Glucocorticoid receptor interactions with glucocorticoids: evaluation by molecular modeling and functional analysis of glucocorticoid receptor mutants
Introduction
Topical glucocorticoids (GCs) are the most effective treatment currently available for atopic dermatitis and other inflammatory skin diseases. GC esters are preferred as the higher lipophilicity improves cutaneous uptake [1], [2]. For GC 17-esters, a high activity correlating with a high glucocorticoid receptor (GR) binding has been demonstrated, too [3]. High potency, however, may even lead to skin atrophy which can become irreversible following prolonged GC application. GC 21-esters reveal only low receptor binding, which suggests difficulties in fitting into the GR ligand-binding domain (LBD). Therefore, GC 21-esters are less frequently used. GC 17,21-double esters penetrate skin even better than monoesters and often serve as prodrugs for the delivery of active GC 17-esters [4], [5]. Prednicarbate (PC) is a prednisolone (PD) double ester [6], for which an improved benefit/risk ratio regarding antiinflammatory versus atrophogenic potential has been demonstrated in man [7], [8], [9] and cell culture [6], [10]. In the skin, PC is metabolized by ester-cleavage resulting in the formation of PD 17-ethylcarbonate (P17EC), which is converted to PD 21-ethylcarbonate (P21EC) and finally to PD [4]. The hydrolytic rate, however, depends on the cell type, in keratinocytes metabolism takes place to a higher extent than in fibroblasts. This does not hold true with ester cleavage of betamethasone 17-valerate (BMV) [1]. The activity of native drugs and metabolites determines the benefit/risk ratio, e.g. in contrast to PC there is no improvement with P17EC,21-phenylacetate (PEP) despite of identical metabolism [6]. The very low rate of GC metabolism in COS-7 cells [11] makes GR-transfected COS-7 cells an ideal model to study receptor binding of GC esters. As we recently described activity of native PC [6], [11], it was of interest to compare the interactions of GR with esterified and non-esterified GC in detail.
The crystal structure of the GR LBD has recently been solved, but the coordinates are not yet available [12]. Resolution of the crystal structures of the GR LBD and the LBDs of other intracellular receptors, however, increased our knowledge about molecular mechanisms of steroid binding. In the liganded crystal structures of the estrogen receptor (ER) α, ER β, progesterone receptor (PR) and GR, a conserved arginine as well as a glutamate (ER) or glutamine (PR, GR) form hydrogen bonds with the C3OH/-keto group of the steroids, which is a main feature of steroid A-ring recognition [13], [14], [15]. Substituents of the D-ring, which is anchored at the opposite end of the ligand-binding pocket show greater variability and also the interacting amino acids of different receptors seem to be more variable. Therefore, D-ring interactions should be involved in binding specificity. PR shows 80% homology to GR regarding identical and physicochemically similar amino acids. A GR homology model based on PR data [14] was chosen to study GC–GR interactions.
Here, we describe GR-binding activities of a series of GCs including various GC esters measured in GR-transfected COS-7 cells and their docking to a GR LBD homology model built by Höltje and Jessen (manuscript in preparation). Moreover, we performed 3D quantitative structure–activity relationship (QSAR) investigations based on the receptor docked GC conformations in order to correlate measured and calculated receptor-binding affinities. These QSAR investigations give information about the fitness of the model and allow to predict binding affinities of other ligands. For a further confirmation, we investigated receptor binding to GR mutants [16] as well as transactivation (TA) and transrepression (TR).
Section snippets
Materials
BMV, betamethasone 17,21-dipropionate (BMDP) and clobetasol 17-propionate (CLP) were obtained from Glaxo Wellcome (Hamburg, Germany), mometasone furoate (MF) from Essex Pharma (München, Germany). Other GCs—PC, P17EC, P21EC, PD, PEP, desoximetasone (DOM), desoximetasone 21-cinnamate (DOMCIN), methylprednisolone aceponate (MPDA), methylprednisolone 17-propionate (MP17P) and methylprednisolone (MPD)—were kindly provided by Aventis Pharma (Frankfurt, Germany). [1,2,4,6,7-] dexamethasone ([
Glucocorticoid structures and receptor-binding data
Structures of the GCs studied by molecular dynamics simulations and their IC50exp values from competitive binding experiments expressed as negative decadic log values are summarized in Table 1A. Structures and receptor-binding data from additionally analyzed GCs are shown in Table 1B.
Model and ligand docking
The GR homology model (Höltje and Jessen, manuscript in preparation) offers a ligand-binding pocket with a size of 618.5 Å3 (SURFNET software, radius: 1–4.5 Å). Applying the PROCHECK software, the monitored
Discussion
While in known steroid receptor LBDs, the A-ring of steroids seems to be anchored in a similar manner, the interaction with the D-ring appears to be more variable [13], [14], [15], [25]. Vayssiere et al. [26] described GCs inducing TR but devoid of TA, which differ only in the D-ring substituents. Therefore, D-ring substituents should influence receptor binding, transcriptional potency and even the benefit/risk ratio [6], [10]. Aim of this study was to characterize the GR-binding properties of
Acknowledgements
The authors thank J. Carlstedt-Duke, M. Danielsen and R. Evans for providing plasmids. Irina Spika was a recipient of a fellowship from the “Berliner Programm zur Förderung der Chancengleicheit von Frauen in Forschung und Lehre”.
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