AKR1C3 Inhibitors Research Articles

3D-QSAR studies of 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acids as AKR1C3 inhibitors: Highlight the importance of molecular docking in conformation generation

AKR1C3 is a promising drug target for castration-resistant prostate cancer (CRPC). Here, 3D-QSAR analysis were performed on 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acids to correlate their chemical structures with their observed AKR1C3 inhibitory activity. Three structural alignment methods employing various conformers were used to scrutinize the effect of conformation selection on the predictive accuracy of QSAR models. Using docked conformation, the best CoMFA and CoMSIA models were developed and validated with a training set of 61 molecules and a test set of 7 molecules. Detailed analysis of contour maps provided helpful structural insights to rational design of AKR1C3 inhibitors with enhanced potency.

In vitro CAPE inhibitory activity towards human AKR1C3 and the molecular basis

AKR1C3 is a critical enzyme for producing testosterone and 5α-DHT in the human body. Inhibiting AKR1C3 is a potential target for treating castration-resistant prostate cancer (CRPC). To find AKR1C3 inhibitors with a new molecular skeleton and binding mode, we analyzed the in vitro inhibitory activity of caffeic acid phenethyl ester (CAPE) and eight other phenolic acid analogues towards AKR1C3 and six other human AKR1 enzymes. We analyzed CAPE and octyl gallate interactions with AKR1C3 using X-ray crystallography, which provided a molecular basis for understanding the phenolic acid inhibitory activity and selectivity towards human AKR1s.

MP62-19 AKR1C3 OVEREXPRESSION IN PROSTATE CANCER PROMOTES EPITHELIAL-MESENCHYMAL TRANSITION AND METASTASIS VIA ACTIVATION OF MAPK/ERK SIGNALING AND UPREGULATION OF ZEB1

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2016MP62-19 AKR1C3 OVEREXPRESSION IN PROSTATE CANCER PROMOTES EPITHELIAL-MESENCHYMAL TRANSITION AND METASTASIS VIA ACTIVATION OF MAPK/ERK SIGNALING AND UPREGULATION OF ZEB1 Bin Wang, Kaijie Wu, Jun Huang, Jer-Tsong Hsieh, and Dalin He Bin WangBin Wang More articles by this author , Kaijie WuKaijie Wu More articles by this author , Jun HuangJun Huang More articles by this author , Jer-Tsong HsiehJer-Tsong Hsieh More articles by this author , and Dalin HeDalin He More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.927AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES AKR1C3, as a crucial steroidogenic enzyme, facilitated intratumoral androgen biosynthesis and androgen receptor (AR) reactivation in the development of castration-resistant prostate cancer. Also, amount of data have shown that AKR1C3 expression was significantly elevated in clinical metastatic prostate cancer (PCa) specimens, indicating an alternative role of AKR1C3 in PCa metastasis. Epithelial-mesenchymal transition (EMT) has been recognized to play pivotal roles in promoting carcinoma invasion and metastasis; however, the roles of AKR1C3 in EMT and PCa metastasis have not been fully elucidated. METHODS C4-2, 22RV1-T and PC-3 cells with higher AKR1C3 expression were selected and treated with several specific AKR1C3 shRNAs or small molecule inhibitor, and the cell migration and invasion abilities were detected by wound healing assay and Transwell assay. The expression of EMT markers (i.e., E-cadherin and vimentin) and the related transcription factors (i.e., Snail, Slug, ZEB1 and Twist1) was examined by Western blot or quantitative PCR assays, and the phosphorylation of AKT or ERK was detected by Western blot. Also, the expression of vimentin, ZEB1 and p-ERK (T202/Y204) was detected by immunohistochemical staining in PC-3 xenograft tissues after AKR1C3 inhibitor treatment. The correlation between AKR1C3 and EMT markers (including EMT-related transcription factors) expression in clinical specimens from tissue microarray or TCGA database was analyzed. RESULTS AKR1C3 was overexpressed in more aggressive PCa cell lines regardless of the AR status. Knock-down of AKR1C3 expression or inhibition of AKR1C3 activity could significantly suppressed cell migration and invasion abilities in vitro. Also, knock-down of AKR1C3 expression or inhibition of AKR1C3 activity could increase E-cadherin expression but decrease vimentin expression, in which the phosphorylation of ERK and the transcription factor ZEB1 expression were specifically downregulated. In consistency, inhibition of AKR1C3 activity by inhibitor treatment could decrease the expression of vimentin, ZEB1 and p-ERK (T202/Y204) in xenograft tissues in vivo. Moreover, there was a significant correlation between AKR1C3 and EMT markers (i.e., E-cadherin, vimentin and ZEB1) in human PCa specimens. CONCLUSIONS AKR1C3 is a novel EMT regulator in PCa metastasis through activation of MAPK/ERK signaling and upregulation of ZEB1 expression. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e820 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Bin Wang More articles by this author Kaijie Wu More articles by this author Jun Huang More articles by this author Jer-Tsong Hsieh More articles by this author Dalin He More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Berberine inhibits androgen synthesis by interaction with aldo-keto reductase 1C3 in 22Rv1 prostate cancer cells.

