Allosteric Modulation of Glucocorticoid Receptor Activity through Key Interactions within the Ligand Binding Pocket

The glucocorticoid receptor (GR) is a transcription factor that influences thousands of genes throughout the body. Activated by steroidal hormones known as glucocorticoids (GCs), the GR-GC complex suppresses inflammation and treats autoimmune diseases and cancer. However, long-term GC use is associated with deleterious effects such as hypertension, osteoporosis, and diabetes. As a result, there is an urgent need for the development of a GC capable of selectively modulating the GR. Unfortunately, much is still unknown regarding how GR conformation is influenced upon ligand binding. To this end, a series of GC analogues were designed, synthesized, and biologically evaluated through testing with both luciferase and gene expression assays. Their resulting activity was explained by docking ligands of interest and probing their impact on GR conformation using molecular dynamic simulations and free-energy calculations. In the first study, DX1, an analogue of dexamethasone with a C21 substituted mercaptobenzothiazole moiety, maintained 90% of the anti-inflammatory potential of its parent molecule, while simultaneously exhibiting a reduced toxicity profile. Additionally, DX4 and PN4 displayed comparable mRNA expression in the attomolar region to dexamethasone at nanomolar concentrations. Molecular dynamic (MD) simulations revealed that this potency results from the formation of an unoccupied channel near the A-ring of each compound. The flexibility of the GR was further probed in the second study through the systematic derivatization of hydrocortisone. Aryl pyrazole addition to the A-ring alone produced compounds 6 and 7 which both exhibited expression of Sgk1 mRNA abundance by nearly 20-fold over control each. Furthermore, these compounds were not selective for any other nuclear receptor than the GR, making them unlikely to produce as many side effects. Finally, a GR dimer, comprising DNA binding domains, hinge regions, and ligand binding domains, was constructed in silico and docked with a variety of both GR agonists and antagonists. Subsequent MD simulations revealed, for the first time, that agonists consistently produce unique interdomain contacts, while antagonists allosterically drive the dimer complex apart. Together, this research offers enhanced alternatives to current GC therapies and establishes a platform for more deeply understanding how different ligands influence GR activity.