Publications by authors named "Chris Ulens"

42 Publications

The structural basis for an on-off switch controlling Gβγ-mediated inhibition of TRPM3 channels.

Proc Natl Acad Sci U S A 2020 11 29;117(46):29090-29100. Epub 2020 Oct 29.

Institut für Physiologie und Pathophysiologie, Philipps-Universität Marburg, 35037 Marburg, Germany;

TRPM3 channels play important roles in the detection of noxious heat and in inflammatory thermal hyperalgesia. The activity of these ion channels in somatosensory neurons is tightly regulated by µ-opioid receptors through the signaling of Gβγ proteins, thereby reducing TRPM3-mediated pain. We show here that Gβγ directly binds to a domain of 10 amino acids in TRPM3 and solve a cocrystal structure of this domain together with Gβγ. Using these data and mutational analysis of full-length proteins, we pinpoint three amino acids in TRPM3 and their interacting partners in Gβ that are individually necessary for TRPM3 inhibition by Gβγ. The 10-amino-acid Gβγ-interacting domain in TRPM3 is subject to alternative splicing. Its inclusion in or exclusion from TRPM3 channel proteins therefore provides a mechanism for switching on or off the inhibitory action that Gβγ proteins exert on TRPM3 channels.
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http://dx.doi.org/10.1073/pnas.2001177117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7682392PMC
November 2020

Editorial: From Peptide and Protein Toxins to Ion Channel Structure/Function and Drug Design.

Front Pharmacol 2020 25;11:548366. Epub 2020 Sep 25.

Department of Molecular Neuroimmune Signaling, Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

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http://dx.doi.org/10.3389/fphar.2020.548366DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7546396PMC
September 2020

Thermodynamic and Evolutionary Coupling between the Native and Amyloid State of Globular Proteins.

Cell Rep 2020 04;31(2):107512

Switch Laboratory, VIB Center for Brain and Disease Research, Herestraat 49, 3000 Leuven, Belgium; Switch Laboratory, Department of Cellular and Molecular Medicine, KU Leuven, Herestraat 49, 3000 Leuven, Belgium. Electronic address:

The amyloid-like aggregation propensity present in most globular proteins is generally considered to be a secondary side effect resulting from the requirements of protein stability. Here, we demonstrate, however, that mutations in the globular and amyloid state are thermodynamically correlated rather than simply associated. In addition, we show that the standard genetic code couples this structural correlation into a tight evolutionary relationship. We illustrate the extent of this evolutionary entanglement of amyloid propensity and globular protein stability. Suppressing a 600-Ma-conserved amyloidogenic segment in the p53 core domain fold is structurally feasible but requires 7-bp substitutions to concomitantly introduce two aggregation-suppressing and three stabilizing amino acid mutations. We speculate that, rather than being a corollary of protein evolution, it is equally plausible that positive selection for amyloid structure could have been a driver for the emergence of globular protein structure.
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http://dx.doi.org/10.1016/j.celrep.2020.03.076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175379PMC
April 2020

Modulation of the ligand-gated ion channel (ELIC) and the 5-HT receptor via a common vestibule site.

Elife 2020 Jan 28;9. Epub 2020 Jan 28.

Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Faculty of Medicine, KU Leuven, Leuven, Belgium.

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote () channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.
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http://dx.doi.org/10.7554/eLife.51511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015668PMC
January 2020

A lipid site shapes the agonist response of a pentameric ligand-gated ion channel.

Nat Chem Biol 2019 12 7;15(12):1156-1164. Epub 2019 Oct 7.

Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABA and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.
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http://dx.doi.org/10.1038/s41589-019-0369-4DOI Listing
December 2019

A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite.

Front Pharmacol 2019 30;10:848. Epub 2019 Jul 30.

Centre for Snakebite Research & Interventions, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.