Aldo-keto reductase family 1 member C3 has recently been regarded as a potential therapeutic target in castrate-resistant prostate cancer. Herein, we investigated whether berberine delayed the progression of castrate-resistant prostate cancer by reducing androgen synthesis through the inhibition of Aldo-keto reductase family 1 member C3. Cell viability and cellular testosterone content were measured in prostate cancer cells. Aldo-keto reductase family 1 member C3 mRNA and protein level were detected by RT-PCR and Western bolt analyses, respectively. Computer analysis with AutoDock Tools explored the molecular interaction of berberine with Aldo-keto reductase family 1 member C3. We found that berberine inhibited 22Rv1 cells proliferation and decreased cellular testosterone formation in a dose-dependent manner. Berberine inhibited Aldo-keto reductase family 1 member C3 enzyme activity, rather than influenced mRNA and protein expressions. Molecular docking study demonstrated that berberine could enter the active center of Aldo-keto reductase family 1 member C3 and form π-π interaction with the amino-acid residue Phe306 and Phe311. In conclusion, the structural interaction of berberine with Aldo-keto reductase family 1 member C3 is attributed to the suppression of Aldo-keto reductase family 1 member C3 enzyme activity and the inhibition of 22Rv1 prostate cancer cell growth by decreasing the intracellular androgen synthesis. Our result provides the experimental basis for the design, research, and development of AKR1C3 inhibitors using berberine as the lead compound.

Abstract 3450: Resistance to P450c17 inhibitors in castration-resistant prostate cancer may result from the DHEA-S depot that remains and can be used by AKR1C3 for intratumoral androgen biosynthesis

Abstract Localized intermediate and high-risk prostate cancer can be treated with androgen deprivation therapy (ADT). Initially, patients undergo remission but inevitably relapse due to the emergence of castration resistant prostate cancer (CRPC). Newer agents such as the P450c17 inhibitor, abiraterone acetate (AA), have gained approval for the treatment of CRPC and increased median survival by 4 months. We have developed a novel and validated stable isotope dilution liquid chromatography selected reaction monitoring mass spectrometry (SID-LC/ESI/SRM/MS) method for the quantification of conjugated and unconjugated keto-androgens in human serum (J Steroid Biochem Mol Biol (2013) 138:281). This method was applied to human serum from patients enrolled in a neoadjuvant AA clinical trial (J Clin Oncol (2014) 32:3705). We found that testosterone (T) and 5α-dihydrotestosterone (DHT) did not always decrease in tandem, which suggests that pathways that bypass T may lead to DHT. These may include the alternative pathway (Δ4-androstene-3,17-dione -> 5α-androstane-3,17-dione -> DHT) or the backdoor pathway (androsterone -> 5α-androstane-3α,17β-diol -> DHT). Second, despite achieving >90% inhibition of P450c17, the level of dehydroepiandrosterone sulfate (DHEA-S) that remains (20,000 ng/dL) is 4,000-fold higher than castrate levels of T achieved in the trial. We hypothesize that the DHEA-S depot that remains is sufficient to feed intratumoral androgen biosynthesis and that this could account for the clinical failure of P450c17 inhibitors in CRPC. Adaptive intratumoral androgen biosynthesis may be facilitated by the up-regulation of AKR1C3 (type 5 17β-hydroxysteroid dehydrogenase). In order to test our hypothesis, we have developed a panel of isogenic prostate cancer cell lines that either express AKR1C3 or are AKR1C3 null. These cell lines are then challenged with post-AA levels of circulating androgens (DHEA-S, DHEA and Δ4-androstene-3,17-dione) to determine whether they can still make sufficient androgens to activate the androgen receptor (AR), where androgen metabolism is measured using SID-LC/ESI/SRM/MS. Using LNCaP and LNCaP-AKR1C3 cells, we find that the level of T detected as T-17β-glucuronide in the AKR1C3 expressing cells could be sufficient to activate the AR. These results would suggest that AKR1C3 inhibitors in combination with AA could benefit patients who might otherwise fail AA treatment. [Supported by grants: R25-CA101871 to DT; P30-ES-13508 to TMP; P01-CA-163337 to SPB, EM, TMP and PSN)] Citation Format: Daniel Tamae, Elahe Mostaghel, Bruce Montgomery, Peter S. Nelson, Steven P. Balk, Philip W. Kantoff, Mary-Ellen Taplin, Trevor M. Penning. Resistance to P450c17 inhibitors in castration-resistant prostate cancer may result from the DHEA-S depot that remains and can be used by AKR1C3 for intratumoral androgen biosynthesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3450. doi:10.1158/1538-7445.AM2015-3450

