Emmanuel Sturchler

Structural mechanism of ligand activation in human calcium-sensing receptor

Human calcium-sensing receptor (CaSR) is a G-protein-coupled receptor (GPCR) that maintains extracellular Ca2+ homeostasis through the regulation of parathyroid hormone secretion. It functions as a disulfide-tethered homodimer composed of three main domains, the Venus Flytrap module, cysteine-rich domain, and seven-helix transmembrane region. Here, we present the crystal structures of the entire extracellular domain of CaSR in the resting and active conformations. We provide direct evidence that L-amino acids are agonists of the receptor. In the active structure, L-Trp occupies the orthosteric agonist-binding site at the interdomain cleft and is primarily responsible for inducing extracellular domain closure to initiate receptor activation. Our structures reveal multiple binding sites for Ca2+ and PO43- ions. Both ions are crucial for structural integrity of the receptor. While Ca2+ ions stabilize the active state, PO43- ions reinforce the inactive conformation. The activation mechanism of CaSR involves the formation of a novel dimer interface between subunits. DOI: http://dx.doi.org/10.7554/eLife.13662.001

Different potencies of angiotensin receptor blockers at suppressing adrenal β-Arrestin1-dependent post-myocardial infarction hyperaldosteronism.

Aldosterone is 1 of the various hormones with detrimental functions for the failing heart, whose circulating levels are elevated post-myocardial infarction (MI) and in patients with chronic heart failure (HF) [(1)][1]. We have recently discovered that angiotensin II, acting through its type 1

Suppression of adrenal βarrestin1-dependent aldosterone production by ARBs: head-to-head comparison

The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion is mediated by either Gq/11 proteins or βarrestin1 (βarr1), both of which can couple to its type 1 receptors (AT1Rs), present in adrenocortical zona glomerulosa (AZG) cell membranes. In the present study, we examined the relative potencies of all the currently used in the clinic AT1R antagonist drugs (angiotensin receptor blockers, ARBs, or sartans) at preventing activation of these two signaling mediators (G proteins and βarrs) at the AngII-bound AT1R and, consequently, at suppression of aldosterone in vitro. All ARBs were found to be potent inhibitors of G protein activation at the AT1R. However, candesartan and valsartan were the most potent at blocking AngII-induced βarr activation at this receptor, among the tetrazolo-biphenyl-methyl derivatives, translating into excellent efficacies at aldosterone suppression in H295R cells. Conversely, irbesartan and losartan were largely G protein-selective inhibitors at the AT1R, with very low potency towards βarr inhibition. As a result, they were very weak suppressors of βarr1-dependent aldosterone production in H295R cells. These findings provide important pharmacological insights into the drug class of ARBs and medicinal chemistry insights for future drug development in the field of AngII antagonism.

Stress-induced nuclear import of apoptosis signal-regulating kinase 1 is mediated by karyopherin α2/β1 heterodimer

The apoptosis signal-regulating kinase 1 (ASK1) is activated in response to a wide variety of extracellular stressors. Consequently, dysregulation of ASK1 is associated with multiple pathologies. Here, we show that ASK1 translocates from the cytoplasm to the nucleus in HEK293 cells and human cardiomyocytes in response to hydrogen peroxide (H(2)O(2)) or angiotensin respectively. Immunoprecipitation and mass spectrometry experiments reveal that ASK1 physically interacts with the karyopherin α2/β1 heterodimer in response to stress and genetic knockdown experiments confirm that this association mediates H(2)O(2)-induced ASK1 nuclear translocation. In addition, we have identified a nuclear localization signal (NLS)-like motif within the primary amino acid sequence of ASK1 composed of two clusters of basic amino acids separated by an intervening 16 amino acid spacer, KR[ACANDLLVDEFLKVSS]KKKK. Mutation of the downstream lysine cluster markedly reduces the H(2)O(2)-induced ASK1-karyopherin α2/β1 interaction and inhibits ASK1 nuclear translocation. Furthermore, we demonstrate that nuclear ASK1 is active and participates in H(2)O(2)-induced ASK1-mediated cell death. Collectively, our findings have identified a functional interaction between ASK1 and the karyopherin α2/β1 heterodimer and have also revealed a novel mechanism by which nuclear trafficking regulates the apoptotic function of ASK1 in response to stress.

Development of a high-throughput screening-compatible cell-based functional assay to identify small molecule probes of the galanin 3 receptor (GalR3).

