Human aldo-keto reductase 1C3 (AKR1C3) is a steroid modifying enzyme involved in cancer progression. Here, A-ring modified 17α-picolyl and 17(E)-picolinylidene androstane derivatives are shown to inhibit AKR1C3 activity in vitro. None of the androstane derivatives have off-target affinity for the androgen receptor, based on a fluorescence assay in yeast cells. The X-ray structure of AKR1C3 in complex with the strongest inhibitor, a 17α-picolyl androstane with a C3-oxime modification, was determined at 1.7 Å resolution. Based on this crystal structure and molecular docking, inhibition of AKR1C3 by the 17α-picolyl or 17(E)-picolinylidene derivatives depends on interactions between the C3 modification and the NADP+ cofactor, while the C17α-picolyl or C17-picolinylidene group anchors the inhibitor to AKR1C3. Because one AKR1C3 inhibitor identified here was also previously reported to inhibit CYP17, it may be possible for future researchers to design dual AKR1C3/CYP17 inhibitors based on a steroid scaffold for potential treatment of advanced prostate cancers.

Department of Biology and Ecology University of Novi Sad Novi Sad Serbia

Department of Chemistry Biochemistry and Environmental Protection University of Novi Sad Novi Sad Serbia

Institute of Organic Chemistry and Biochemistry The Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Byrns MC, Jin Y, Penning TM.. Inhibitors of type 5 17β-hydroxysteroid dehydrogenase (AKR1C3): Overview and structural insights. J Steroid Biochem Mol Biol. 2011;125(1–2):95–104. PubMed PMC

Brožič P, Turk S, Rižner TL, Gobec S.. Inhibitors of aldo-keto reductases AKR1C1-AKR1C4. Curr Med Chem. 2011;18(17):2554–2565. PubMed

Penning TM. The aldo-keto reductases (AKRs): overview. Chem Biol Interact. 2015;234:236–246. PubMed PMC

Penning TM, Wangtrakuldee P, Auchus RJ.. Structural and functional biology of aldo-keto reductase steroid-transforming enzymes. Endocr Rev. 2019;40(2):447–475. PubMed PMC

Yepuru M, Wu Z, Kulkarni A, Yin F, Barrett CM, Kim J, Steiner MS, Miller DD, Dalton JT, Narayanan R, et al. Steroidogenic enzyme AKR1C3 is a novel androgen receptor-selective coactivator that promotes prostate cancer growth. Clin Cancer Res. 2013;19(20):5613–5625. PubMed

Liu Y, He S, Chen Y, Liu Y, Feng F, Liu W, Guo Q, Zhao L, Sun H.. Overview of AKR1C3: inhibitor achievements and disease insights. J Med Chem. 2020;63(20):11305–11329. PubMed

Xing S, Jiang J, Chu X, Wang X, Wang Z, Li X, Lv B, Guo C, He S, Wang L, et al. Discovery of highly potent AKR1C3 inhibitors treating sorafenib-resistant hepatocellular carcinoma. J Med Chem. 2025;68(7):7367–7389. PubMed

Maddeboina K, Jonnalagadda SK, Morsy A, Duan L, Chhonker YS, Murry DJ, Penning TM, Trippier PC.. Aldo-keto reductase 1C3 inhibitor prodrug improves pharmacokinetic profile and demonstrates in vivo efficacy in a prostate cancer xenograft model. J Med Chem. 2023;66(14):9894–9915. PubMed PMC

Khanim F, Davies N, Veliça P, Hayden R, Ride J, Pararasa C, Chong MG, Gunther U, Veerapen N, Winn P, et al. Selective AKR1C3 inhibitors do not recapitulate the anti-leukaemic activities of the pan-AKR1C inhibitor medroxyprogesterone acetate. Br J Cancer. 2014;110(6):1506–1516. PubMed PMC

Verma K, Zang T, Penning TM, Trippier PC.. Potent and highly selective aldo-keto reductase 1C3 (AKR1C3) inhibitors act as chemotherapeutic potentiators in acute myeloid leukemia and T-cell acute lymphoblastic leukemia. J Med Chem. 2019;62(7):3590–3616. PubMed PMC