Synthesis of non-prenyl analogues of baccharin as selective and potent inhibitors for aldo-keto reductase 1C3

Inhibitors of a human member (AKR1C3) of the aldo-keto reductase superfamily are regarded as promising therapeutics for the treatment of prostatic and breast cancers. Baccharin [3-prenyl-4-(dihydrocinnamoyloxy)cinnamic acid], a component of propolis, was shown to be both potent (Ki 56nM) and highly isoform-selective inhibitor of AKR1C3. In this study, a series of derivatives of baccharin were synthesized by replacing the 3-prenyl moiety with aryl and alkyl ether moieties, and their inhibitory activities for the enzyme were evaluated. Among them, two benzyl ether derivatives, 6m and 6n, showed an equivalent inhibitory potency to baccharin. The molecular docking of 6m in AKR1C3 has allowed the design and synthesis of (E)-3-{3-[(3-hydroxybenzyl)oxy]-4-[(3-phenylpropanoyl)oxy]phenyl}acrylic acid (14) with improved potency (Ki 6.4nM) and selectivity comparable to baccharin. Additionally, 14 significantly decreased the cellular metabolism of androsterone and cytotoxic 4-oxo-2-nonenal by AKR1C3 at much lower concentrations than baccharin.

In vitro and in vivo characterisation of ASP9521: a novel, selective, orally bioavailable inhibitor of 17β-hydroxysteroid dehydrogenase type 5 (17βHSD5; AKR1C3).

Aldo-keto reductase 1C3 [AKR1C3;17β-hydroxysteroid dehydrogenase type 5 (17βHSD5)], plays a crucial role in persistent production of androgens despite castration, by catalysing conversion of the adrenal androgens dehydroepiandrosterone and androstenedione (AD) into androstenediol and testosterone (T). Hence, AKR1C3 is a promising therapeutic target in castration-resistant prostate cancer, as combination of an AKR1C3 inhibitor and a gonadotropin-releasing hormone analogue may lead to complete androgen blockade. This study describes the preclinical characterisation of the novel AKR1C3 inhibitor ASP9521. The inhibitory effect of ASP9521 on AKR1C3-mediated conversion from AD into T was evaluated both in vitro and in vivo, using CWR22R xenografted mice. The effect of ASP9521 on PSA production and cell proliferation was tested using LNCaP cells stably expressing human AKR1C3 (LNCaP-AKR1C3). Pharmacokinetics of ASP9521 were studied in rats, dogs and cynomolgus monkeys. ASP9521 inhibited conversion of AD into T by recombinant human or cynomolgus monkey AKR1C3 in a concentration-dependent manner (IC50,human: 11 nmol/L; IC50,monkey: 49 nmol/L). ASP9521 showed >100-fold selectivity for AKR1C3 over the isoform AKR1C2. In LNCaP-AKR1C3 cells, ASP9521 suppressed AD-dependent PSA production and cell proliferation. In CWR22R xenografts, single oral administration of ASP9521 (3 mg/kg) inhibited AD-induced intratumoural T production and this inhibitory effect was maintained for 24 h. After oral administration, ASP9521 was rapidly eliminated from plasma, while its intratumoural concentration remained high. The bioavailability of ASP9521 after oral administration (1 mg/kg) was 35 %, 78 % and 58 % in rats, dogs and monkeys, respectively. ASP9521 is a potent, selective, orally bioavailable AKR1C3 inhibitor.

