Domain Of Human Glucocorticoid Receptor Research Articles

Drug repositioning to propose alternative modulators for glucocorticoid receptor through structure-based virtual screening

Drug repositioning has recently become one of the widely used drug design approaches in proposing alternative compounds with potentially fewer side effects. In this study, structure-based pharmacophore modelling and docking was used to screen existing drug molecules to bring forward potential modulators for ligand-binding domain of human glucocorticoid receptor (hGR). There exist several drug molecules targeting hGR, yet their apparent side effects still persist. Our goal was to disclose new compounds via screening existing drug compounds to bring forward fast and explicit solutions. The so-called shared pharmacophore model was created using the most persistent pharmacophore features shared by several crystal structures of the receptor. The shared model was first used to screen a small database of 75 agonists and 300 antagonists/decoys, and exhibited a successful outcome in its ability to distinguish agonists from antagonists/decoys. Then, it was used to screen a database of over 5000 molecules composed of FDA-approved, worldwide used and investigational drug compounds. A total of 110 compounds satisfying the pharmacophore requirements were subjected to different docking experiments for further assessment of their binding ability. In the final hit list of 54 compounds which fulfilled all scoring criteria, 19 of them were nonsteroidal and when further investigated, each presented a unique scaffold with little structural resemblance to any known nonsteroidal GR modulators. Independent 100 ns long MD simulations conducted on three selected drug candidates in complex with hGR displayed stable conformations incorporating several hydrogen bonds common to all three compounds and the reference molecule dexamethasone. Communicated by Ramaswamy H. Sarma

An NMR study on the intrinsically disordered core transactivation domain of human glucocorticoid receptor.

A large number of transcriptional activation domains (TADs) are intrinsically unstructured, meaning they are devoid of a three-dimensional structure. The fact that these TADs are transcriptionally active without forming a 3-D structure raises the question of what features in these domains enable them to function. One of two TADs in human glucocorticoid receptor (hGR) is located at its N-terminus and is responsible for ~70% of the transcriptional activity of hGR. This 58-residue intrinsically-disordered TAD, named tau1c in an earlier study, was shown to form three helices under trifluoroethanol, which might be important for its activity. We carried out heteronuclear multi-dimensional NMR experiments on hGR tau1c in a more physiological aqueous buffer solution and found that it forms three helices that are ~30% pre-populated. Since pre-populated helices in several TADs were shown to be key elements for transcriptional activity, the three pre-formed helices in hGR tau1c delineated in this study should be critical determinants of the transcriptional activity of hGR. The presence of pre-structured helices in hGR tau1c strongly suggests that the existence of pre-structured motifs in target-unbound TADs is a very broad phenomenon.

Investigating Allosteric Coupling Mechanism in Constitutively Active Human Glucocorticoid Receptor Two Domain Construct

Glucocorticoid receptor (GR) is a hormone dependent nuclear transcription factor. It plays key roles in organ development, metabolite homeostasis, and stress and inflammatory response. GR is composed of three major domains, the intrinsically disordered (ID) N-terminal domain (NTD), the DNA binding domain (DBD) and the ligand binding domain (LBD). Several human GR translational isoforms differing only in the lengths of their ID NTDs with finely tunable transcription activity have been identified.In my project, through thermodynamic analysis of the ID NTD constructs of different translational isoforms, we found that full length GR NTD can be viewed as having at least two thermodynamically coupled regions, a functional (F) region, indispensable for GR transcriptional activity, and a regulatory (R) region, whose length serves to regulate the stability of NTD, and thus the activity of GR. F region and R region are negatively coupled to each other [1].Truncation of LBD from the full length receptor removes the hormone dependence of GR transcription activity, and generates the constitutively active GR NTD-DBD two domain constructs. From investigating the NTD-DBD two domain constructs of different translational isoforms by in vitro biophysics assays and in vivo luciferase activity assay, we are on the way figuring out the coupling mechanism between the DBD and ID NTD.Allosteric coupling mechanisms derived from our experimental data makes it possible to build the Ensemble Allosteric Coupling Model (EAM) to explain the finely tunable activity of different GR translational isoforms, which cannot be accounted for by the binding energetics between the different isoforms and the GR response element.[1] Jing Li, et al. Thermodynamic dissection of the intrinsically disordered N-terminal domain of human glucocorticoid receptor. J Biol Chem. 2012.

Cysteines 638 and 665 in the hormone binding domain of human glucocorticoid receptor define the specificity to glucocorticoids.

To understand the function of cysteines, we have substituted cysteines 638, 643, and 665 by serine in the hormone-binding domain (HBD) of the human glucocorticoid receptor (hGR). In hormone-binding assays using [3H]dexamethasone, hGR C643S and hGR C665S exhibited wild type receptor Kd of 2.5 nM and hGR C665SM666L displayed a Kd of 3.7 nM, while hGR C638S exhibited a Kd of 162 pM, a 15-fold higher affinity. The affinity of hGR C638S for RU486 was 10-fold higher, and the mutants C643S and C665S bound RU486 with a 10-fold lower affinity when compared to wild type GR. While C665S bound aldosterone with very high relative affinity, the double mutant C665SM666L failed to bind aldosterone. The expression of wild type, mutant, and truncated hGRs in vitro showed an identical level of expression of the cloned receptors. Similar levels of expression of the receptors were observed in transfected cells, both by immunoprecipitation and by Western blotting. Transcription activation of the chimeric reporter gene mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) with hGR C638S was 4-fold higher than the level observed with wild type hGR in the presence of dexamethasone. In the presence of RU486, hGR C638S induced MMTV-CAT 25-fold compared to the highest levels observed with wild type hGR and RU486. Even though the hGR C665S stimulated transcription with aldosterone, hGR C665SM666L did not. DNA-receptor interaction analyses by gel mobility shift assay demonstrated that the increased transactivation potential of hGR C638S was due to its intense interaction with DNA. These findings suggest that C638 and C665 are involved in maintaining specificity to glucocorticoids.

Hormone binding domain of human glucocorticoid receptor. Enhancement of transactivation function by substitution mutants M565R and A573Q.

To determine the importance of specific amino acids in the hormone-binding domain of the human glucocorticoid receptor (hGR), we have generated mutants M565R, G567A, and A573Q. In hormone binding assays using [3H]cortisol, half-maximal saturation of dexamethasone competition was achieved at 10 pM with hGR M565R and hGRA573Q compared to 10 nM with wild type hGR. Similar results were obtained in competition assays with [3H]dexamethasone and the glucocorticoid antagonist RU 486. The substitution mutants M565R and A573Q demonstrated a higher relative affinity for aldosterone compared to the wild type hGR. In CV-1 cells cotransfected with the mutant receptors, hGR M565R and A573Q showed a remarkable 6-fold elevated transcription activation of the chimeric reporter gene mouse mammary tumor virus-chloramphenicol acetyl-transferase (MMTV-CAT). The mutant hGR G567A failed to bind agonists and antagonists efficiently. Immunoblotting with hGR specific antibodies of the whole cell extract from transfected CV-1 cells revealed that these differences in hormone binding and transcription activation were not due to the decreased levels of expression. These data support that idea that Gly567 in the hGR hormone-binding domain lies in a region crucial to ligand binding and transactivation function.