Snakebite is a neglected tropical disease that causes 138,000 deaths each year. Neurotoxic snake venoms contain small neurotoxins, including three-finger toxins (3FTxs), which can cause rapid paralysis in snakebite victims by blocking postsynaptic transmission nicotinic acetylcholine receptors (nAChRs). These toxins are typically weakly immunogenic and thus are often not effectively targeted by current polyclonal antivenom therapies. We investigated whether nAChR mimics, also known as acetylcholine binding proteins (AChBPs), could effectively capture 3FTxs and therefore be developed as a novel class of snake-generic therapeutics for combatting neurotoxic envenoming. First, we identified the binding specificities of 3FTx from various medically important elapid snake venoms to nAChR using two recombinant nAChR mimics: the AChBP from and a humanized neuronal α7 version (α7-AChBP). We next characterized these AChBP-bound and unbound fractions using SDS-PAGE and mass spectrometry. Interestingly, both mimics effectively captured long-chain 3FTxs from multiple snake species but largely failed to capture the highly related short-chain 3FTxs, suggesting a high level of binding specificity. We next investigated whether nAChR mimics could be used as snakebite therapeutics. We showed that while α7-AChBP alone did not protect against (Egyptian cobra) venom lethality , it significantly prolonged survival times when coadministered with a nonprotective dose of antivenom. Thus, nAChR mimics are capable of neutralizing specific venom toxins and may be useful adjunct therapeutics for improving the safety and affordability of existing snakebite treatments by reducing therapeutic doses. Our findings justify exploring the future development of AChBPs as potential snakebite treatments.
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http://dx.doi.org/10.3389/fphar.2019.00848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6683245PMC
July 2019

Structure-Guided Exploration of SDS22 Interactions with Protein Phosphatase PP1 and the Splicing Factor BCLAF1.

Structure 2019 03 17;27(3):507-518.e5. Epub 2019 Jan 17.

Laboratory of Biosignaling & Therapeutics, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium. Electronic address:

SDS22 is an ancient regulator of protein phosphatase-1 (PP1). Our crystal structure of SDS22 shows that its twelve leucine-rich repeats adopt a banana-shaped fold that is shielded from solvent by capping domains at its extremities. Subsequent modeling and biochemical studies revealed that the concave side of SDS22 likely interacts with PP1 helices α5 and α6, which are distal from the binding sites of many previously described PP1 interactors. Accordingly, we found that SDS22 acts as a "third" subunit of multiple PP1 holoenzymes. The crystal structure of SDS22 also revealed a large basic surface patch that enables binding of a phosphorylated form of splicing factor BCLAF1. Taken together, our data provide insights into the formation of PP1:SDS22 and the recruitment of additional interaction proteins, such as BCLAF1.
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http://dx.doi.org/10.1016/j.str.2018.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6538287PMC
March 2019

Structure of a transporter domain emerges.

Authors:
Chris Ulens

J Biol Chem 2018 12;293(52):20008-20009

From the Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, KU Leuven, 3000 Leuven, Belgium

Magnesium homeostasis relies on transporters like the CNNM family, but little information on these proteins' structure and regulation limits our understanding of their biology and functions in disease. New characterization of a conserved cytoplasmic domain now confirms the presence of a self-liganded architecture that is indispensable for Mg efflux and suggests a possible role for a dimeric assembly.
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http://dx.doi.org/10.1074/jbc.H118.006869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6311507PMC
December 2018

Mutations in the voltage-sensing domain affect the alternative ion permeation pathway in the TRPM3 channel.

J Physiol 2018 06 25;596(12):2413-2432. Epub 2018 Apr 25.

Laboratory of Experimental Gynecology and G-PURE, KU Leuven, Department of Development and Regeneration, Herestraat 49 box 611, B-3000, Leuven, Belgium.

