Elsevier

Chemico-Biological Interactions

Volume 276, 1 October 2017, Pages 174-181
Chemico-Biological Interactions

Structural basis for the inhibition of AKR1B10 by the C3 brominated TTNPB derivative UVI2008

https://doi.org/10.1016/j.cbi.2017.01.026Get rights and content

Highlights

  • The X-ray structure of human AKR1B10 with RARβ/γ agonist UVI2008 has been solved.

  • C3 halogen addition to pan-RAR agonist TTNPB enables RARβ/γ and AKR1B10 selectivity.

  • Selectivity of UVI2008 for AKR1B10 is facilitated by Trp112 native conformation.

Abstract

UVI2008, a retinoic acid receptor (RAR) β/γ agonist originated from C3 bromine addition to the parent RAR pan-agonist 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoic acid (TTNPB), is also a selective inhibitor of aldo-keto reductase family member 1B10 (AKR1B10). Thus, it might become a lead drug for the design of compounds targeting both activities, as an AKR1B10 inhibitor and RAR agonist, which could constitute a novel therapeutic approach against cancer and skin-related diseases. Herein, the X-ray structure of the methylated Lys125Arg/Val301Leu AKR1B10 (i.e. AKME2MU) holoenzyme in complex with UVI2008 was determined at 1.5 Å resolution, providing an explanation for UVI2008 selectivity against AKR1B10 (IC50 = 6.1 μM) over the closely related aldose reductase (AR, IC50 = 70 μM). The carboxylic acid group of UVI2008 is located in the anion-binding pocket, at hydrogen-bond distance of catalytically important residues Tyr49 and His111. The inhibitor bromine atom can only fit in the wider active site of AKR1B10, mainly because of the native Trp112 side-chain orientation, not possible in AR. In AKR1B10, Trp112 native conformation, and thus UVI2008 binding, is facilitated through interaction with Gln114. IC50 analysis of the corresponding Thr113Gln mutant in AR confirmed this hypothesis. The elucidation of the binding mode of UVI2008 to AKR1B10, along with the previous studies on the retinoid specificity of AKR1B10 and on the stilbene retinoid scaffold conforming UVI2008, could indeed be used to foster the drug design of bifunctional antiproliferative compounds.

Introduction

The NADPH-dependent aldo-keto reductase (AKR) superfamily consists of approximately 150 phase I drug-metabolizing enzymes that share high sequence similarities. In humans, the subfamily 1B includes the closely related enzymes aldose reductase (AR or AKR1B1), AKR1B10 and AKR1B15, the first two being of biomedical interest. AR has been deeply studied regarding its implication in the polyol pathway and recently also in relation to inflammatory pathologies, including colon cancer [1]. In contrast to the ubiquitous AR, AKR1B10 has a restricted expression in healthy tissues (mostly in colon and small intestine), and is a promising antineoplastic target, as it is overexpressed in several cancer types, including different hepatic, lung, breast and pancreatic tumors [2], [3], [4], [5], [6]. AKR1B10 might impact the carcinogenesis process via the reduction of a wide range of substrates, involved in different pathways: Retinoic acid (RA) signaling control [6], detoxification of reactive lipid aldehydes [5], anticancer drugs [6], lipid synthesis promotion [5] and regulation of protein isoprenylation [7]. Many AR inhibitors (ARIs) have been developed, but most of them have been withdrawn in the clinical stage because of their side effects and toxicity. Those adverse effects might have been caused in several cases by cross-inhibition with close enzymes, mostly AKR1B10 and aldehyde reductase (AKR1A1) [8], [9]. In the recent years, the focus has been switched from the latter enzyme to AKR1B10, given its higher sequence identity with AR [8], [9]. On the other hand, the emergency of AKR1B10 as a drug target has fostered the research for selective inhibitors not cross-reacting with AR. In this regard, about a dozen of AKR1B10 structures have been obtained in the last couple of years [8], [9], [10], [11]. UVI2008 is a RAR β/γ agonist originated from a collection of arotinoids with a stilbenoid structure based on the pan-agonist TTNPB, only differing in a C3 bromine atom addition [12], [13]. Moreover, we previously showed that UVI2008 had the highest potency and selectivity for AKR1B10 among a series of RAR/RXR synthetic retinoids. However, at that time, we could only provide a docking model to support the inhibitory properties of UVI2008, using the V301L AKR1B10 complex with the inhibitor fidarestat as a receptor molecule (PDB ID 4GAB) [14].

Here, we report the X-ray structure of an AKR1B10-NADP+-UVI2008 ternary complex at 1.5 Å resolution, by means of the methylated Lys125Arg/Val301Leu AKR1B10 mutant (i.e. AKME2MU) system previously described [9], showing unequivocally how this synthetic retinoid interacts with AKR1B10. We also show that the parent compound TTNPB is not inhibiting AKR1B10 and that the addition of the C3 halogen atom (either Br or Cl) provides an AKR1B10 specific interaction. Interestingly, this same feature has been recognized as a structural selectivity determinant contributing to RARβ-selectivity [15]. Overall, this three-dimensional structure serves as a framework for the development of more potent compounds bearing two antiproliferative activities, RAR agonism and AKR1B10 inhibition.

Section snippets

Chemicals

UVI2007 and UVI2008 were synthesized as previously described [13]. TTNPB and other chemicals were obtained from Sigma–Aldrich.

Site-directed mutagenesis

All cDNAs (wild-type and mutants) coding for the proteins used in this work were generated as reported in Refs. [9], [16], [17], except for the AR Thr113Gln mutant. The latter was obtained using the wild-type AR cDNA cloned into pET-16b as a template and following the manufacturer's instructions for the Quickchange™ Site-Directed Mutagenesis Kit method (Stratagene. Two

Structure-Activity Relationships (SAR) in the AKME2MU-NADP+-UVI2008 complex

In a previous work, we screened a set of different synthetic retinoids versus AR and AKR1B10 and found out that, among them, the C3 halogenated TTNPB derivatives UVI2007 and UVI2008 were the most active and selective AKR1B10 inhibitors [14]. As both were already described RAR agonists, extensive SAR have already been performed in relation to that biological activity [13], [15], [25]. In order to deepen on the AKR1B10 SAR of the former, first we assayed the parent compound TTNPB against AR and

Conclusions and perspectives

We previously explored the inhibitory properties of some synthetic retinoids against AR and AKR1B10 [14] and here we present a high resolution structure (1.5 Å) of the hit UVI2008 in complex with the AKME2MU holoenzyme. The binding pocket displays hydrophobic interactions, H-bonds to its carboxylic acid and, most remarkably, an aromatic C⋯Br interaction. This C⋯halogen interaction provides affinity and selectivity for AKR1B10, as AR Trp111 locked conformation would clash against the Br atom.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

We thank the IGBMC Structural Genomics Platform staff (in particular Dr. Alastair McEwen and Pierre Poussin-Courmontagne). The crystallographic experiments were performed on the X06DA beamline at the Swiss Light Source synchrotron, Paul Scherrer Institut, Villigen, Switzerland. We thank in particular Dr. Vincent Olieric for his help in data collection. This work has been funded by the CNRS, the INSERM, the Université de Strasbourg, the Région Alsace, the Hôpital Civil de Strasbourg, Instruct

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