The galanin 3 receptor (GalR3) belongs to the large G protein-coupled receptor (GPCR) family of proteins. GalR3 and two other closely related receptors, GalR1 and GalR2, together with their endogenous ligand galanin, are involved in a variety of physiological and pathophysiological processes. GalR3 in particular has been strongly implicated in addiction and mood-related disorders such as anxiety and depression. It has been the target of many drug discovery programs within the pharmaceutical industry, but despite the significant resources and effort devoted to discovery of galanin receptor subtype selective small molecule modulators, there have been very few reports for the discovery of such molecules. GalR3 has proven difficult to enable in cell-based functional assays due to its apparent poor cell surface expression in recombinant systems. Here, we describe the generation of a modified GalR3 that facilitates its cell surface expression while maintaining wild-type receptor pharmacology. The modified GalR3 has been used to develop a high-throughput screening-compatible, cell-based, cAMP biosensor assay to detect selective small molecule modulators of GalR3. The performance of the assay has been validated by challenging it against a test library of small molecules with known pharmacological activities (LOPAC; Sigma Aldrich). This approach will enable identification of GalR3 selective modulators (chemical probes) that will facilitate dissection of the biological role(s) that GalR3 plays in normal physiological processes as well as in disease states.

Mechanism of Oxidative Stress-Induced ASK1-Catalyzed MKK6 Phosphorylation

Apoptosis signal-regulating kinase 1 (ASK1) is a serine/threonine kinase that responds to a plethora of stress-inducing signals. In turn, activation of ASK1 is associated with a number of human pathological conditions, including neurodegenerative disease, inflammation, and heart failure. In response to oxidative stress, ASK1 activates the cell death-associated p38 MAPK pathway by phosphorylating MKK6. Here, we investigated the regulation of oxidative stress-induced ASK1-catalyzed phosphorylation of MKK6. MKK6 phosphorylation levels increased immediately after H(2)O(2) treatment in intact cells and decreased following treatment for 30 min. When expressed in HEK293T cells, ASK1 was reproducibly purified within a high-molecular mass complex ( approximately 1500 kDa) known as the ASK1 signalosome. Measurement of the in vitro kinetic parameters revealed that the catalytic efficiency (k(cat)/K(m)) of ASK1 was 4000-fold greater in cells treated with H(2)O(2) for 3 min than in untreated cells. Interestingly, although the K(m(ATP)) values were found to be unchanged, the K(m(MKK6)) was dramatically decreased ( approximately 1000-fold). The increased affinity was specific for MKK6 and short-lived, as the K(m(MKK6)) returned to basal levels 30 min after treatment. Consistently, endogenous MKK6 was found within the ASK1 signalosome in intact cells and in addition copurified with ASK1 following treatment for 3 min. In contrast, proteins modulating ASK1 activity and degradation were found to interact with the ASK1 signalosome once MKK6 activation was completed. Taken together, these data suggest that oxidative stress rapidly increases ASK1 catalytic efficiency for MKK6 phosphorylation by increasing MKK6 binding affinity within the ASK1 signalosome prior to induction of inactivation and degradation of the complex.

Structure–activity relationship study of angiotensin II analogs in terms of β‐arrestin‐dependent signaling to aldosterone production

The known angiotensin II (AngII) physiological effect of aldosterone synthesis and secretion induction, a steroid hormone that contributes to the pathology of postmyocardial infarction (MI) heart failure (HF), is mediated by both Gq/11 proteins and β‐arrestins, both of which couple to the AngII type 1 receptors (AT1Rs) of adrenocortical zona glomerulosa (AZG) cells. Over the past several years, AngII analogs with increased selectivity (“bias”) toward β‐arrestin‐dependent signaling at the AT1R have been designed and described, starting with SII, the gold‐standard β‐arrestin‐”biased” AngII analog. In this study, we examined the relative potencies of an extensive series of AngII peptide analogs at relative activation of G proteins versus β‐arrestins by the AT1R. The major structural difference of these peptides from SII was their varied substitutions at position 5, rather than position 4 of native AngII. Three of them were found biased for β‐arrestin activation and extremely potent at stimulating aldosterone secretion in AZG cells in vitro, much more potent than SII in that regard. Finally, the most potent of these three ([Sar1, Cys(Et)5, Leu8]‐AngII, CORET) was further examined in post‐MI rats progressing to HF and overexpressing adrenal β‐arrestin1 in vivo. Consistent with the in vitro studies, CORET was found to exacerbate the post‐MI hyperaldosteronism, and, consequently, cardiac function of the post‐MI animals in vivo. Finally, our data suggest that increasing the size of position 5 of the AngII peptide sequence results in directly proportional increases in AT1R‐dependent β‐arrestin activation. These findings provide important insights for AT1R pharmacology and future AngII‐targeted drug development.