Key Points: Mutagenesis at positively charged amino acids (arginines and lysines) (R1-R4) in the voltage-sensor domain (transmembrane segment (S) 4) of voltage-gated Na , K and Ca channels can lead to an alternative ion permeation pathway distinct from the central pore. Recently, a non-canonical ion permeation pathway was described in TRPM3, a member of the transient receptor potential (TRP) superfamily. The non-canonical pore exists in the native TRPM3 channel and can be activated by co-stimulation of the endogenous agonist pregnenolone sulphate and the antifungal drug clotrimazole or by stimulation of the synthetic agonist CIM0216. Alignment of the voltage sensor of Shaker K channels with the entire TRPM3 sequence revealed the highest degree of similarity in the putative S4 region of TRPM3, and suggested that only one single gating charge arginine (R2) in the putative S4 region is conserved. Mutagenesis studies in the voltage-sensing domain of TRPM3 revealed several residues in the voltage sensor (S4) as well as in S1 and S3 that are crucial for the occurrence of the non-canonical inward currents. In conclusion, this study provides evidence for the involvement of the voltage-sensing domain of TRPM3 in the formation of an alternative ion permeation pathway.

Abstract: Transient receptor potential (TRP) channels are cationic channels involved in a broad array of functions, including homeostasis, motility and sensory functions. TRP channel subunits consist of six transmembrane segments (S1-S6), and form tetrameric channels with a central pore formed by the region encompassing S5 and S6. Recently, evidence was provided for the existence of an alternative ion permeation pathway in TRPM3, which allows large inward currents upon hyperpolarization independently of the central pore. However, very little knowledge is available concerning the localization of this alternative pathway in the native TRPM3 channel protein. Guided by sequence homology with Shaker K channels, in which mutations in S4 can create an analogous 'omega' pore, we performed site-directed mutagenesis studies and patch clamp experiments to identify amino acid residues involved in the formation of the non-canonical pore in TRPM3. Based on our results, we pinpoint four residues in S4 (W982, R985, D988 and G991) as crucial determinants of the properties of the alternative ion permeation pathway.
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http://dx.doi.org/10.1113/JP274124DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6002228PMC
June 2018

An allosteric binding site of the α7 nicotinic acetylcholine receptor revealed in a humanized acetylcholine-binding protein.

J Biol Chem 2018 02 13;293(7):2534-2545. Epub 2017 Dec 13.

From the Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, Faculty of Medicine, KU Leuven, 3000 Leuven, Belgium,

Nicotinic acetylcholine receptors (nAChRs) belong to the family of pentameric ligand-gated ion channels and mediate fast excitatory transmission in the central and peripheral nervous systems. Among the different existing receptor subtypes, the homomeric α7 nAChR has attracted considerable attention because of its possible implication in several neurological and psychiatric disorders, including cognitive decline associated with Alzheimer's disease or schizophrenia. Allosteric modulators of ligand-gated ion channels are of particular interest as therapeutic agents, as they modulate receptor activity without affecting normal fluctuations of synaptic neurotransmitter release. Here, we used X-ray crystallography and surface plasmon resonance spectroscopy of α7-acetylcholine-binding protein (AChBP), a humanized chimera of a snail AChBP, which has 71% sequence similarity with the extracellular ligand-binding domain of the human α7 nAChR, to investigate the structural determinants of allosteric modulation. We extended previous observations that an allosteric site located in the vestibule of the receptor offers an attractive target for receptor modulation. We introduced seven additional humanizing mutations in the vestibule-located binding site of AChBP to improve its suitability as a model for studying allosteric binding. Using a fragment-based screening approach, we uncovered an allosteric binding site located near the β8-β9 loop, which critically contributes to coupling ligand binding to channel opening in human α7 nAChR. This work expands our understanding of the topology of allosteric binding sites in AChBP and, by extrapolation, in the human α7 nAChR as determined by electrophysiology measurements. Our insights pave the way for drug design strategies targeting nAChRs involved in ion channel-mediated disorders.
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http://dx.doi.org/10.1074/jbc.M117.815316DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818190PMC
February 2018

Novel mechanism of modulation at a ligand-gated ion channel; action of 5-Cl-indole at the 5-HT A receptor.

Br J Pharmacol 2016 12 1;173(24):3467-3479. Epub 2016 Nov 1.

Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK.

Background And Purpose: The 5-HT receptor is a prototypical member of the Cys-loop ligand-gated ion channel (LGIC) superfamily and an established therapeutic target. In addition to activation via the orthosteric site, receptor function can be modulated by allosteric ligands. We have investigated the pharmacological action of Cl-indole upon the 5-HT A receptor and identified that this positive allosteric modulator possesses a novel mechanism of action for LGICs.