The Activity of SN33638, an Inhibitor of AKR1C3, on Testosterone and 17β-Estradiol Production and Function in Castration-Resistant Prostate Cancer and ER-Positive Breast Cancer.

AKR1C3 is a novel therapeutic target in castration-resistant prostate cancer (CRPC) and estrogen receptor (ER)-positive breast cancer because of its ability to produce testosterone and 17β-estradiol intratumorally, thus promoting nuclear receptor signaling and tumor progression. A panel of CRPC, ER-positive breast cancer and high/low AKR1C3-expressing cell lines were treated with SN33638, a selective inhibitor of AKR1C3, in the presence of hormone or prostaglandin (PG) precursors, prior to evaluation of cell proliferation and levels of 11β-PG F2α (11β-PGF2α), testosterone, 17β-estradiol, and prostate-specific antigen (PSA). A meta-analysis of AKR1C3 mRNA expression in patient samples was also conducted, which revealed that AKR1C3 mRNA was upregulated in CRPC, but downregulated in ER-positive breast cancer. 11β-PGF2α and testosterone levels in the cell line panel correlated with AKR1C3 protein expression. SN33638 prevented 11β-PGF2α formation in cell lines that expressed AKR1C3, but partially inhibited testosterone formation and subsequently cell proliferation and/or PSA expression only in high (LAPC4 AKR1C3-overexpressing cells) or moderate (22RV1) AKR1C3-expressing cell lines. SN33638 had little effect on 17β-estradiol production or estrone-stimulated cell proliferation in ER-positive breast cancer cell lines. Although SN33638 could prevent 11β-PGF2α formation, its ability to prevent testosterone and 17β-estradiol production and their roles in CRPC and ER-positive breast cancer progression was limited due to AKR1C3-independent steroid hormone production, except in LAPC4 AKR1C3 cells where the majority of testosterone was AKR1C3-dependent. These results suggest that inhibition of AKR1C3 is unlikely to produce therapeutic benefit in CRPC and ER-positive breast cancer patients, except possibly in the small subpopulation of CRPC patients with tumors that have upregulated AKR1C3 expression and are dependent on AKR1C3 to produce the testosterone required for their growth.

Isoquinoline alkaloids as a novel type of AKR1C3 inhibitors

AKR1C3 is an important human enzyme that participates in the reduction of steroids and prostaglandins, which leads to proliferative signalling. In addition, this enzyme also participates in the biotransformation of xenobiotics, such as drugs and procarcinogens. AKR1C3 is involved in the development of both hormone-dependent and hormone-independent cancers and was recently demonstrated to confer cell resistance to anthracyclines. Because AKR1C3 is frequently upregulated in various cancers, this enzyme has been suggested as a therapeutic target for the treatment of these pathological conditions. In this study, nineteen isoquinoline alkaloids were examined for their ability to inhibit a recombinant AKR1C3 enzyme. As a result, stylopine was demonstrated to be the most potent inhibitor among the tested compounds and exhibited moderate selectivity towards AKR1C3. In the follow-up cellular studies, stylopine significantly inhibited the AKR1C3-mediated reduction of daunorubicin in intact cells without considerable cytotoxic effects. This inhibitor could therefore be used as a model AKR1C3 inhibitor in research or evaluated as a possible therapeutic anticancer drug. Furthermore, based on our results, stylopine can serve as a model compound for the design and future development of structurally related AKR1C3 inhibitors.

Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate

Background:We and others have identified the aldo-keto reductase AKR1C3 as a potential drug target in prostate cancer, breast cancer and leukaemia. As a consequence, significant effort is being invested in the development of AKR1C3-selective inhibitors.Methods:We report the screening of an in-house drug library to identify known drugs that selectively inhibit AKR1C3 over the closely related isoforms AKR1C1, 1C2 and 1C4. This screen initially identified tetracycline as a potential AKR1C3-selective inhibitor. However, mass spectrometry and nuclear magnetic resonance studies identified that the active agent was a novel breakdown product (4-methyl(de-dimethylamine)-tetracycline (4-MDDT)).Results:We demonstrate that, although 4-MDDT enters AML cells and inhibits their AKR1C3 activity, it does not recapitulate the anti-leukaemic actions of the pan-AKR1C inhibitor medroxyprogesterone acetate (MPA). Screens of the NCI diversity set and an independently curated small-molecule library identified several additional AKR1C3-selective inhibitors, none of which had the expected anti-leukaemic activity. However, a pan AKR1C, also identified in the NCI diversity set faithfully recapitulated the actions of MPA.Conclusions:In summary, we have identified a novel tetracycline-derived product that provides an excellent lead structure with proven drug-like qualities for the development of AKR1C3 inhibitors. However, our findings suggest that, at least in leukaemia, selective inhibition of AKR1C3 is insufficient to elicit an anticancer effect and that multiple AKR1C inhibition may be required.

