threatened and habitual abortion and postmenopausal hormone replacement therapy. Although progestins have a promiscuous nature, dydrogesterone does not have clinically relevant androgenic, estrogenic, glucocorticoid or mineralocorticoid activities. To date, systematic biochemical characterization of this progestin and its active main metabolite, 20α-dihydrodydrogesterone, has not been performed in comparison to progesterone. The objective of this study was to evaluate the selectivity and potential androgenic/antiandrogenic effects of dydrogesterone and its metabolite in comparison to progesterone and medroxyprogesterone acetate by analyzing their interference with AR signaling in vitro. We characterized dydrogesterone and its metabolite for their binding and transactivation of androgen and other steroid hormone receptors and for their potential inhibitory effects against androgen biosynthetic enzymes, 17ß-hydroxysteroid dehydrogenase types 3 and 5 and 5α-reductase types 1 and 2. We found that dydrogesterone resembled progesterone mainly in its progestogenic effects and less in its androgenic, anti-androgenic, glucocorticoid and antiglucocorticoid effects; whereas, 20α-dihydrodydrogesterone showed reduced progestogenic potency with no androgenic, glucocorticoid and mineralocorticoid effects. Effects on the androgen and glucocorticoid receptor differed depending on the technology used to investigate transactivation. Progesterone, but not dydrogesterone and 20α-dihydrodydrogesterone, exerted anti-androgenic effects at the pre-receptor level by inhibiting 5α-reductase type 2. Dydrogesterone, 20α-dihydrodydrogesterone and progesterone inhibited the biosynthesis of testosterone catalyzed by 17ß-hydroxysteroid dehydrogenase types 3 and 5; however, due to their micromolar K(i) values, these activities appeared to be not of relevance at therapeutic levels. Overall, our data show that the anti-androgenic potential of dydrogesterone and 20α-dihydrodydrogesterone is less pronounced compared to progesterone.

Synthesis and biological evaluation of (6- and 7-phenyl) coumarin derivatives as selective nonsteroidal inhibitors of 17ß-hydroxysteroid dehydrogenase type 1.

17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is an enzyme that catalyzes NADPH-dependent reduction of the weak estrogen, estrone, into the most potent estrogen, estradiol, which exerts proliferative effects via the estrogen receptors. Overexpression of 17ß-HSD1 in estrogen-responsive tissues is related to the development of hormone-dependent diseases, such as breast cancer and endometriosis; thus, 17ß-HSD1 represents an attractive target for the development of new therapies. We have discovered that simple coumarines 1 and 2 significantly inhibit 17ß-HSD1 in a recombinant enzyme assay, with high selectivity against 17ß-HSD2. We postulated that the introduction of various p-substituted phenyl moieties to position 6 or 7 of the coumarin core using the Suzuki-Miyaura cross-coupling reaction would provide mimetics of steroidal structures with improved inhibition of 17ß-HSD1. The best inhibitor in the series proved to be 6a, with an IC(50) of 270 nM, and with exceptional selectivity for 17ß-HSD1 over 17ß-HSD2 and against the α and ß estrogen receptors.

New cyclopentane derivatives as inhibitors of steroid metabolizing enzymes AKR1C1 and AKR1C3.

A series of cyclopentane derivatives was synthesized and evaluated for inhibition of the steroid metabolizing enzymes AKR1C1 and AKR1C3. Selective inhibitors that are active in the low micromolar range were identified. These compounds represent promising starting points in the development of new anticancer agents for the treatment of hormone-dependent forms of cancer and other diseases where AKR1C1 and AKR1C3 are involved.

Discovery of new inhibitors of aldo-keto reductase 1C1 by structure-based virtual screening.

Aldo-keto reductase 1C1 is a hydroxysteroid dehydrogenase that inactivates progesterone by converting it to 20alpha-hydroxyprogesterone. It also inactivates 3alpha,5alpha-tetrahydroprogesterone, an allosteric modulator of the gamma-aminobutyric acid receptor that has anaesthetic, analgesic, anxiolytic and anti-convulsant effects. Inhibitors of aldo-keto reductase 1C1 are thus very interesting as potential agents for the treatment of endometrial cancer, premenstrual syndrome, catamenial epilepsy, and depressive disorders, and for the maintenance of pregnancy. We have used the molecular docking program eHiTS for virtual screening of 1990 compounds from the National Cancer Institute "Diversity Set". Fifty compounds with the highest predicted binding energies were then evaluated in vitro. Three structurally diverse hits were obtained that inhibit aldo-keto reductase 1C1 in the low micromolar range of IC(50) values. These hits represent promising starting points for structural optimization in hit-to-lead development.