GABAB receptor allosteric modulators exhibit pathway‐dependent and species‐selective activity

Positive modulation of the GABAB receptor (GABABR) represents a potentially useful therapeutic approach for the treatment of nicotine addiction. The positive allosteric modulators (PAMs) of GABABR GS39783 and BHF177 enhance GABA‐stimulated [35S]GTPγS‐binding, and have shown efficacy in a rodent nicotine self‐administration procedure reflecting aspects of nicotine dependence. Interestingly, the structural related analog, NVP998, had no effect on nicotine self‐administration in rats despite demonstrating similar pharmacokinetic properties. Extensive in vitro characterization of GS39783, BHF177, and NVP998 activity on GABABR‐regulated signaling events, including modulation of cAMP, intracellular calcium levels, and ERK activation, revealed that these structurally related molecules display distinct pathway‐specific signaling activities that correlate with the dissimilarities observed in rodent models and may be predictive of in vivo efficacy. Furthermore, these GABABR allosteric modulators exhibit species‐dependent activity. Collectively, these data will be useful in guiding the development of GABABR allosteric modulators that display optimal in vivo efficacy in a preclinical model of nicotine dependence, and will identify those that have the potential to lead to novel antismoking therapies.

ASK1 and Its Role in Neurodegenerative Diseases

The apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed serine/threonine protein kinase and one of more than 20 members that make up the triple MAP kinase (MAP3K) family of enzymes. Over the past decade, genetic studies have revealed that ASK1 plays a pivitol role in the cellular response to a wide variety of environmental and biological stressors including; reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), endoplasmic reticulum (ER) stress caused by protein aggregation, influx of calcium ions, and receptor-mediated signals transduced via lipopolysaccharides (LPS), Fas ligand, cytokines (TNFα) and certain G protein-coupled receptor (GPCR) agonists [1-5]. In addition, exogenous expression of ASK1 in cells has shown that ASK1 signaling engages the intrinsic apoptosis pathway promoting cytochrome c release from mitochodria and subsequent activation of caspase 3 and 9 [1, 6, 7]. Conversely, ASK1 deficient cells are resistant to cell death induced by oxidative and ER stress, indicating that ASK1 acts as the lynch pin in certain forms of stress-induced cell death [8]. Once activated, ASK1 relays cellular stress signals via the classical three tierd mitogen activated protein kinase (MAPK) signaling cascade, whereby a MAP3K phosphorylates and activates a MAP2K, that in turn phosphorylates and activates a MAPK [9] (Figure 1). More specifically, the ASK1 signaling axis activates the p38 and the c-jun NH2-terminal kinases (JNK) family of MAPKs, via activation of MKK3/MKK6 and MKK4/MKK7 respectively [1, 2, 4]. In addition to its role in the cellular stress response, ASK1 also regulates physiological processes including neuronal differentiation, synaptic plasticity and the innate immune response [10-13]. Thus, ASK1 acts as an important regulator of several important biological processess and not surprisingly, ASK1 activation is under tight regulatory control. Regulation of ASK1 activity is accomplished via a number of mechanisms including; protein-protein interactions as well as both spatial and temporal control. Firstly, more than 30 ASK1 interacting partners have been shown to regulate ASK1 activity (either positively or negatively) by posttranslational modifications and/or by inducing conformational changes through protein-protein interactions. Secondly, ASK1 signaling complexes are located in both the cytoplasm and mitochondria [14], with nuclear translocation observed upon stress induction indicating that ASK1 localization might also dictate the biological outcome [15,16] and thirdly, duration of ASK1 signaling can influence the nature of the

KK-92A, a novel GABAB receptor positive allosteric modulator, attenuates nicotine self-administration and cue-induced nicotine seeking in rats

RationaleGABAB receptors (GABABR) play a critical role in GABAergic neurotransmission in the brain and are thought to be one of the most promising targets for the treatment of drug addiction. GABABR positive allosteric modulators (PAMs) have shown promise as potential anti-addictive therapies, as they lack the sedative and muscle relaxant properties of full GABAB receptor agonists such as baclofen.ObjectivesThe present study was aimed at developing novel, selective, and potent GABABR PAMs with efficacy on abuse-related effects of nicotine.ResultsWe synthetized ~100 analogs of BHF177, a GABABR PAM that has been shown to inhibit nicotine taking and seeking, and tested their activity in multiple cell-based functional assays. Among these compounds, KK-92A displayed superior PAM properties at the GABABR. Interestingly, our results revealed the existence of pathway-selective differential modulation of GABABR signaling by the structurally related GABABR allosteric modulators BHF177 and KK-92A. In vivo, similarly to BHF177, KK-92A inhibited intravenous nicotine self-administration under both fixed- and progressive-ratio schedules of reinforcement in rats. In contrast to BHF177, KK-92A had no effect on food self-administration. Furthermore, KK-92A decreased cue-induced nicotine-seeking behavior without affecting food seeking.ConclusionsThese results indicate that KK-92A is a selective GABABR PAM with efficacy in inhibition of the primary reinforcing and incentive motivational effects of nicotine, and attenuation of nicotine seeking, further confirming that GABABR PAMs may be useful antismoking medications.