Chirumamilla Chandra S, Palagani Ajay, Kamaraj Balu, Declerck Ken, Verbeek Marinus W C, Oksana Ryabtsova, De Bosscher Karolien, Bougarne Nadia, Ruttens Bart, Gevaert Kris, Houtman René, De Vos Winnok H, Joossens Jurgen, Van Der Veken Pieter, Augustyns Koen, Van Ostade Xaveer, Bogaerts Annemie, De Winter Hans, Vanden Berghe Wim
Laboratory of Protein Chemistry, Proteomics and Epigenetic Signalling, Department of Biomedical Sciences, University of Antwerp, Antwerp, Belgium.
Research Group PLASMANT, Department of Chemistry, University of Antwerp, Antwerp, Belgium.
Front Immunol. 2017 Nov 1;8:1324. doi: 10.3389/fimmu.2017.01324. eCollection 2017.
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.
合成糖皮质激素（GC）是治疗急慢性炎症性疾病的主要药物。由于长期使用会带来较高的不良反应，自九十年代以来，GC药理学一直专注于更具选择性的GC配体结合策略，分为选择性糖皮质激素受体（GR）激动剂（SEGRA）或选择性糖皮质激素受体调节剂（SEGRM）。在本研究中，开发了带有亲电共价结合弹头的GSK866类似物，其具有潜在的SEGRA特性，可改善其在持久局部皮肤疾病应用中的临床安全性。由于共价结合药物的解离速率与非共价药物相比可忽略不计，其治疗效果可以延长，通常，达到相同治疗效果水平所需的药物剂量较小，从而有可能减少全身副作用。在各种生化和细胞试验中，对与半胱氨酸反应性弹头偶联的SEGRA GSK866的不同类似物进行了GR效力和选择性表征。GR和NFκB依赖性报告基因研究表明，两种非甾体GSK866类似物UAMC-1217和UAMC-1218具有良好的抗炎特性，同时GR反式激活作用降低，而UAMC-1158和UAMC-1159化合物未能调节细胞GR活性。GR免疫定位和S211磷酸化GR Western分析进一步支持了这些结果，表明在使用GSK866、UAMC-1217或UAMC-1218处理后，GR有明显的磷酸化激活和核转位，但在UAMC-1158或UAMC-1159处理时未出现这种情况。此外，对与UAMC-1217或UAMC-1218结合的重组GR配体结合域（LBD）的胰蛋白酶肽段进行质谱分析，证实了共价半胱氨酸依赖性GR结合。最后，分子动力学模拟以及与GSK866或其共价结合类似物UAMC-1217或UAMC-1218结合的GR-LBD的糖皮质激素受体配体结合域（GR-LBD）共调节因子相互作用谱分析揭示了可能是其SEGRA特性基础的细微构象差异。总之，GSK866类似物UAMC-1217和UAMC-1218有望成为一类新型的共价结合SEGRA配体，用于治疗局部炎症性皮肤病。
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