Experimental Approach: The impact of Cl-indole upon the 5-HT receptor was assessed using single cell electrophysiological recordings and [ H]-granisetron binding in HEK293 cells stably expressing the 5-HT receptor.

Key Results: Cl-indole failed to evoke 5-HT A receptor-mediated responses (up to 30 μM) or display affinity for the [ H]-granisetron binding site. However, in the presence of Cl-indole, termination of 5-HT application revealed tail currents mediated via the 5-HT A receptor that were independent of the preceding 5-HT concentration but were antagonized by the 5-HT receptor antagonist, ondansetron. These tail currents were absent in the 5-HT AB receptor. Furthermore, the presence of 5-HT revealed a concentration-dependent increase in the affinity of Cl-indole for the orthosteric binding site of the human 5-HT A receptor.

Conclusions And Implications: Cl-indole acts as both an orthosteric agonist and an allosteric modulator, but the presence of an orthosteric agonist (e.g. 5-HT) is a prerequisite to reveal both actions. Precedent for ago-allosteric action is available, yet the essential additional presence of an orthosteric agonist is now reported for the first time. This widening of the pharmacological mechanisms to modulate LGICs may offer further therapeutic opportunities.
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http://dx.doi.org/10.1111/bph.13638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5120152PMC
December 2016

Allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine.

Proc Natl Acad Sci U S A 2016 10 10;113(43):E6696-E6703. Epub 2016 Oct 10.

Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium;

Pentameric ligand-gated ion channels or Cys-loop receptors are responsible for fast inhibitory or excitatory synaptic transmission. The antipsychotic compound chlorpromazine is a widely used tool to probe the ion channel pore of the nicotinic acetylcholine receptor, which is a prototypical Cys-loop receptor. In this study, we determine the molecular determinants of chlorpromazine binding in the Erwinia ligand-gated ion channel (ELIC). We report the X-ray crystal structures of ELIC in complex with chlorpromazine or its brominated derivative bromopromazine. Unexpectedly, we do not find a chlorpromazine molecule in the channel pore of ELIC, but behind the β8-β9 loop in the extracellular ligand-binding domain. The β8-β9 loop is localized downstream from the neurotransmitter binding site and plays an important role in coupling of ligand binding to channel opening. In combination with electrophysiological recordings from ELIC cysteine mutants and a thiol-reactive derivative of chlorpromazine, we demonstrate that chlorpromazine binding at the β8-β9 loop is responsible for receptor inhibition. We further use molecular-dynamics simulations to support the X-ray data and mutagenesis experiments. Together, these data unveil an allosteric binding site in the extracellular ligand-binding domain of ELIC. Our results extend on previous observations and further substantiate our understanding of a multisite model for allosteric modulation of Cys-loop receptors.
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http://dx.doi.org/10.1073/pnas.1603101113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5087063PMC
October 2016

Palonosetron-5-HT Receptor Interactions As Shown by a Binding Protein Cocrystal Structure.

ACS Chem Neurosci 2016 12 22;7(12):1641-1646. Epub 2016 Sep 22.

Department of Biochemistry, University of Cambridge , Tennis Court Road, Cambridge CB2 1QW, United Kingdom.

Palonosetron is a potent 5-HT receptor antagonist and an effective therapeutic agent against emesis. Here we identify the molecular determinants of compound recognition in the receptor binding site by obtaining a high resolution structure of palonosetron bound to an engineered acetylcholine binding protein that mimics the 5-HT receptor binding site, termed 5-HTBP, and by examining the potency of palonosetron in a range of 5-HT receptors with mutated binding site residues. The structural data indicate that palonosetron forms a tight and effective wedge in the binding pocket, made possible by its rigid tricyclic ring structure and its interactions with binding site residues; it adopts a binding pose that is distinct from the related antiemetics granisetron and tropisetron. The functional data show many residues previously shown to interact with agonists and antagonists in the binding site are important for palonosetron binding, and indicate those of particular importance are W183 (a cation-π interaction and a hydrogen bond) and Y153 (a hydrogen bond). This information, and the availability of the structure of palonosetron bound to 5-HTBP, should aid the development of novel and more efficacious drugs that act via 5-HT receptors.
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http://dx.doi.org/10.1021/acschemneuro.6b00132DOI Listing
December 2016

Functional and Biochemical Characterization of Alvinella pompejana Cys-Loop Receptor Homologues.

