Unbinding Pathways from the Glucocorticoid Receptor Shed Light on

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Unbinding pathways from glucocorticoid receptor shed light on reduced sensitivity of glucocorticoid ligands to a naturally occurring, clinically relevant, mutant receptor Gabriele Costantino, Anna Maria Capelli, Agostino Bruno, and Antonio Jose Entrena Guadix J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400802b • Publication Date (Web): 08 Aug 2013 Downloaded from http://pubs.acs.org on August 10, 2013

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Journal of Medicinal Chemistry

Unbinding pathways from glucocorticoid receptor shed light on reduced sensitivity of glucocorticoid ligands to a naturally occurring, clinically relevant, mutant receptor Anna Maria Capelli,1,2,a Agostino Bruno,2,a Antonio Entrena Guadix,3 Gabriele Costantino2 1

Chemistry Research and Drug Design Department, Chiesi Farmaceutici S.p.A., Largo F. Belloli, Parma,

Italy; 2Dipartimento di Farmacia, Universita’ degli Studi di Parma, viale Area delle Scienze 27/A, Parma, Italy. 3Departamento de Quımica Farmaceutica y Organica, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja s/n, 18071 Granada, Spain a

These authors equally contributed to the work.

ABSTRACT Glucocorticoids are endogenous steroid hormones that regulate essential biological functions including metabolism, growth, and apoptosis. Glucocorticoids represent the most effective anti-inflammatory agents to treat several inflammatory conditions. However, the clinical use of such drugs is hampered by severe side effects. Therefore, the development of novel glucocorticoids receptor (GRs) modulators with increased therapeutic index is impelling. Herein, using steered molecular dynamics (SMD) simulations we provide a detailed picture of the unbinding process of three clinically relevant GR modulators from GR ligand binding domains. The SMD protocol here described can be used to prioritize the synthesis of structural analogues on the basis of their potential of mean forces (PMFs) and calculated unbinding energies. Moreover, our results are instrumental to explain at atomic resolution the reduced ability of dexamethasone to activate the naturally occurring mutant I747M-GR, which is implicated in rare familial glucocorticoid resistance, clinically characterized by glucocorticoids insensitivity.

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INTRODUCTION Glucocorticoids receptors (GR) belong to the steroid-thyroid-retinoid family of the nuclear receptors (NRs). These are globular proteins which act as ligand-dependent transcription factors controlling specific gene expression in most mammalian cells.1,2 NRs share a common domain organization, and the most conserved regions are represented by the DNA-binding domains and the ligand binding domain (LBD).1,2 Several X-ray crystal structures of the LBD of different NRs were solved, and three common characteristics are present: (i) the folding into a canonical three-layer helical sandwich, (ii) a hydrophobic pocket for ligand binding, and (iii) an active conformation induced by agonist binding that facilitates its association with co-activator proteins.3 Moreover, NRs share a common cascade of functional events, which are conserved among the different NR families.1,4 As a matter of fact, on ligand binding, NRs undergo conformational changes, which promote receptor dimerization, co-factor interaction and the binding at the DNA response element.1,4–6 Glucocorticoids (GCs) are a class of endogenous steroid hormones that regulate a variety of cell, tissue-, and organ-specific biological functions including metabolism, growth, development behavior, and apoptosis.1,4–6 GCs represent the most effective anti-inflammatory agents to treat several chronic and acute inflammatory conditions such as asthma, psoriasis, rheumatoid arthritis and irritable bowel disease.5,7,8 Despite the great benefits of GCs severe side effects such as osteoporosis, diabetes, hypertension, and obesity can occur, thus, hampering the GCs clinical use.8,9 Therefore, the development of novel modulators that can dissociate the therapeutic effects from the undesired adverse effects is impelling.8,9Drug design strategies of new GR modulators require a deep understanding of how GCs act at molecular level. The biological action of glucocorticoids is mediated by the glucocorticoid nuclear receptor (GR). Upon ligand binding, GR translocates into the nucleus and binds to DNA at glucocorticoid response elements (GRE) in the promoter region of steroid-responsive genes (transactivation).5 GR may also negatively modulate (transrepression) inflammatory responses through inhibitory interactions with pro-inflammatory transcription factors such as Activator Protein (AP)-1 and Nuclear Factor (NF)-kB, without binding to DNA; in this case the GR controls gene expression by modulating the transcriptional activities of those factors.8 Dexamethasone (Dexa, Figure 1) is a classical glucocorticosteroid characterized by nanomolar affinity for GR and high oral bioavailability (30%).6,10 A considerable amount of research has been done by medicinal


