Yes / The aldo-keto reductase 1C3 isoform (AKR1C3) plays a vital role in the biosynthesis of androgens, making this enzyme an attractive target for castration-resistant prostate cancer therapy. Although AKR1C3 is a promising drug target, no AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid, a non-steroidal anti-inflammatory drug, is known to potently inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. To diminish off-target effects, we have applied a scaffold hopping strategy replacing the benzoic acid moiety of flufenamic acid with an acidic hydroxyazolecarbonylic scaffold. In particular, differently N-substituted hydroxylated triazoles were designed to simultaneously interact with both subpockets 1 and 2 in the active site of AKR1C3, larger for AKR1C3 than other AKR1Cs isoforms. Through computational design and iterative rounds of synthesis and biological evaluation, novel compounds are reported, sharing high selectivity (up to 230-fold) for AKR1C3 over 1C2 isoform and minimal COX1 and COX2 off-target inhibition. A docking study of compound 8, the most interesting compound of the series, suggested that its methoxybenzyl substitution has the ability to fit inside subpocket 2, being involved in π-π staking interaction with Trp227 (partial overlapping) and in a T-shape π-π staking with Trp86. This compound was also shown to diminish testosterone production in the AKR1C3-expressing 22RV1 prostate cancer cell line while synergistic effect was observed when 8 was administered in combination with abiraterone or enzalutamide. / University of Turin (Ricerca Locale grant 2014 and 2015) and Prostate Cancer UK grant S12-027

Yes / The aldo-keto reductase 1C3 (AKR1C3) enzyme is considered an attractive target in Castration Resistant Prostate Cancer (CRPC) because of its role in the biosynthesis of androgens. Flufenamic acid, a non-selective AKR1C3 inhibitor, has previously been subjected to bioisosteric modulation to give rise to a series of compounds with the hydroxytriazole core. In this work, the hit compound of the previous series has been modulated further, and new, more potent, and selective derivatives have been obtained. The poor solubility of the most active compound (cpd 5) has been improved by substituting the triazole core with an isoxazole heteronucleous, with similar enzymatic activity being retained. Potent AKR1C3 inhibition is translated into antiproliferative effects against the 22RV1 CRPC cellular model, and the in-silico design, synthesis and biological activity of new compounds is described herein. Compounds have also been assayed in combination with two approved antitumor drugs, abiraterone and enzalutamide. / This research was financially supported by the University of Turin (Ricerca Locale grants BOSD_RILO_20_01, LOLM_RILO_21_01, PIPA_RILO_20_01 and PIPA_RILO_21_01), Fondazione Cassa di Risparmio di Torino (Grant BOSD_CRT_17_2) and TUBITAK (The Scientific and Technological Research Council of Turkey-2219 program).

Yes / The aldo-keto reductase 1C3 (AKR1C3) isoform plays a vital role in the biosynthesis of androgens and is considered an attractive target in prostate cancer (PCa). No AKR1C3-targeted agent has to date been approved for clinical use. Flufenamic acid and indomethacine are non-steroidal anti-inflammatory drugs known to inhibit AKR1C3 in a non-selective manner as COX off-target effects are also observed. Recently, we employed a scaffold hopping approach to design a new class of potent and selective AKR1C3 inhibitors based on a N-substituted hydroxylated triazole pharmacophore. Following a similar strategy, we designed a new series focused around an acidic hydroxybenzoisoxazole moiety, which was rationalised to mimic the benzoic acid role in the flufenamic scaffold. Through iterative rounds of drug design, synthesis and biological evaluation, several compounds were discovered to target AKR1C3 in a selective manner. The most promising compound of series (6) was found to be highly selective (up to 450-fold) for AKR1C3 over the 1C2 isoform with minimal COX1 and COX2 off-target effects. Other inhibitors were obtained modulating the best example of hydroxylated triazoles we previously presented. In cell-based assays, the most promising compounds of both series reduced the cell proliferation, prostate specific antigen (PSA) and testosterone production in AKR1C3-expressing 22RV1 prostate cancer cells and showed synergistic effect when assayed in combination with abiraterone and enzalutamide. Structure determination of AKR1C3 co-crystallized with one representative compound from each of the two series clearly identified both compounds in the androstenedione binding site, hence supporting the biochemical data. / University of Turin (Ricerca Locale grant 2015-2017) and Prostate Cancer UK grant S12-027.