PLoS One 2016 21;11(3):e0151183. Epub 2016 Mar 21.

Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, University of Leuven, Leuven, Belgium.

Cys-loop receptors are membrane spanning ligand-gated ion channels involved in fast excitatory and inhibitory neurotransmission. Three-dimensional structures of these ion channels, determined by X-ray crystallography or electron microscopy, have revealed valuable information regarding the molecular mechanisms underlying ligand recognition, channel gating and ion conductance. To extend and validate the current insights, we here present promising candidates for further structural studies. We report the biochemical and functional characterization of Cys-loop receptor homologues identified in the proteome of Alvinella pompejana, an extremophilic, polychaete annelid found in hydrothermal vents at the bottom of the Pacific Ocean. Seven homologues were selected, named Alpo1-7. Five of them, Alpo2-6, were unidentified prior to this study. Two-electrode voltage clamp experiments revealed that wild type Alpo5 and Alpo6, both sharing remarkably high sequence identity with human glycine receptor α subunits, are anion-selective channels that can be activated by glycine, GABA and taurine. Furthermore, upon expression in insect cells fluorescence size-exclusion chromatography experiments indicated that four homologues, Alpo1, Alpo4, Alpo6 and Alpo7, can be extracted out of the membrane by a wide variety of detergents while maintaining their oligomeric state. Finally, large-scale purification efforts of Alpo1, Alpo4 and Alpo6 resulted in milligram amounts of biochemically stable and monodisperse protein. Overall, our results establish the evolutionary conservation of glycine receptors in annelids and pave the way for future structural studies.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0151183PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4801368PMC
August 2016

Structural hot spots for the solubility of globular proteins.

Nat Commun 2016 Feb 24;7:10816. Epub 2016 Feb 24.

VIB Switch Laboratory, Flanders Institute for Biotechnology (VIB), 3000 Leuven, Belgium.

Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function.
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http://dx.doi.org/10.1038/ncomms10816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4770091PMC
February 2016

Development of Plate Reader and On-Line Microfluidic Screening to Identify Ligands of the 5-Hydroxytryptamine Binding Protein in Venoms.

Toxins (Basel) 2015 Jun 24;7(7):2336-53. Epub 2015 Jun 24.

AIMMS Division of BioAnalytical Chemistry, Faculty of Sciences, VU University Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.

The 5-HT3 receptor is a ligand-gated ion channel, which is expressed in the nervous system. Its antagonists are used clinically for treatment of postoperative- and radiotherapy-induced emesis and irritable bowel syndrome. In order to better understand the structure and function of the 5-HT3 receptor, and to allow for compound screening at this receptor, recently a serotonin binding protein (5HTBP) was engineered with the Acetylcholine Binding Protein as template. In this study, a fluorescence enhancement assay for 5HTBP ligands was developed in plate-reader format and subsequently used in an on-line microfluidic format. Both assay types were validated using an existing radioligand binding assay. The on-line microfluidic assay was coupled to HPLC via a post-column split which allowed parallel coupling to a mass spectrometer to collect MS data. This high-resolution screening (HRS) system is well suitable for compound mixture analysis. As a proof of principle, the venoms of Dendroapsis polylepis, Pseudonaja affinis and Pseudonaja inframacula snakes were screened and the accurate masses of the found bioactives were established. To demonstrate the subsequent workflow towards structural identification of bioactive proteins and peptides, the partial amino acid sequence of one of the bioactives from the Pseudonaja affinis venom was determined using a bottom-up proteomics approach.
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http://dx.doi.org/10.3390/toxins7072336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4516916PMC
June 2015

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor.

