Selective Glucocorticoid Receptor Properties of GSK866 Analogs with Cysteine Reactive Warheads.

Chandra S Chirumamilla,Ken Declerck,Marinus W C Verbeek,Koen Augustyns,Pieter Van Der Veken,Nadia Bougarne,Jurgen Joossens,Hans De Winter,Karolien De Bosscher,Winnok H De Vos,Balu Kamaraj,Wim Vanden Berghe,Kris Gevaert,Annemie Bogaerts,Ryabtsova Oksana,Ajay Palagani,Xaveer Van Ostade,Bart Ruttens,René Houtman

doi:10.3389/fimmu.2017.01324

Abstract

Synthetic glucocorticoids (GC) are the mainstay therapy for treatment of acute and chronic inflammatory disorders. Due to the high adverse effects associated with long-term use, GC pharmacology has focused since the nineties on more selective GC ligand-binding strategies, classified as selective glucocorticoid receptor (GR) agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). In the current study, GSK866 analogs with electrophilic covalent-binding warheads were developed with potential SEGRA properties to improve their clinical safety profile for long-lasting topical skin disease applications. Since the off-rate of a covalently binding drug is negligible compared to that of a non-covalent drug, its therapeutic effects can be prolonged and typically, smaller doses of the drug are necessary to reach the same level of therapeutic efficacy, thereby potentially reducing systemic side effects. Different analogs of SEGRA GSK866 coupled to cysteine reactive warheads were characterized for GR potency and selectivity in various biochemical and cellular assays. GR- and NFκB-dependent reporter gene studies show favorable anti-inflammatory properties with reduced GR transactivation of two non-steroidal GSK866 analogs UAMC-1217 and UAMC-1218, whereas UAMC-1158 and UAMC-1159 compounds failed to modulate cellular GR activity. These results were further supported by GR immuno-localization and S211 phospho-GR western analysis, illustrating significant GR phosphoactivation and nuclear translocation upon treatment of GSK866, UAMC-1217, or UAMC-1218, but not in case of UAMC-1158 or UAMC-1159. Furthermore, mass spectrometry analysis of tryptic peptides of recombinant GR ligand-binding domain (LBD) bound to UAMC-1217 or UAMC-1218 confirmed covalent cysteine-dependent GR binding. Finally, molecular dynamics simulations, as well as glucocorticoid receptor ligand-binding domain (GR-LBD) coregulator interaction profiling of the GR-LBD bound to GSK866 or its covalently binding analogs UAMC-1217 or UAMC-1218 revealed subtle conformational differences that might underlie their SEGRA properties. Altogether, GSK866 analogs UAMC-1217 and UAMC-1218 hold promise as a novel class of covalent-binding SEGRA ligands for the treatment of topical inflammatory skin disorders.

Highlights

Synthetic glucocorticoids (GCs) such as prednisolone, dexamethasone, or fluticasone esters remain the frontline treatment for inflammatory disorders, autoimmune diseases, and/or hematological malignancies [1]
The crystal structure of the ligand-binding domain of the glucocorticoid hormone receptor (GR) has been solved in complex with selective glucocorticoid receptor agonists (SEGRAs) compound GSK866 (Figure 1), and this structure shows the location of a glucocorticoid receptor ligand-binding domain (GR-LBD) cysteine residue (C643) in close proximity to the dichlorophenyl group of SEGRA compound GSK866 (Figure 2A)
Based on our semi-flexible docking models, we found that spatial occupation of GSK866, and analogs University of Antwerp Medicinal Chemistry (UAMC)-1217 or UAMC-1218 in the GR-LBD differ from the binding mode of Dex in the GR-LBD (Figures S3A–E in Supplementary Material)

Summary

Introduction

Synthetic glucocorticoids (GCs) such as prednisolone, dexamethasone, or fluticasone esters remain the frontline treatment for (chronic) inflammatory disorders, autoimmune diseases, and/or hematological malignancies [1]. Binding of GCs to the GR activates and translocates to the nucleus by causing a conformational change in the GR followed by a dissociation of the bound HSPs. The activated GR can regulate gene activation by GR dimerization (transactivation) and binding to glucocorticoid response elements (GREs) in the genome, or regulate gene repression by GR monomers (transrepression) which interfere with the activity of pro-inflammatory transcription factors, such as NFκB and AP1 [3, 4]. New GC pharmacological approaches are being developed that attempt to amplify the therapeutic beneficial anti-inflammatory (transrepression) actions and to minimize adverse metabolic (transactivation) activities. These compounds are classified as selective glucocorticoid receptor agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs) [7, 13, 14]. In vivo efficacy for SEGRA/SEGRM compounds with reduced side effects has already been demonstrated for treatment of acute infections, such as rheumatoid arthritis, asthma, and colitis, in the clinic [20,21,22]

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