Interaction of severe acute respiratory syndrome-coronavirus and NL63 coronavirus spike proteins with angiotensin converting enzyme-2

Although in different groups, the coronaviruses severe acute respiratory syndrome-coronavirus (SARS-CoV) and NL63 use the same receptor, angiotensin converting enzyme (ACE)-2, for entry into the host cell. Despite this common receptor, the consequence of entry is very different; severe respiratory distress in the case of SARS-CoV but frequently only a mild respiratory infection for NL63. Using a wholly recombinant system, we have investigated the ability of each virus receptor-binding protein, spike or S protein, to bind to ACE-2 in solution and on the cell surface. In both assays, we find that the NL63 S protein has a weaker interaction with ACE-2 than the SARS-CoV S protein, particularly in solution binding, but the residues required for contact are similar. We also confirm that the ACE-2-binding site of NL63 S lies between residues 190 and 739. A lower-affinity interaction with ACE-2 might partly explain the different pathological consequences of infection by SARS-CoV and NL63.

Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells

What is already known about this subject center dot Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins. center dot In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets. center dot Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid-TP interaction inhibits signalling downstream TP has not been shown. What this study adds center dot This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells. center dot Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types. Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TP alpha- and TP beta-transfected HEK 293T cells was explored using binding assays and the TP antagonist H-3-SQ29548. Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10-30 mu M). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by > 50% H-3-SQ29548 binding to different cell types. These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.

Inhibition of coxsackie B virus infection by soluble forms of its receptors: Binding affinities, altered particle formation, and competition with cellular receptors

We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus 133 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37 degrees C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed.

Comparative study of oestrogenic properties of eight phytoestrogens in MCF7 human breast cancer cells

Previous studies have compared the oestrogenic properties of phytoestrogens in a wide variety of disparate assays. Since not all phytoestrogens have been tested in each assay, this makes inter-study comparisons and ranking oestrogenic potency difficult. In this report, we have compared the oestrogen agonist and antagonist activity of eight phytoestrogens (genistein, daidzein, equol, miroestrol, deoxymiroestrol, 8-prenylnaringenin, coumestrol and resveratrol) in a range of assays all based within the same receptor and cellular context of the MCF7 human breast cancer cell line. The relative binding of each phytoestrogen to oestrogen receptor (ER) of MCF7 cytosol was calculated from the molar excess needed for 50 % inhibition of [H-3]oestradiol binding (IC50), and was in the order coumestrol (35x)/8-prenylnaringenin (45 x)/deoxymiroestrol (50 x) > miroestrol (260x) > genistein (1000x) > equol (4000x) > daidzein (not achieved: 40 % inhibition at 10(4)-fold molar excess) > resveratrol (not achieved: 10 % inhibition at 10(5)-fold molar excess). For cell-based assays, the rank order of potency (estimated in terms of the concentration needed to achieve a response equivalent to 50 % of that found with 17 beta-oestradiol (IC50)) remained very similar for all the assays whether measuring ligand ability to induce a stably transfected oestrogen-responsive ERE-CAT reporter gene, cell growth in terms of proliferation rate after 7 days or cell growth in terms of saturation density after 14 days. The IC50 values for these three assays in order were for 17 beta-oestradiol (1 x 10-(11) M, 1 x 10-(11) M, 2 x 10(-11) M), and in rank order of potency for the phytoestrogens, deoxymiroestrol (1 x 10(-10) M, 3 x 10(-11) M, 2 x 10(-11) M) > miroestrol (3 x 10(-10) M, 2 x 10(-11) M, 8 x 10(-11) M) > 8-prenylnaringenin (1 x 10(-9) M, 3 x 10(-10) M, 3 x 10(-10) M) > cournestrol (3 x 10(-8) M, 2 x 10(-8) M, 3 x 10(-8) M) > genistein (4 x 10(-8) M, 2 x 10(-8) M, 1 x 10(-8) M)/equol (1 x 10(-7) M, 3 x 10(-8) M, 2 x 10(-8) M) > daidzein (3 x 10(-7) M, 2 x 10(-7) M, 4 x 10(-8) M) > resveratrol (4 x 10(-6) M, not achieved, not achieved). Despite using the same receptor context of the MCF7 cells, this rank order differed from that determined from receptor binding. The most marked difference was for cournestrol and 8-prenylnaringenin which both displayed a relatively potent ability to displace [3H]oestradiol from cytosolic ER compared with their much lower activity in the cell-based assays. Albeit at varying concentrations, seven of the eight phytoestrogens (all except resveratrol) gave similar maximal responses to that given by 17 beta-oestradiol in cell-based assays which makes them full oestrogen agonists. We found no evidence for any oestrogen antagonist action of any of these phytoestrogens at concentrations of up to 10(-6) M on either reporter gene induction or on stimulation of cell growth. (c) 2005 Elsevier Ltd. All rights reserved.