A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells

Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.

Morpholylureas are a new class of potent and selective inhibitors of the type 5 17-β-hydroxysteroid dehydrogenase (AKR1C3)

Inhibitors of the aldo–keto reductase enzyme AKR1C3 are of interest as potential drugs for leukemia and hormone-related cancers. A series of non-carboxylate morpholino(phenylpiperazin-1-yl)methanones were prepared by palladium-catalysed coupling of substituted phenyl or pyridyl bromides with the known morpholino(piperazin-1-yl)methanone, and shown to be potent (IC50∼100nM) and very isoform-selective inhibitors of AKR1C3. Lipophilic electron-withdrawing substituents on the phenyl ring were positive for activity, as was an H-bond acceptor on the other terminal ring, and the ketone moiety (as a urea) was essential. These structure–activity relationships are consistent with an X-ray structure of a representative compound bound in the AKR1C3 active site, which showed H-bonding between the carbonyl oxygen of the drug and Tyr55 and His117 in the ‘oxyanion hole’ of the enzyme, with the piperazine bridging unit providing the correct twist to allow the terminal benzene ring to occupy the lipophilic pocket and align with Phe311.

Highlights of This Issue

The presence of an immunosuppressive microenvironment induced by tumors and host regulatory cells can limit the full potential of adoptive T-cell immunotherapy. John and colleagues demonstrated that specifically blocking PD-1 immunosuppression can potently enhance tumor eradication in mice following adoptive transfer of CAR T cells in the absence of pathology. Therapeutic responses were strongly correlated with increased CAR T-cell function and a concomitant decrease in MDSC's at the tumor site. These results are likely to increase the effectiveness of immunotherapy against a number of cancers that are currently resistant to first-line treatment.Although steroidogenic enzyme AKR1C3 is consistently upregulated in castration-resistant prostate cancer (CRPC). Its role, if any, in progression to castration resistance is unknown. Yepuru and colleagues demonstrate that AKR1C3 not only contributes to this recognized role as a steroidogenic enzyme but also plays a dual role as an AR-selective coactivator. While overexpression of AKR1C3 increases androgen-dependent prostate cancer and CRPC growth, inhibition of AKR1C3 reduces CRPC growth. These effects were mediated by both its enzyme and coactivator functions. The findings depict, for the first time, a functional role for AKR1C3 and propose AKR1C3 as pharmacologically targetable AR-selective coactivator.The MET pathway is frequently activated in human cancer and is implicated as a resistance mechanism to targeted therapeutics. Wu and colleagues demonstrated that treatment with LY2801653, a novel oncokinase inhibitor with MET as one of its targets, inhibited tumor growth in non–small-cell lung cancer cell line and patient tumor-derived xenograft mouse models. In the MET-dependent orthotopic model (NCI-H441), LY2801653 treatment inhibited both primary tumor growth and metastasis, and prolonged survival of tumor-bearing animals. Mechanistic studies demonstrated that LY2801653 inhibited MET-pathway signaling, anchorage-independent growth, migration, and invasion. These results support the ongoing clinical development of LY2801653 and suggest that differences in the MET activation status of tumors may be predictive of response.A mechanism of resistance to FLT3 inhibitors in patients with FLT3-ITD-positive AML is the acquisition of secondary FLT3 tyrosine kinase domain (KD) mutations. To explore the evolution of KD mutations during tyrosine kinase inhibitor (TKI) therapy, Baker and colleagues performed deep amplicon sequencing of FLT3 in leukemic blast samples from patients during treatment with sorafenib and sunitinib. KD mutations at residues D835 and F691 could be detected early after commencement of therapy in morphologic remission samples, and subsequently monitored in response to individual TKIs. Application of sensitive sequencing methods during therapy may allow individualized treatment with currently available FLT3 inhibitors.

