Abstract
The evolution of glucocorticoid drugs was driven by the demand of lowering the unwanted side effects, while keeping the beneficial anti-inflammatory effects. Potency is an important aspect of this evolution as many undesirable side effects are associated with use of high-dose glucocorticoids. The side effects can be minimized by highly potent glucocorticoids that achieve the same treatment effects at lower doses. This demand propelled the continuous development of synthetic glucocorticoids with increased potencies, but the structural basis of their potencies is poorly understood. To determine the mechanisms underlying potency, we solved the X-ray structures of the glucocorticoid receptor (GR) ligand-binding domain (LBD) bound to its endogenous ligand, cortisol, which has relatively low potency, and a highly potent synthetic glucocorticoid, mometasone furoate (MF). The cortisol-bound GR LBD revealed that the flexibility of the C1-C2 single bond in the steroid A ring is primarily responsible for the low affinity of cortisol to GR. In contrast, we demonstrate that the very high potency of MF is achieved by its C-17α furoate group completely filling the ligand-binding pocket, thus providing additional anchor contacts for high-affinity binding. A single amino acid in the ligand-binding pocket, Q642, plays a discriminating role in ligand potency between MF and cortisol. Structure-based design led to synthesis of several novel glucocorticoids with much improved potency and efficacy. Together, these results reveal key structural mechanisms of glucocorticoid potency and provide a rational basis for developing novel highly potent glucocorticoids.
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Acknowledgements
We thank staff members of the Life Science Collaborative Access Team of the Advanced Photon Source (APS) for assistance with data collection at the beam lines of sector 21, which is in part funded by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (085P1000817). Use of APS was supported by the Office of Science of the US Department of Energy, under contract No. DE-AC02-06CH11357. This study was supported by NIDDK/NIH fund (DK066202 and DK071662), American Asthma Foundation fund (2010), Amway (China), the National Natural Science Foundation of China (91217311), the Chinese Postdoctoral Science Foundation (2012M511158 and 2013T60477), and the Jay and Betty Van Andel Foundation.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Figure S1
Protein sequence alignment of the LBDs of GR, PR, MR, and AR (PDF 462 kb)
Supplementary information, Figure S2
Protein expression and purification of selected GR LBD mutant proteins (PDF 123 kb)
Supplementary information, Figure S3
Protein crystals and diffraction images of cortisol- and MF- bound GR LBD (PDF 302 kb)
Supplementary information, Figure S4
The transcriptional activities of GR AYVTI mutant proteins (PDF 96 kb)
Supplementary information, Figure S5
Detailed structural comparisons of the steroid C-9α group and C-16 group of Dex and cortisol in the ligand binding pocket of the GR LBD (PDF 300 kb)
Supplementary information, Figure S6
The F623 and M639 mutations cannot separate the transcriptional activities of MF- and DEX-bound GR (PDF 213 kb)
Supplementary information, Figure S7
In vitro binding assay of GR Q642A mutant protein (PDF 309 kb)
Supplementary information, Figure S8
Transrepression properties of compounds with a C-17α furoate group (PDF 344 kb)
Supplementary information, Table S1
Statistics of data sets and structure refinement (PDF 188 kb)
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He, Y., Yi, W., Suino-Powell, K. et al. Structures and mechanism for the design of highly potent glucocorticoids. Cell Res 24, 713–726 (2014). https://doi.org/10.1038/cr.2014.52
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DOI: https://doi.org/10.1038/cr.2014.52
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