Pharmacological characterisation of the agonist radioligand binding site of 5-HT2A, 5-HT2B and 5-HT2C receptors

In the present study we compared the affinity of various drugs for the high affinity "agonist-preferring" binding site of human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors stably expressed in monoclonal mammalian cell lines. To ensure that the "agonist-preferring" conformation of the receptor was preferentially labelled in competition binding experiments, saturation analysis was conducted using antagonist and agonist radiolabels at each receptor. Antagonist radiolabels ([H-3]-ketanserin for 5-HT2A receptor and [H-3]-mesulergine for 5-HT2B and 5-HT2C receptor) bound to a larger population of receptors in each preparation than the corresponding agonist radiolabel ([I-125]-DOI for 5-HT2A receptor binding and [H-3]-5-HT for 5-HT2B and 5-HT2C receptor binding). Competition experiments were subsequently conducted against appropriate concentrations of the agonist radiolabels bound to the "agonist-preferring" subset of receptors in each preparation. These studies confirmed that there are a number of highly selective antagonists available to investigate 5-HT2 receptor subtype function (for example, MDL 100907, RS-127445 and RS-102221 for 5-HT2A, 5-HT2B and 5-HT2C receptors respectively). There remains, however, a lack of highly selective agonists. (-)DOI is potent and moderately selective for 5-HT2A receptors, BW723C86 has poor selectivity for human 5-HT2B receptors, while Org 37684 and VER-3323 display some selectivity for the 5-HT2C receptor. We report for the first time in a single study, the selectivity of numerous serotonergic drugs for 5-HT2 receptors from the same species, in mammalian cell lines and using, exclusively, agonist radiolabels. The results indicate the importance of defining the selectivity of pharmacological tools, which may have been over-estimated in the past, and highlights the need to find more selective agonists to investigate 5-HT2 receptor pharmacology.

Investigation of the mechanism of agonist and inverse agonist action at D-2 dopamine receptors

This study investigated, for the D-2 dopamine receptor, the relation between the ability of agonists and inverse agonists to stabilise different states of the receptor and their relative efficacies. K-i values for agonists were determined in competition, versus the binding of the antagonist [H-3]spiperone. Competition data were fitted best by a two-binding site model (with the exception of bromocriptine, for which a one-binding site model provided the best fit) and agonist affinities for the higher (K-h) (G protein-coupled) and lower affinity (K-l) (G protein-uncoupled) sites determined. Ki values for agonists were also determined in competition versus the binding of the agonist [H-3]N-propylnorapomorphine (NPA) to provide a second estimate of K-h,. Maximal agonist effects (E-max) and their potencies (EC50) were determined from concentration-response curves for agonist stimulation of guanosine-5'-O-(3-[S-32] thiotriphosphate) ([S-35]GTPgammaS) binding. The ability of agonists to stabilise the G protein-coupled state of the receptor (K-l/K-h, determined from ligand-binding assays) did not correlate with either of two measures of relative efficacy (relative E-max, Kl/EC50) of agonists determined in [S-35]GTPgammaS-binding assays, when the data for all of the compounds tested were analysed For a subset of compounds, however, there was a relation between K-l/K-h and E-max.. Competition-binding data versus [H-3]spiperone and [H-3]NPA for a range of inverse agonists were fitted best by a one-binding site model. K-i values for the inverse agonists tested were slightly lower in competition versus [H-3]NPA compared to [H-3]spiperone. These data do not provide support for the idea that inverse agonists act by binding preferentially to the ground state of the receptor. (C) 2004 Elsevier Inc. All rights reserved.

Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host

Objectives: Influenza A H3N2 viruses isolated recently have characteristic receptor binding properties that may decrease susceptibility to neuraminidase inhibitor drugs. A panel of clinical isolates and recombinant viruses generated by reverse genetics were characterized and tested for susceptibility to zanamivir. Methods: Plaque reduction assays and neuraminidase enzyme inhibition assays were used to assess susceptibility to zanamivir. Receptor binding properties of the viruses were characterized by differential agglutination of red blood cells (RBCs) from different species. Sequence analysis of the haemagglutinin (HA) and neuraminidase (NA) genes was carried out. Results: Characterization of a panel of H3N2 clinical isolates from 1968 to 2000 showed a gradual decrease in agglutination of chicken and guinea pig RBCs over time, although all isolates could agglutinate turkey RBCs equally. Sequence analysis of the HA and NA genes identified mutations in conserved residues of the HA1 receptor binding site, in particular Leu-226 --> Ile-226/Val-226, and modification of potential glycosylation site motifs. This may be indicative of changes in virus binding to sialic acid (SA) receptors in recent years. Although recent isolates had reduced susceptibility to zanamivir in MDCK cell based plaque reduction assays, no difference was found in an NA enzyme-inhibition assay. Assays with recombinant isogenic viruses showed that the recent HA, but not the NA, conferred reduced susceptibility to zanamivir. Conclusion: This study demonstrates that recent clinical isolates of influenza A H3N2 virus no longer agglutinate chicken RBCs, but despite significant receptor binding changes as a result of changes in HA, there was little variation in sensitivity of the NA to zanamivir.

Cleavage and serum reactivity of the severe acute respiratory syndrome coronavirus spike protein

Severe acute respiratory syndrome (SARS) coronavirus (SCoV) spike (S) protein is the major surface antigen of the virus and is responsible for receptor binding and the generation of neutralizing antibody. To investigate SCoV S protein, full-length and individual domains of S protein were expressed on the surface of insect cells and were characterized for cleavability and reactivity with serum samples obtained from patients during the convalescent phase of SARS. S protein could be cleaved by exogenous trypsin but not by coexpressed furin, suggesting that the protein is not normally processed during infection. Reactivity was evident by both flow cytometry and Western blot assays, but the pattern of reactivity varied according to assay and sequence of the antigen. The antibody response to SCoV S protein involves antibodies to both linear and conformational epitopes, with linear epitopes associated with the carboxyl domain and conformational epitopes associated with the amino terminal domain. Recombinant SCoV S protein appears to be a suitable antigen for the development of an efficient and sensitive diagnostic test for SARS, but our data suggest that assay format and choice of S antigen are important considerations.

Protein-disulfide isomerase-mediated reduction of two disulfide bonds of HIV envelope glycoprotein 120 occurs post-CXCR4 binding and is required for fusion

The human immunodeficiency virus (HIV) envelope (Env) glycoprotein (gp) 120 is a highly disulfide-bonded molecule that attaches HIV to the lymphocyte surface receptors CD4 and CXCR4. Conformation changes within gp120 result from binding and trigger HIV/cell fusion. Inhibition of lymphocyte surface-associated protein-disulfide isomerase (PDI) blocks HIV/cell fusion, suggesting that redox changes within Env are required. Using a sensitive assay based on a thiol reagent, we show that (i) the thiol content of gp120, either secreted by mammalian cells or bound to a lymphocyte surface enabling CD4 but not CXCR4 binding, was 0.5-1 pmol SH/pmol gp120 (SH/gp120), whereas that of gp120 after its interaction with a surface enabling both CD4 and CXCR4 binding was raised to 4 SH/gp120; (ii) PDI inhibitors prevented this change; and (iii) gp120 displaying 2 SH/gp120 exhibited CD4 but not CXCR4 binding capacity. In addition, PDI inhibition did not impair gp120 binding to receptors. We conclude that on average two of the nine disulfides of gp120 are reduced during interaction with the lymphocyte surface after CXCR4 binding prior to fusion and that cell surface PDI catalyzes this process. Disulfide bond restructuring within Env may constitute the molecular basis of the post-receptor binding conformational changes that induce fusion competence.

