Screening, synthesis, crystal structure, and molecular basis of 6-amino-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitriles as novel AKR1C3 inhibitors

Highlights

• Substituted pyranopyrazoles as novel AKR1C3 inhibitors.

• Crystal structure revealed a unique binding pattern.

• Pyranopyrazoles change conformations to suit AKR1C3 and AKR1C1.

• Meclofenamic acid reshapes itself to fit AKR1C3 and AKR1C1.

• Conformational changes of ligand should be considered in design of AKR1C3 inhibitor.

Abstract

AKR1C3 is a promising therapeutic target for castration-resistant prostate cancer. Herein, an evaluation of in-house library discovered substituted pyranopyrazole as a novel scaffold for AKR1C3 inhibitors. Preliminary SAR exploration identified its derivative 19d as the most promising compound with an IC₅₀ of 0.160 μM among the 23 synthesized molecules. Crystal structure studies revealed that the binding mode of the pyranopyrazole scaffold is different from the current inhibitors. Hydroxyl, methoxy and nitro group at the C4-phenyl substituent together anchor the inhibitor to the oxyanion site, while the core of the scaffold dramatically enlarges but partially occupies the SP pockets with abundant hydrogen bond interactions. Strikingly, the inhibitor undergoes a conformational change to fit AKR1C3 and its homologous protein AKR1C1. Our results suggested that conformational changes of the receptor and the inhibitor should both be considered during the rational design of selective AKR1C3 inhibitors. Detailed binding features obtained from molecular dynamics simulations helped to finally elucidate the molecular basis of 6-amino-4-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitriles as AKR1C3 inhibitors, which would facilitate the future rational inhibitor design and structural optimization.

Introduction

AKR1C3, one member of aldo-keto reductase (AKR) superfamily, has also been designated as 17β-hydroxysteroid dehydrogenase type 5 (17β-HSD5) due to its 17- ketosteroid reductase activity. The substrates of AKR1C3 include the precursors in all pathways to the potent androgens testosterone (T) and 5α-dihydrotestosterone (DHT) in the prostate, such as dehydroepiandrosterone, △4-androstene-3,17-dione, androstan-3,17-dione and androsterone.¹ Recent studies have established that the reactivation of the androgen axis contributes to castrate resistant prostate cancer (CRPC), and led to FDA approval of abiraterone (steroidogenic enzyme CYP17A1 inhibitor) and enzalutamide (new androgen receptor antagonist) for the treatment of patients with CRPC.2, 3, 4 The involvement of AKR1C3 in downstream androgen biosynthesis suggests that AKR1C3 plays a critical role in CRPC. Recent investigations have indicated that AKR1C3 is overexpressed in cell lines deprived of androgens, in prostate tumor xenografts in castrate mice, and in CRPC patients. Knockdown or inhibition of AKR1C3 resulted in suppression of tumor cell growth within a castrate environment and a decrease in intra-tumoral testosterone production in castrated nude mice induced by androstenedione.5, 6, 7, 8, 9, 10, 11, 12, 13 Yepuru et al. have identified that AKR1C3 is also a unique AR-selective coactivator to promote CRPC growth and AKR1C3-selective competitive inhibitors inhibit this coactivator function.¹⁴ Moreover, AKR1C3 is implicated in resistance to abiraterone and enzalutamide therapies and enzalutamide resistance in vitro and in vivo can be overcome with AKR1C3 competitive inhibitors.7, 8 Collectively, these data suggest that AKR1C3 inhibition may have distinct advantages over the current therapeutics for the treatment of CRPC.

Considerable efforts have been invested into the discovery of compounds inhibiting AKR1C3 first for research purposes, but later also for therapeutic applications.1, 15, 16, 17, 18, 19, 20, 21, 22, 23 Penning et al. have extensively studied the inhibitory activities of nonsteroidal anti-inflammatory drugs (NSAIDs) and discovered N-phenylanthranilate and indomethacin analogs as AKR1C3 inhibitors with high selectivity versus COX-1,2.17, 18, 19, 20, 21, 22, 23 The high-throughput screening conducted by Jamieson et al. identified 3-(3,4-dihydroisoquinolin-2(1H)-ylsulfonyl)benzoic acid as a new carboxylate inhibitor of AKR1C3.²⁴ Several non-carboxylate inhibitors including isoquinolines, morpholylureas, pyrrolidine, and indole derivative have also been reported.14, 25, 26, 27 At the same time, there have been significant efforts to explore the interaction of existing inhibitors with the enzyme by single-crystal techniques. Studies conducted by us and other groups have reported 43 crystal structures of AKR1C3 in complex with different inhibitors.28, 29, 30, 31, 32, 33, 34, 35 The ligand binding pocket of AKR1C3 can be divided into oxyanion site, steroid channel, and three sub-pockets, SP1, SP2, and SP3.¹ Most of inhibitors are anchored to the oxyanion site by hydrogen bonding with Tyr55 and His117 and occupy the SP1 pocket, where usually result in conformational changes of Trp227, Phe306 and Phe311.¹⁵ Nevertheless, no AKR1C3 inhibitors are currently in clinical use for the treatment of CRPC, and superior AKR1C3 inhibitors are still in need.

To seek novel AKR1C3 inhibitors, in the current study, we conducted a screening on an in-house compound library consisting of 298 small molecules by using enzymatic assay. Nine inhibitors were identified with new scaffolds compared to the current AKR1C3 inhibitors. Preliminary SAR studies were performed on the best compound and identified 19d as the most potent AKR1C3 inhibitor with an IC₅₀ of 0.160 μM among the synthesized 23 6-Amino-4-phenyl-1,4-dihydropyrano-[2,3-c]pyrazole-5-carbonitriles. Crystal structure, molecular docking, together with binding free-energies and per-residue contributions studies revealed a new insight into the molecular basis for selective inhibition of AKR1C3, which would be helpful in future design and optimization of new AKR1C3 inhibitors.