Khanim FL, Hayden RE, Birtwistle J, Lodi A, Tiziani S, Davies NJ, Ride JP, Viant MR, Gunther UL, Mountford JC, et al. Combined bezafibrate and medroxyprogesterone acetate: potential novel therapy for acute myeloid leukaemia. PLoS One. 2009;4(12):e8147. PubMed PMC

Hertzog JR, Zhang Z, Bignan G, Connolly PJ, Heindl JE, Janetopoulos CJ, Rupnow BA, McDevitt TM.. AKR1C3 mediates pan-AR antagonist resistance in castration-resistant prostate cancer. Prostate. 2020;80(14):1223–1232. PubMed

Zhao J, Ning S, Lou W, Yang JC, Armstrong CM, Lombard AP, D’Abronzo LS, Evans CP, Gao AC, Liu C, et al. Cross-resistance among next-generation antiandrogen drugs through the AKR1C3/AR-V7 axis in advanced prostate cancer. Mol Cancer Ther. 2020;19(8):1708–1718. PubMed PMC

Heibein AD, Guo B, Sprowl JA, MacLean DA, Parissenti AM.. Role of aldo-keto reductases and other doxorubicin pharmacokinetic genes in doxorubicin resistance, DNA binding, and subcellular localization. BMC Cancer. 2012;12(1):381. PubMed PMC

Liu C, Armstrong CM, Lou W, Lombard A, Evans CP, Gao AC.. Inhibition of AKR1C3 activation overcomes resistance to abiraterone in advanced prostate cancer. Mol Cancer Ther. 2017;16(1):35–44. PubMed PMC

Shiiba M, Yamagami H, Yamamoto A, Minakawa Y, Okamoto A, Kasamatsu A, Sakamoto Y, Uzawa K, Takiguchi Y, Tanzawa H, et al. Mefenamic acid enhances anticancer drug sensitivity via inhibition of aldo-keto reductase 1C enzyme activity. Oncol Rep. 2017;37(4):2025–2032. PubMed

Bauman DR, Rudnick SI, Szewczuk LM, Jin Y, Gopishetty S, Penning TM.. 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. Mol Pharmacol. 2005;67(1):60–68. PubMed

Brožič P, Turk S, Adeniji AO, Konc J, Janežič D, Penning TM, Lanišnik Rižner T, Gobec S.. Selective inhibitors of aldo-keto reductases AKR1C1 and AKR1C3 discovered by virtual screening of a fragment library. J Med Chem. 2012;55(17):7417–7424. PubMed PMC

Savić MP, Ajduković JJ, Plavša JJ, Bekić SS, Ćelić AS, Klisurić OR, Jakimov DS, Petri ET, Djurendić EA.. Evaluation of A-ring fused pyridine d-modified androstane derivatives for antiproliferative and aldo-keto reductase 1C3 inhibitory activity. Medchemcomm. 2018;9(6):969–981. PubMed PMC

Flanagan JU, Yosaatmadja Y, Teague RM, Chai MZL, Turnbull AP, Squire CJ.. Crystal structures of three classes of non-steroidal anti-inflammatory drugs in complex with aldo-keto reductase 1C3. PLoS One. 2012;7(8):e43965. PubMed PMC

Rebello RJ, Oing C, Knudsen KE, Loeb S, Johnson DC, Reiter RE, Gillessen S, Van der Kwast T, Bristow RG.. Prostate cancer. Nat Rev Dis Primers. 2021;7(1):9. PubMed

Dai C, Heemers H, Sharifi N.. Androgen signaling in prostate cancer. Cold Spring Harb Perspect Med. 2017;7(9):a030452. PubMed PMC

Ajduković JJ, Djurendić EA, Petri ET, Klisurić OR, Celić AS, Sakač MN, Jakimov DS, Gaši KMP.. 17(E)-picolinylidene androstane derivatives as potential inhibitors of prostate cancer cell growth: antiproliferative activity and molecular docking studies. Bioorg Med Chem. 2013;21(23):7257–7266. PubMed