Mathematical modelling of competitive LDL/VLDL binding and uptake by hepatocytes

Elevated levels of low-density-lipoprotein cholesterol (LDL-C) in the plasma are a well-established risk factor for the development of coronary heart disease. Plasma LDL-C levels are in part determined by the rate at which LDL particles are removed from the bloodstream by hepatic uptake. The uptake of LDL by mammalian liver cells occurs mainly via receptor-mediated endocytosis, a process which entails the binding of these particles to specific receptors in specialised areas of the cell surface, the subsequent internalization of the receptor-lipoprotein complex, and ultimately the degradation and release of the ingested lipoproteins' constituent parts. We formulate a mathematical model to study the binding and internalization (endocytosis) of LDL and VLDL particles by hepatocytes in culture. The system of ordinary differential equations, which includes a cholesterol-dependent pit production term representing feedback regulation of surface receptors in response to intracellular cholesterol levels, is analysed using numerical simulations and steady-state analysis. Our numerical results show good agreement with in vitro experimental data describing LDL uptake by cultured hepatocytes following delivery of a single bolus of lipoprotein. Our model is adapted in order to reflect the in vivo situation, in which lipoproteins are continuously delivered to the hepatocyte. In this case, our model suggests that the competition between the LDL and VLDL particles for binding to the pits on the cell surface affects the intracellular cholesterol concentration. In particular, we predict that when there is continuous delivery of low levels of lipoproteins to the cell surface, more VLDL than LDL occupies the pit, since VLDL are better competitors for receptor binding. VLDL have a cholesterol content comparable to LDL particles; however, due to the larger size of VLDL, one pit-bound VLDL particle blocks binding of several LDLs, and there is a resultant drop in the intracellular cholesterol level. When there is continuous delivery of lipoprotein at high levels to the hepatocytes, VLDL particles still out-compete LDL particles for receptor binding, and consequently more VLDL than LDL particles occupy the pit. Although the maximum intracellular cholesterol level is similar for high and low levels of lipoprotein delivery, the maximum is reached more rapidly when the lipoprotein delivery rates are high. The implications of these results for the design of in vitro experiments is discussed.

Endocannabinoid receptor antagonists: potential for obesity treatment

Obesity has been described as a global epidemic. Its increasing prevalence is matched by growing costs, not only to the health of the individual, but also to the medical services required to treat a range of obesity-related diseases. In most instances, obesity is a product of progressively less energetic lifestyles and the over-consumption of readily available, palatable, and highly caloric foods. Past decades have seen massive investment in the search for effective anti-obesity therapies, so far with limited success. An important part of the process of developing new pharmacologic treatments for obesity lies in improving our understanding of the psychologic and physiologic processes that govern appetite and bodyweight regulation. Recent discoveries concerning the endogenous cannabinoids are beginning to give greater insight into these processes. Current research indicates that endocannabinoids may be key to the appetitive and consummatory aspects of eating motivation, possibly mediating the craving for and enjoyment of the most desired, most fattening foods. Additionally, endocannabinoids appear to modulate central and peripheral processes associated with fat and glucose metabolism. Selective cannabinoid receptor antagonists have been shown to suppress the motivation to eat, and preferentially reduce the consumption of palatable, energy-dense foods. Additionally, these agents act to reduce adiposity through metabolic mechanisms that are independent of changes in food intake. Given the current state of evidence, we conclude that the endocannabinoids represent an exciting target for new anti-obesity therapies.

Photoperiod and testosterone modulate growth and melanogenesis of S91 murine melanoma

In vivo and in vitro assays were performed with S91 murine melanoma cells aiming to investigate the effects of testosterone and photoperiod on tumor growth and melanogenesis (tyrosinase activity). In vivo assays were performed by inducing melanoma tumors in castrated mice receiving increasing concentrations of testosterone and submitted to varying photoperiod regimens. The results demonstrated that the increase of melanin content was higher in animals submitted to the longest days, thus demonstrating the importance of photoperiod length in melanin synthesis. Increase in tumor growth and protein content was observed in testosterone-treated animals submitted to 12L:12D; in testosterone-treated animals submitted to 4L:20D and 20L:4D tumor growth was significantly smaller. In S91 cultured cells, testosterone increased cell proliferation and reduced tyrosinase activity in a dose-dependent manner. Radioactive binding assays demonstrated that the hormone was acting through low affinity testosterone receptors, since the presence of aromatase inhibitor did not affect the binding assay in a statistically significant way, and all the in vitro experiments were performed in the presence of the inhibitor. Our in vivo data added to the in vitro results corroborate the hypothesis that S91 melanoma cells directly respond to testosterone and that this effect is modulated by light.

