Elsevier

Steroids

Volume 68, Issue 4, April 2003, Pages 329-339
Steroids

Glucocorticoid receptor interactions with glucocorticoids: evaluation by molecular modeling and functional analysis of glucocorticoid receptor mutants

https://doi.org/10.1016/S0039-128X(03)00030-8Get rights and content

Abstract

In the treatment of inflammatory skin diseases, there are some glucocorticoid (GC) double esters combining pronounced antiinflammatory activity and minor atrophogenic side effects. The reason, however, is only poorly understood. To investigate interactions of GCs with the ligand-binding domain of the glucocorticoid receptor (GR), we measured receptor-binding potency of a series of GC esters including their metabolites and performed a molecular modeling study using progesterone receptor crystal structure data. Ligand docking to the GR-binding pocket showed good fitting of GC 17-esters corresponding to their high receptor-binding affinity, and unfavorable sterical interactions for GC 21-esters with substituents larger than propionate. Molecular dynamics simulations served to visualize induced fit procedures. Ligand docked GC conformations after dynamics simulations were used for generation of a 3D quantitative structure–activity relationship model. Using a set of 11 steroids, this model showed a correlation coefficient (r2) of 0.98, a leave-one-out cross validation (q2) of 0.79 and was able to predict binding affinity of further six ligands with a standard error of prediction of 0.33. Moreover, interactions of Asn-564 and Met-639 with the steroids were investigated by studying GR mutants of these amino acids. Met-639 participates in hydrophobic interactions mainly with GC side chains, while Asn-564 forms a hydrogen bond to the C11OH group of the steroid. Asn-564 is shown to be very important for ligand binding and even more for target gene activation and transcription factor repression.

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 C3OH/-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). [1,2,4,6,7-3H] dexamethasone ([3H

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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