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chemists to improve the therapeutic index of glucocorticosteroids by increasing their potency and reducing their systemic side effects.1,4–6,9,10 These efforts culminated in the discovery of Fluticasone Furoate (FF, Figure 1) and Fluticasone Propionate (FP, Figure 1) for topic administration.11 In particular, FF shows subnanomolar potency and longer duration of action with respect to Dexa and FP.12 Furthermore, FF is characterized by slow dissociation rate (koff) from GR, comparable to that of FP, but slower than that showed by Dexa.10 koff can be translated into dissociative half-life (dissociative t1/2) for the receptor-ligand complex and this is a direct measure of the residence time in vitro and a possible indicator for (i) in vivo duration of action and (ii) non-target related toxicity due to sub-optimal selectivity profile.13,14 Considering that either the lack of efficacy or off-target effects represent the main reasons for drug attrition,13–16 the optimization of t1/2 dissociative parameter might be taken into account in drug design strategies and during the lead optimization process.13,14 The disclosure of the X-ray crystal structures of Dexa and FF bound to human GR LBD in complex with the co-activator TIF2 (transcriptional intermediary factor 2)6,11 provided, for the first time, important atomistic details about the binding modes of such drugs. Indeed, the crystal structures revealed that the furoate chain is nicely accommodated in the lipophilic 17α pocket of the receptor.6,11 On the contrary the hydroxyl group at C17 of Dexa does not completely fill this hydrophobic cavity and is involved in a hydrogen bond interaction with Q642.6,11 Although of foremost importance, these X-ray crystal structures represent static pictures of ligand-receptor complexes and do not provide apparent explanation for the different binding kinetic properties of Dexa, FF and FP and the mechanism underlying the unbinding process. Hence, the mechanism which regulates the koff kinetic parameters of glucocorticoids for GR is still elusive. In this regard, computational tools have proven to be useful in deciphering the structural details underlying the different koff values for a series of ligand analogues.17,18 In particular Steered Molecular Dynamics (SMD) simulations have demonstrated to be a flexible and powerful tool for providing information about the energy landscape driving the ligand-receptor unbinding processes, and information about time-resolved complex dissociation,17–22 also in the case of another NR. 23,24 Herein, by using SMD simulations we provide a detailed description of the unbinding process of Dexa, FF and FP, respectively, from GR LBD. The information thus gathered is instrumental to propose an atomistic


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explanation for the high reduction in efficacy of Dexa in the naturally occurring I747M-GR, a mutated receptor implicated in rare familial glucocorticoid resistance, clinically characterized by generalized, partial, target tissue insensitivity to glucocorticoids25,26. As a result of such condition, hypothalamic-pituitary-adrenal (HPA) axis is activated leading to elevation in the concentration of circulating cortisol and adrenocorticotropin hormone (ACTH), giving rise to adrenal hyperplasia and increase in the production of adrenal steroids with mineralocorticoid and androgenic activity.25,26 This syndrome is currently treated by pharmacologically reducing the endogenous secretion of ACTH and by adequately suppressing the HPA axis as corticotroph hyperstimulation coupled to decrease in glucocorticoid negative feedback inhibition at hypothalamic and pituitary levels may result in ACTH-secreting adenoma. Current treatments consist in the administration of high doses of Dexamethasone (1-3mg/d), which has to be carefully titrated according to the clinical manifestation and biochemical profiles of the patients.27,28 Therefore, the possibility of relying on more potent and selective glucocorticoids to treat such a rare condition would be highly desirable in order to reduce the doses and minimize side-effects of the administered drug. The SMD protocol here described can be used to prioritize the synthesis of novel glucocorticosteroids, structurally-related to Dexa, on the basis of their calculated Potential of Mean Forces (PMFs) and unbinding energies. RESULTS (i) PMF along the Unbinding Reaction Coordinates In Figure 2a is reported the PMF calculated applying the Jarzynski’s equality (eq. 3) and the cumulative expansion up to the second order (eq. 4). The typical end-to-end distance vs. constrained center is plotted in Figure 2b, demonstrating that the stiff-approximation is fulfilled under our simulations conditions by using a very small pulling velocity and a force constant of 15 kcal/mol*Å2. The analysis of Figure 2a reveals that the PMF of FF (88.36 ± 6.33 kcal/mol) is higher than that of Dexa (63.87 ± 10.23 kcal/mol), but it is in the twilight zone of statistically significance with respect to that of FP (78.01 ± 3.65 kcal/mol) according to their standard deviations values (Figure 2a, orange and grey shadow regions, respectively). This result is qualitatively in agreement with the experimental values reported in the Inset table. Furthermore, the PMF of Dexa shows higher variability with respect to the other compounds (Figure 2a, green shadow region) and the energy basins of FF and FP are narrower than that of Dexa, suggesting that Dexa can dissociate from the GR