Szabó N, Ajduković JJ, Djurendić EA, Sakač MN, Ignáth I, Gardi J, Mahmoud G, Klisurić OR, Jovanović-Šanta S, Penov Gaši KM, et al. Determination of 17α-hydroxylase-C17,20-lyase (P45017α) enzyme activities and their inhibition by selected steroidal picolyl and picolinylidene compounds. Acta Biol Hung. 2015;66(1):41–51. PubMed

Harshman LC, Taplin M-E.. Abiraterone acetate: targeting persistent androgen dependence in castration-resistant prostate cancer. Adv Ther. 2013;30(8):727–747. PubMed PMC

Swami U, McFarland TR, Nussenzveig R, Agarwal N.. Advanced prostate cancer: treatment advances and future directions. Trends Cancer. 2020;6(8):702–715. PubMed

Mostaghel EA, Marck BT, Plymate SR, Vessella RL, Balk S, Matsumoto AM, Nelson PS, Montgomery RB.. Resistance to CYP17A1 inhibition with abiraterone in castration-resistant prostate cancer: induction of steroidogenesis and androgen receptor splice variants. Clin Cancer Res. 2011;17(18):5913–5925. PubMed PMC

Liu C, Lou W, Zhu Y, Yang JC, Nadiminty N, Gaikwad NW, Evans CP, Gao AC.. Intracrine androgens and AKR1C3 activation confer resistance to enzalutamide in prostate cancer. Cancer Res. 2015;75(7):1413–1422. PubMed PMC

Kafka M, Mayr F, Temml V, Möller G, Adamski J, Höfer J, Schwaiger S, Heidegger I, Matuszczak B, Schuster D, et al. Dual inhibitory action of a novel AKR1C3 inhibitor on both full-length AR and the variant AR-V7 in enzalutamide resistant metastatic castration resistant prostate cancer. Cancers (Basel). 2020;12(8):2092. PubMed PMC

Armstrong CM, Gao AC.. Dysregulated androgen synthesis and anti-androgen resistance in advanced prostate cancer. Am J Clin Exp Urol. 2021;9(4):292–300. PubMed PMC

Liedtke AJ, Adeniji AO, Chen M, Byrns MC, Jin Y, Christianson DW, Marnett LJ, Penning TM.. 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. J Med Chem. 2013;56(6):2429–2446. PubMed PMC

Veitch ZW, Guo B, Hembruff SL, Bewick AJ, Heibein AD, Eng J, Cull S, Maclean DA, Parissenti AM.. Induction of 1C aldoketoreductases and other drug dose-dependent genes upon acquisition of anthracycline resistance. Pharmacogenet Genomics. 2009;19(6):477–488. PubMed

Djurendić E, Daljev J, Sakac M, Canadi J, Santa SJ, Andrić S, Klisurić O, Kojić V, Bogdanović G, Djurendić-Brenesel M, et al. Synthesis of some epoxy and/or N-oxy 17-picolyl and 17-picolinylidene-androst-5-ene derivatives and evaluation of their biological activity. Steroids. 2008;73(1):129–138. PubMed

Ajduković JJ, Jakimov DS, Rárová L, Strnad M, Dzichenka YU, Usanov S, Škorić DĐ, Jovanović-Šanta SS, Sakač MN.. Novel alkylaminoethyl derivatives of androstane 3-oximes as anticancer candidates: synthesis and evaluation of cytotoxic effects. RSC Adv. 2021;11(59):37449–37461. PubMed PMC

Marinović MA, Petri ET, Grbović LM, Vasiljević BR, Jovanović-Šanta SS, Bekić SS, Ćelić AS.. Investigation of the potential of bile acid methyl esters as inhibitors of aldo-keto reductase 1C2: insight from molecular docking, virtual screening, experimental assays and molecular dynamics. Mol Inform. 2022;41(10):e2100256. PubMed