Development of potent and selective indomethacin analogues for the inhibition of AKR1C3 (Type 5 17β-hydroxysteroid dehydrogenase/prostaglandin F synthase) in castrate-resistant prostate cancer.

Castrate-resistant prostate cancer (CRPC) is a fatal, metastatic form of prostate cancer. CRPC is characterized by reactivation of the androgen axis due to changes in androgen receptor signaling and/or adaptive intratumoral androgen biosynthesis. AKR1C3 is upregulated in CRPC where it catalyzes the formation of potent androgens. This makes AKR1C3 a target for the treatment of CRPC. AKR1C3 inhibitors should not inhibit AKR1C1/AKR1C2, which inactivate 5α-dihydrotestosterone. Indomethacin, used to inhibit cyclooxygenase, also inhibits AKR1C3 and displays selectivity over AKR1C1/AKR1C2. Parallel synthetic strategies were used to generate libraries of indomethacin analogues, which exhibit reduced cyclooxygenase inhibitory activity but retain AKR1C3 inhibitory potency and selectivity. The lead compounds inhibited AKR1C3 with nanomolar potency, displayed >100-fold selectivity over AKR1C1/AKR1C2, and blocked testosterone formation in LNCaP-AKR1C3 cells. The AKR1C3·NADP+·2′-des-methyl-indomethacin crystal structure was determined, and it revealed a unique inhibitor binding mode. The compounds reported are promising agents for the development of therapeutics for CRPC.

Synthesis and structure–activity relationships for 1-(4-(piperidin-1-ylsulfonyl)phenyl)pyrrolidin-2-ones as novel non-carboxylate inhibitors of the aldo-keto reductase enzyme AKR1C3

High expression of the aldo-keto reductase enzyme AKR1C3 in the human prostate and breast has implicated it in the development and progression of leukemias and of prostate and breast cancers. Inhibitors are thus of interest as potential drugs. Most inhibitors of AKR1C3 are carboxylic acids, whose transport into cells is likely dominated by carrier-mediated processes. We describe here a series of (piperidinosulfonamidophenyl)pyrrolidin-2-ones as potent (<100 nM) and isoform-selective non-carboxylate inhibitors of AKR1C3. Structure–activity relationships identified the sulfonamide was critical, and a crystal structure showed the 2-pyrrolidinone does not interact directly with residues in the oxyanion hole. Variations in the position, co-planarity or electronic nature of the pyrrolidinone ring severely diminished activity, as did altering the size or polarity of the piperidino ring. There was a broad correlation between the enzyme potencies of the compounds and their effectiveness at inhibiting AKR1C3 activity in cells.

2,3-Diarylpropenoic acids as selective non-steroidal inhibitors of type-5 17β-hydroxysteroid dehydrogenase (AKR1C3)

The aldo–keto reductase AKR1C3 is an important target for the development of new drugs. Selective inhibitors of this enzyme are needed because they should not inhibit other, structurally closely related AKR1C isoforms. A comprehensive series of 2,3-diarylpropenoic acids was synthesized and evaluated for the inhibition of AKR1C1–AKR1C3. We found that the 4-methylsulfonylphenyl substituent at position 2 of these acids is required to exhibit the selective inhibition of AKR1C3. The best results were obtained for the compounds that fulfill the above requirement and possess a 4-bromophenyl, 4-methylthiophenyl, 4-methylphenyl or 4-ethylphenyl substituent at position 3 of the substituted propenoic acids (i.e., acids 28, 29, 37, and 39, respectively). These compounds represent an important step toward the development of drug candidates for a treatment of the hormone-dependent and hormone-independent forms of prostate and breast cancers.