Flavonoids and cinnamic acid derivatives as inhibitors of 17beta-hydroxysteroid dehydrogenase type 1.

17beta-Hydroxysteroid dehydrogenase (17beta-HSD) type 1 converts estrone to estradiol, a potent ligand for estrogen receptors. It represents an important target for the development of drugs for treatment of estrogen-dependent diseases. In the present study, we have examined the inhibitory activities of some flavonoids, their biosynthetic precursors (cinnamic acids and coumaric acid), and their derivatives. The proliferative activity of flavonoids on the T-47D estrogen-receptor-positive breast cancer cell line was also evaluated. Among 10 flavonoids, 7,4'-dihydroxyflavone, diosmetin, chrysoeriol, scutellarein, genkwanin and fisetin showed more than 70% inhibition of 17beta-HSD type 1 at 6microM. In a series of 18 derivatives of cinnamic acid, the best inhibitor was 4'-cyanophenyl 3,4-methylenedioxycinnamate, with more than 70% inhibition of 17beta-HSD type 1. None of flavonoids affected the proliferation of T-47D breast cancer cells.

Phytoestrogens as inhibitors of the human progesterone metabolizing enzyme AKR1C1.

Phytoestrogens are plant-derived, non-steroidal constituents of our diets. They can act as agonists or antagonists of estrogen receptors, and they can modulate the activities of the key enzymes in estrogen biosynthesis. Much less is known about their actions on the androgen and progesterone metabolizing enzymes. We have examined the inhibitory action of phytoestrogens on the key human progesterone-metabolizing enzyme, 20alpha-hydroxysteroid dehydrogenase (AKR1C1). This enzyme inactivates progesterone and the neuroactive 3alpha,5alpha-tetrahydroprogesterone, to form their less active counterparts, 20alpha-hydroxyprogesterone and 5alpha-pregnane-3alpha,20alpha-diol, respectively. We overexpressed recombinant human AKR1C1 in Escherichia coli, purified it to homogeneity, and examined the selected phytoestrogens as inhibitors of NADPH-dependent reduction of a common AKR substrate, 9,10-phenantrenequinone, and progesterone. The most potent inhibitors were 7-hydroxyflavone, 3,7-dihydroxyflavone and flavanone naringenin with IC(50) values in the low microM range. Docking of the flavones in the active site of AKR1C1 revealed their possible binding modes, in which they are sandwiched between the Leu308 and Trp227 of AKR1C1.

Cinnamic acids as new inhibitors of 17beta-hydroxysteroid dehydrogenase type 5 (AKR1C3).

17Beta-hydroxysteroid dehydrogenase type 5 (AKR1C3) that is involved in the pre-receptor regulation of androgen and estrogen action in the human is an emerging therapeutic target in the treatment of hormone-dependent forms of cancer, such as prostate cancer, breast cancer and endometrial cancer. To discover novel inhibitors, we tested the effect of a series of cinnamic acids on the reductive activity of the human recombinant AKR1C3. The compounds were evaluated in a spectrophotometric assay using 9,10-phenanthrenequinone as a substrate. The best inhibitor in the series was alpha-methylcinnamic acid (IC50=6.4 microM). Also, unsubstituted cinnamic acid was a good inhibitor of AKR1C3 (IC50=50 microM). Small hydrophobic substituents of the phenyl ring did not alter the activity; however, substitution with polar groups decreased the potency of inhibition. The most active compounds in this series represent promising starting points for further structural modifications in the search for more potent inhibitors of AKR1C3.

Nonsteroidal anti-inflammatory drugs and their analogues as inhibitors of aldo-keto reductase AKR1C3: new lead compounds for the development of anticancer agents.

Nonsteroidal anti-inflammatory drugs (NSAIDs) like indomethacin, flufenamic acid, and related compounds have been recently identified as potent inhibitors of AKR1C3. We report that some other NSAIDs (diclofenac and naproxen) also inhibit AKR1C3, with the IC(50) values in the low micromolar range. In order to obtain more information about the structure-activity relationship and to identify new leads, a series of compounds designed on the basis of NSAIDs were synthesized and screened on AKR1C3. The most active compounds were 2-[(2,2-diphenylacetyl)amino]benzoic acid 4 (IC(50)=11microM) and 3-phenoxybenzoic acid 10 (IC(50)=0.68microM). These compounds represent promising starting points for the development of new anticancer agents.