Plavša JJ, Řezáčová P, Kugler M, Pachl P, Brynda J, Voburka Z, Ćelić A, Petri ET, Škerlová J.. In situ proteolysis of an N-terminal His tag with thrombin improves the diffraction quality of human aldo-keto reductase 1C3 crystals. Acta Crystallogr F Struct Biol Commun. 2018;74(Pt 5):300–306. PubMed PMC

Gorrec F. The MORPHEUS protein crystallization screen. J Appl Crystallogr. 2009;42(Pt 6):1035–1042. PubMed PMC

Mueller U, Darowski N, Fuchs MR, Förster R, Hellmig M, Paithankar KS, Pühringer S, Steffien M, Zocher G, Weiss MS, et al. Facilities for macromolecular crystallography at the Helmholtz-Zentrum Berlin. J Synchrotron Radiat. 2012;19(Pt 3):442–449. PubMed PMC

Gerlach M, Mueller U, Weiss MS.. The MX beamlines BL14. 1-3 at BESSY II. JLSRF. 2016;2:A47.

Kabsch W. XDS. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 2):125–132. PubMed PMC

McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ.. Phaser crystallographic software. J Appl Crystallogr. 2007;40(Pt 4):658–674. PubMed PMC

Qiu W, Zhou M, Mazumdar M, Azzi A, Ghanmi D, Luu-The V, Labrie F, Lin S-X.. Structure-based inhibitor design for an enzyme that binds different steroids: a potent inhibitor for human type 5 17beta-hydroxysteroid dehydrogenase. J Biol Chem. 2007;282(11):8368–8379. PubMed

Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A, et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67(Pt 4):235–242. PubMed PMC

Murshudov GN, Vagin AA, Dodson EJ.. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr. 1997;53(Pt 3):240–255. PubMed

Emsley P, Lohkamp B, Scott WG, Cowtan K.. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 4):486–501. PubMed PMC

Winn MD, Murshudov GN, Papiz MZ.. Macromolecular TLS refinement in REFMAC at moderate resolutions. In: Macromolecular crystallography, part D. Vol 374. Methods in Enzymology. Cambridge, MA: Academic Press; 2003: p. 300–321. PubMed

Pedretti A, Villa L, Vistoli G.. Atom-type description language: a universal language to recognize atom types implemented in the VEGA program. Theor Chem Acc. 2003;109(4):229–232.

Pedretti A, Mazzolari A, Gervasoni S, Fumagalli L, Vistoli G.. The VEGA suite of programs: an versatile platform for cheminformatics and drug design projects. Bioinformatics. 2021;37(8):1174–1175. PubMed

Trott O, Olson AJ.. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem. 2010;31(2):455–461. PubMed PMC

Hanwell MD, Curtis DE, Lonie DC, Vandermeersch T, Zurek E, Hutchison GR.. Avogadro: An advanced semantic chemical editor, visualization, and analysis platform. J Cheminform. 2012;4(1):17. PubMed PMC

Dallakyan S, Olson AJ.. Small-molecule library screening by docking with PyRx. In: Methods in molecular biology. Vol 1263. New York: Springer; 2015: p. 243–250. PubMed

DeLano WL. The case for open-source software in drug discovery. Drug Discov Today. 2005;10(3):213–217. PubMed

Muddana SS, Peterson BR.. Fluorescent cellular sensors of steroid receptor ligands. Chembiochem. 2003;4(9):848–855. PubMed

Gietz D, Jean AS, Woods RA, Schiestl RH.. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992;20(6):1425–1425. PubMed PMC

Brozic P, Smuc T, Gobec S, Rizner TL.. Phytoestrogens as inhibitors of the human progesterone metabolizing enzyme AKR1C1. Mol Cell Endocrinol. 2006;259(1–2):30–42. PubMed

Byrns MC, Steckelbroeck S, Penning TM.. An indomethacin analogue, N-(4-chlorobenzoyl)-melatonin, is a selective inhibitor of aldo-keto reductase 1C3 (type 2 3alpha-HSD, type 5 17beta-HSD, and prostaglandin F synthase), a potential target for the treatment of hormone dependent and hormone indep. Biochem Pharmacol. 2008;75(2):484–493. PubMed PMC