Retinal removal up-regulates cannabinoid CB1 receptors in the chick optic tectum

The endocannabinoid system has been implicated in several neurobiological processes, including neurodegeneration and neuro protection. The aim of this study was to evaluate the effects of unilateral retinal ablation on the expression of the cannabinoid receptor subtype 1 (CB1) at both protein and mRNA levels in the optic tectum of the adult chick brain. After different survival times postlesion (2-30 days), the chick brains were subjected to immunohistochemical, immunoblotting, and real-time PCR procedures to evaluate CB1 expression. TUNEL and Fluoro-Jade B were used to verify the possible occurrence of cell death, and immunostaining for the microtubule-associated protein MAP-2 was performed to verify possible dendritic remodeling after lesions. No cell death could be observed in the deafferented tectum, at least up to 30 days postlesion, although Fluoro-Jade B could reveal degenerating axons and terminals. Retinal ablation seems to generate an increase of CB1 protein in the optic tectum and other retinorecipient visual areas, which paralleled an increase in MAP-2 staining. On the other hand, CB, mRNA levels were not changed after retinal ablation. Our results reveal that CB, expression in visual structures of the adult chick brain may be negatively regulated by the retinal innervation. The increase of CB1 receptor expression observed after retinal removal indicates that these receptors are not presynaptic in retinal axons projecting to the tectum and suggests a role of the cannabinoid system in plasticity processes ensuing after lesions. (c) 2008 Wiley-Liss, Inc.

Long-term nitric oxide deficiency causes muscarinic supersensitivity and reduces beta(3)-adrenoceptor-mediated relaxation, causing rat detrusor overactivity

Background and purpose: Overactive bladder is a complex and widely prevalent condition, but little is known about its physiopathology. We have carried out morphological, biochemical and functional assays to investigate the effects of long-term nitric oxide (NO) deficiency on muscarinic receptor and beta-adrenoceptor modulation leading to overactivity of rat detrusor muscle. Experimental approach: Male Wistar rats received No-nitro-L-arginine methyl ester (L-NAME) in drinking water for 7-30 days. Functional responses to muscarinic and b-adrenoceptor agonists were measured in detrusor smooth muscle (DSM) strips in Krebs-Henseleit solution. Measurements of [H-3] inositol phosphate, NO synthase (NOS) activity, [H-3] quinuclidinyl benzilate ([H-3]QNB) binding and bladder morphology were also performed. Key results: Long-term L-NAME treatment significantly increased carbachol-induced DSM contractile responses after 15 and 30 days; relaxing responses to the beta(3)-adrenoceptor agonist BRL 37-344 were significantly reduced at 30 days. Constitutive NOS activity in bladder was reduced by 86% after 7 days and maintained up to 30 days of L-NAME treatment. Carbachol increased sixfold the [H-3] inositol phosphate in bladder tissue from rats treated with L-NAME. [H-3] QNB was bound with an apparent KD twofold higher in bladder membranes after L-NAME treatment compared with that in control. No morphological alterations in DSM were found. Conclusions and implications: Long-term NO deficiency increased rat DSM contractile responses to a muscarinic agonist, accompanied by significantly enhanced KD values for muscarinic receptors and [H-3] inositol phosphate accumulation in bladder. This supersensitivity for muscarinic agonists along with reductions of beta(3)-adrenoceptor-mediated relaxations indicated that overactive DSM resulted from chronic NO deficiency.

Ligand dissociation from estrogen receptor is mediated by receptor dimerization: Evidence from molecular dynamics simulations

Estrogen Receptor (ER) is an important target for pharmaceutical design. Like other ligand-dependent transcription factors, hormone binding regulates ER transcriptional activity. Nevertheless, the mechanisms by which ligands enter and leave ERs and other nuclear receptors remain poorly understood. Here, we report results of locally enhanced sampling molecular dynamics simulations to identify dissociation pathways of two ER ligands [the natural hormone 17 beta-estradiol (E-2) and the selective ER modulator raloxifene (RAL)] from the human ER alpha ligand-binding domain in monomeric and dimeric forms. E-2 dissociation occurs via three different pathways in ER monomers. One resembles the mousetrap mechanism (Path I), involving repositioning of helix 12 (H12), others involve the separation of H8 and H11 (Path II), and a variant of this pathway at the bottom of the ligand-binding domain (Path II`). RAL leaves the receptor through Path I and a Path I variant in which the ligand leaves the receptor through the loop region between H11 and H12 (Path I`). Remarkably, ER dimerization strongly suppresses Paths II and II` for E-2 dissociation and modifies RAL escape routes. We propose that differences in ligand release pathways detected in the simulations for ER monomers and dimers provide an explanation for previously observed effects of ER quaternary state on ligand dissociation rates and suggest that dimerization may play an important, and hitherto unexpected, role in regulation of ligand dissociation rates throughout the nuclear receptor family.