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LBD following a slightly different undocking path. Interestingly, multiple dissociating paths were reported for other NRs.29,30 The dissipated work (∆Wdiss, see eq. 4 in SMD simulations and Potential of Mean Forces Calculation section) refers to the strength of friction, due to non-equilibrium conditions, thus representing the irreversible (dissipative) work. The convergence of the estimated PMFs can be improved by reducing the dissipation.19,21,22 The ∆Wdiss values of FF (5.10 kcal/mol) and Dexa (8.25 kcal/mol) as well as that of FP (2.87 kcal/mol) are much lower than the corresponding differences for the calculated PMFs (∆FFF-Dexa=24.48; kcal/mol; ∆FFF-FP=10.34 kcal/mol; and ∆FFP-Dexa=14.14 kcal/mol) and therefore indicate the reliability of the differences observed for the calculated PMFs in our SMD calculations. Finally, the computed PMF are qualitatively in agreement with those reported in the literature for related NRs.23,24 (ii.) Analysis of the dissociating pathways 1. Comparison of the dissociating pathway of Dexa and FF The dissociation path characterized by the lowest work profile of each receptor complex was selected with the aim to rationalize, at atomic level resolution, the different biological behavior of Dexa, FF, and FP. Conformations belonging to the selected paths were clustered as described in the Material and Methods section (see Description of the Unbinding Pathway section), and the representative structures were used to reconstruct the representative dissociation path of each GR-ligand/TIF2 complex. Finally, the structures characterizing crucial dissociation steps were projected back on the corresponding force profiles of the selected SMD runs. The atomistic details were analyzed in the context of the biological and computational data available in literature. The analysis of the PMF profiles (Figure 2a) and of the different SMD runs reveals that Dexa could dissociate from GR LBD following slightly different escaping pathways (Figure 3a). The main differences observed take place when Dexa has already left the hydrophobic pocket of the LBD and is approaching the H11-H12 connecting loop (Figure 3b). This behavior is unique for Dexa bound to the wild type receptor as it was not observed either for FF and FP (Figure 3c and d, respectively) or for Dexa in complex with the I747M mutated receptor (this will be discuss later in the text). The different behavior of FF and FP with respect to Dexa can be related to the presence of a bulky substituent on the C17 carbon (furoate ester for FF, ethyl ester for FP, Figure 3a-c and 4, respectively), which can behave as an anchor reducing the ligand freedom during the unbinding process. On the contrary, Dexa lacks this bulky fragment and can freely


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explore different unbinding pathways. In Figure 4 are reported the representative structures describing these crucial steps in the dissociation pathways for Dexa and FF, respectively. Structures #1 show that Dexa and FF make different interactions with the residues lining up the binding pocket of the LBD. In particular, both ligands make hydrogen bonds with N564 and Q570. However, FF does not interact with Q642 due to the lack of a free OH group at C17. Moreover, the furoate group modifies the orientation of Q642 side chain (Figure 4, structure #1 in the red box), in agreement with the crystal structure conformation (3CLD). The pattern of interactions observed for FF does not immediately affect its unbinding path, as its force profile along the first 2 Å is similar to that of Dexa. However, it will have important consequences on the next steps of the process. Furthermore, a water molecule (W1) was identified inside the hydrophobic pocket of the LBD in both representative structures #1 (Figure 1, structures in green and red boxes). While in the Dexa complex W1 bridges the carbonyl group of the ligand to R611, in the FF one W1 is not always involved in any hydrogen bond interactions and fluctuates nearby R611. It is worth mentioning that the presence of water molecules in this binding site region of GR had been previously observed.3,31 In particular, computational simulations performed by Alvarez et al.31 revealed the presence of a water molecule bridging R611 and Q570 only. In the X-ray crystal structure of GR-FF/TIF2 (3CLD) two water molecules lie near R661 and Q570 (HOH784 and HOH783 chain A, See Supplementary Materials Figure S1). HOH784 could be involved in a network of interactions involving receptor residues R611 and Q570 only. In light of these observations we can speculate that these water molecules might assist the unbinding process of the ligands from the LBD of GR. In the next step of the dissociation path illustrated by the representative structures #2 at r= 4 Å, the interactions with R611 are lost. In the representative structure #2 of Dexa the H11-H12 connecting loop is approaching the ligand, while in the FF complex the furoate group is involved in a network of hydrogen bonds with T739, Q642 and a second water molecule (W2) (Figure 4, representative structure #2 red box). At the same time it interacts with H11-H12 loop residues. These interactions seem to trap FF inside the hydrophobic pocket of LBD, while Dexa is free to move towards the solvent. These observations can explain the different behavior observed in the force profile of the two SMD runs considered (2

Unbinding Pathways from the Glucocorticoid Receptor Shed Light on

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