Adeniji AO, Chen M, Penning TM.. AKR1C3 as a target in castrate resistant prostate cancer. J Steroid Biochem Mol Biol. 2013;137:136–149. PubMed PMC

Bekić SS, Marinović MA, Petri ET, Sakač MN, Nikolić AR, Kojić VV, Ćelić AS.. Identification of D-seco modified steroid derivatives with affinity for estrogen receptor α and β isoforms using a non-transcriptional fluorescent cell assay in yeast. Steroids. 2018;130:22–30. PubMed

Vasiljević BR, Petri ET, Bekić SS, Ćelić AS, Grbović LM, Pavlović KJ.. Microwave-assisted green synthesis of bile acid derivatives and evaluation of glucocorticoid receptor binding. RSC Med Chem. 2021;12(2):278–287. PubMed PMC

Endo S, Matsunaga T, Kanamori A, Otsuji Y, Nagai H, Sundaram K, El-Kabbani O, Toyooka N, Ohta S, Hara A, et al. Selective inhibition of human type-5 17β-hydroxysteroid dehydrogenase (AKR1C3) by baccharin, a component of Brazilian propolis. J Nat Prod. 2012;75(4):716–721. PubMed

Laskowski RA, Swindells MB.. LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model. 2011;51(10):2778–2786. PubMed

Wallace AC, Laskowski RA, Thornton JM.. LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng. 1995;8(2):127–134. PubMed

Marinović MA, Bekić SS, Kugler M, Brynda J, Škerlová J, Škorić DĐ, Řezáčová P, Petri ET, Ćelić AS.. X-ray structure of human aldo-keto reductase 1C3 in complex with a bile acid fused tetrazole inhibitor: experimental validation, molecular docking and structural analysis. RSC Med Chem. 2023;14(2):341–355. PubMed PMC

Paggi JM, Pandit A, Dror RO.. The art and science of molecular docking. Annu Rev Biochem. 2024;93(1):389–410. PubMed

Saikia S, Bordoloi M.. Molecular docking: challenges, advances and its use in drug discovery perspective. Curr Drug Targets. 2019;20(5):501–521. PubMed

Lovering AL, Ride JP, Bunce CM, Desmond JC, Cummings SM, White SA.. Crystal structures of prostaglandin D2 11-ketoreductase (AKR1C3) in complex with the nonsteroidal anti-inflammatory drugs flufenamic acid and indomethacin. Cancer Res. 2004;64(5):1802–1810. PubMed

Jespers W, Åqvist J, Gutiérrez-de-Terán H.. Free energy calculations for protein-ligand binding prediction. Methods Mol Biol. 2021;2266:203–226. PubMed

Fu H, Chipot C, Shao X, Cai W.. Standard binding free-energy calculations: how far are we from automation? J Phys Chem B. 2023;127(49):10459–10468. PubMed

Gaillard P, Carrupt PA, Testa B, Boudon A.. Molecular lipophilicity potential, a tool in 3D QSAR: method and applications. J Comput Aided Mol Des. 1994;8(2):83–96. PubMed

Diederichs K, Karplus PA.. Improved R-factors for diffraction data analysis in macromolecular crystallography. Nat Struct Biol. 1997;4(4):269–275. PubMed

Karplus PA, Diederichs K.. Linking crystallographic model and data quality. Science. 2012;336(6084):1030–1033. PubMed PMC

The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr. 1994;50(Pt 5):760–763. PubMed

Brünger AT. Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. Nature. 1992;355(6359):472–475. PubMed

Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC.. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010;66(Pt 1):12–21. PubMed PMC

Chen M, Adeniji AO, Twenter BM, Winkler JD, Christianson DW, Penning TM.. Crystal structures of AKR1C3 containing an N-(aryl)amino-benzoate inhibitor and a bifunctional AKR1C3 inhibitor and androgen receptor antagonist. Therapeutic leads for castrate resistant prostate cancer. Bioorg Med Chem Lett. 2012;22(10):3492–3497. PubMed PMC