Editorial on research topic: aldo-keto reductases and role in human disease

Research on Aldo-keto reductases (AKRs) over the years has revealed a remarkable diversity in the reactivity of these soluble NAD(P)(H) oxidoreductases. The AKR enzymes primarily catalyze the reduction of aldehydes and ketones to primary and secondary alcohols, respectively. The 10 known human AKR enzymes can turnover a vast range of endogenous and exogenous substrates, including glucose, steroids, carcinogens, reactive aldehydes, and a variety of carbonyl-containing drugs. These enzymes have been implicated in a number of human diseases, including cancer and disorders of endocrinology and metabolism. AKRs are involved in the development and progression of many cancers, as well as chemotherapeutic drug resistance. AKR1B1 and AKR1B10 are overexpressed in tumors, such as liver, breast, and lung cancer. Several AKRs (AKR1A1, AKR1B10, and AKR1C1-3) are involved in tobacco-carcinogenesis, but they also catalyze the detoxication of nicotine derived nitrosamino ketones. In addition, AKR1C1-3 enzymes play a key role in the regulation of proliferative signaling in hormone dependent cancers. AKR1B1 has long been known for its involvement in diabetic complications. Recent studies now suggest AKR1B enzymes play a key role in inflammatory diseases such as atherosclerosis, sepsis, asthma, uveitis, and colon cancer by mediating oxidative stress-induced inflammatory signals. In recognition of the role of AKR in various diseases, significant efforts have been made in the development of specific/selective AKR1B1 and AKR1C3 inhibitors. The aim of this Research Topic forum is to celebrate the advancements seen in the AKR field via review papers and original articles, as well as to promote future research in understanding the underlying mechanism(s) of the role of AKRs in human disease. Eight review articles are included in this issue, and are grouped into disease areas (cancer, disorders of endrocrinology and metabolism) after the artile written by Chen and Zhang (2012) which describes the current understanding of the regulation of all human AKR members in relation to disease. The significance of the AKR enzymes for research in various cancers is discussed in four review articles. The article by Matsunaga et al. (2012) focuses on the role AKR1B10 in drug resistance of cancer cells and the development of AKR1B10 inhibitors to reverse chemoresistance. The article by Ruiz et al. (2012) describes the retinaldehyde reductase activity of AKRs in relation to proliferation and tumorigenesis. The paper from the Penning group (Zhang et al., 2012) summarizes the role of AKR in the metabolic activation and detoxication of polycyclic aromatic hydrocarbons and the relevance of phase II conjugation reactions to human lung carcinogenesis. Rižner (2012) provides a review on the role played by AKR1B and AKR1C members in the pathogenesis of endometrial and cervical cancers, as well as other uterine diseases. The aspect that members of AKRs can mediate the timing of parturition through metabolism of progesterone and prostaglandins is discussed by Byrns (2012). The role of AKR1B members in modification and production of signaling molecules is emphasized by Pastel et al. (2012) for a better understanding of the physiological and pathological role of AKR1B enzymes. The article by Tang et al. (2012) focuses on the increases in oxidative stress by AKR1B1 in pathogenesis of diabetic complications such as cardiovascular diseases. Three Original Research Articles follow the review articles. The article by Fortier and coworkers (Bresson et al., 2012) reports on the prostaglandin F synthase activity of AKR1B1 in different models of living cells and tissues. This activity of AKR1B1 represents a novel target to regulate ischemic and inflammatory responses associated with several human pathologic conditions. The article by Laffin and Petrash (2012) examines the expression of AKR1B1 and AKR1B10 across all major human cancer types. Their findings point to the great potential of AKR1B1 inhibition as novel cancer therapeutics. Schulze et al. (2012) describe their studies on basal and regulatory promoter of AKR1C3 in relation to prostate cancer. As pointed out in the review article by Pastel et al. (2012) there is a “marked contrast between the abundance of enzymatic data and the small number of reports dedicated to functional studies in a physiological context.” It is my hope that with an emphasis on the role of AKRs in human disease the articles included in this issue will benefit a broad audience and the researchers in the rapidly growing AKR filed.

New enzymatic assay for the AKR1C enzymes

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.

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.

3-(3,4-Dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic Acids: Highly Potent and Selective Inhibitors of the Type 5 17-β-Hydroxysteroid Dehydrogenase AKR1C3

A high-throughput screen identified 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acid as a novel, highly potent (low nM), and isoform-selective (1500-fold) inhibitor of aldo-keto reductase AKR1C3: a target of interest in both breast and prostate cancer. Crystal structure studies showed that the carboxylate group occupies the oxyanion hole in the enzyme, while the sulfonamide provides the correct twist to allow the dihydroisoquinoline to bind in an adjacent hydrophobic pocket. SAR studies around this lead showed that the positioning of the carboxylate was critical, although it could be substituted by acid isosteres and amides. Small substituents on the dihydroisoquinoline gave improvements in potency. A set of "reverse sulfonamides" showed a 12-fold preference for the R stereoisomer. The compounds showed good cellular potency, as measured by inhibition of AKR1C3 metabolism of a known dinitrobenzamide substrate, with a broad rank order between enzymic and cellular activity, but amide analogues were more effective than predicted by the cellular assay.