Selective Glucocorticoid Receptor Properties of GSK866 Analogs with Cysteine Reactive Warheads

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.

Variation in glucocorticoid sensitivity and the relation with obesity

“…In the past, novel synthetic derivatives of GCs have been developed that distinguish between GR signaling modes. These compounds have been termed “dissociated ligands,” selective GR modulators, or more recently selective GR agonists (SEGRAs) 117–121 . These compounds are currently being investigated for improvement in therapeutic efficacy, that is, anti‐inflammatory effects coupled with minimal adverse side effects.…”

In Vitro Anti-Inflammatory, Anti-Oxidant, and Cytotoxic Activities of Four Curcuma Species and the Isolation of Compounds from Curcuma aromatica Rhizome

“…Cell viability and cytotoxicity of crude extracts and pure compounds were assessed by MTT assay and the CytoTox-ONE™ Homogeneous Membrane Integrity Assay using HaCaT keratinocyte cells, respectively. The MTT colorimetric assay estimates the number of viable cells based on the ability of mitochondrial enzymes to reduce the tetrazolium dye MTT to a purple colored formazan [37], whereas the CytoTox-ONE™ assay is a fluorometric-based method to detect loss of membrane integrity of dying cells. MTT results showed that exposure to 200 µg/mL of C. aeruginosa, C. comosa, C. aromatica, or C. longa extract inhibited the growth of cells, with relative percentages of cell viability being 12.1 ± 2.9, 14.4 ± 4.1, 28.6 ± 4.1, and 46.9 ± 8.6, respectively (Figure 3a).…”

Standardized Echinacea purpurea extract primes an IFN specific innate immune monocyte response via JAK1-STAT1 dependent gene expression and epigenetic control of endogenous retroviral sequences

“…In contrast to pharmacological drugs (for example glucocorticoids (GC)) which can trigger drastic expression changes of GC-responsive genes (typically, log 2 fold > 1), many bioactive phytochemicals rather induce moderate transcriptional changes (typically log 2 fold > 0,4) of multiple genes converging on the same pathway [44][45][46]. Genes differentially expressed (DEG) by Echinaforce® treatment were enriched for IPA canonical pathways related to innate immune responses including interferon signaling, interferon regulatory factor (IRF) activation and the role of pattern recognition receptors, among others ( Figure 1B-C and Supplementary table 2).…”