Proc Natl Acad Sci U S A 2015 May 27;112(19):E2543-52. Epub 2015 Apr 27.

Laboratory of Structural Neurobiology, Katholieke Universiteit Leuven, Leuven B-3000, Belgium;

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.
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http://dx.doi.org/10.1073/pnas.1418289112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4434711PMC
May 2015

Different ligands of the TRPV3 cation channel cause distinct conformational changes as revealed by intrinsic tryptophan fluorescence quenching.

J Biol Chem 2015 May 31;290(20):12964-74. Epub 2015 Mar 31.

From the Laboratory of Structural Neurobiology and TRP Research Platform Leuven (TRPLe), Department of Cellular and Molecular Medicine, University of Leuven, Herestraat 49 Box 601, 3000 Leuven, Belgium.

TRPV3 is a thermosensitive ion channel primarily expressed in epithelial tissues of the skin, nose, and tongue. The channel has been implicated in environmental thermosensation, hyperalgesia in inflamed tissues, skin sensitization, and hair growth. Although transient receptor potential (TRP) channel research has vastly increased our understanding of the physiological mechanisms of nociception and thermosensation, the molecular mechanics of these ion channels are still largely elusive. In order to better comprehend the functional properties and the mechanism of action in TRP channels, high-resolution three-dimensional structures are indispensable, because they will yield the necessary insights into architectural intimacies at the atomic level. However, structural studies of membrane proteins are currently hampered by difficulties in protein purification and in establishing suitable crystallization conditions. In this report, we present a novel protocol for the purification of membrane proteins, which takes advantage of a C-terminal GFP fusion. Using this protocol, we purified human TRPV3. We show that the purified protein is a fully functional ion channel with properties akin to the native channel using planar patch clamp on reconstituted channels and intrinsic tryptophan fluorescence spectroscopy. Using intrinsic tryptophan fluorescence spectroscopy, we reveal clear distinctions in the molecular interaction of different ligands with the channel. Altogether, this study provides powerful tools to broaden our understanding of ligand interaction with TRPV channels, and the availability of purified human TRPV3 opens up perspectives for further structural and functional studies.
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http://dx.doi.org/10.1074/jbc.M114.628925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4432310PMC
May 2015

Varenicline Interactions at the 5-HT3 Receptor Ligand Binding Site are Revealed by 5-HTBP.

ACS Chem Neurosci 2015 Jul 19;6(7):1151-7. Epub 2015 Feb 19.

†Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, United Kingdom.

Cys-loop receptors are the site of action of many therapeutic drugs. One of these is the smoking cessation agent varenicline, which has its major therapeutic effects at nicotinic acetylcholine (nACh) receptors but also acts at 5-HT3 receptors. Here, we report the X-ray crystal structure of the 5-HT binding protein (5-HTBP) in complex with varenicline, and test the predicted interactions by probing the potency of varenicline in a range of mutant 5-HT3 receptors expressed in HEK293 cells and Xenopus oocytes. The structure reveals a range of interactions between varenicline and 5-HTBP. We identified residues within 5 Å of varenicline and substituted the equivalent residues in the 5-HT3 receptor with Ala or a residue with similar chemical properties. Functional characterization of these mutant 5-HT3 receptors, using a fluorescent membrane potential dye in HEK cells and voltage clamp in oocytes, supports interactions between varenicline and the receptor that are similar to those in 5-HTBP. The structure also revealed C-loop closure that was less than in the 5-HT-bound 5-HTBP, and hydrogen bonding between varenicline and the complementary face of the binding pocket via a water molecule, which are characteristics consistent with partial agonist behavior of varenicline in the 5-HT3 receptor. Together, these data reveal detailed insights into the molecular interaction of varenicline in the 5-HT3 receptor.
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http://dx.doi.org/10.1021/cn500369hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4505686PMC
July 2015

Structure of the SthK carboxy-terminal region reveals a gating mechanism for cyclic nucleotide-modulated ion channels.

PLoS One 2015 27;10(1):e0116369. Epub 2015 Jan 27.

Laboratory of Structural Neurobiology, KU Leuven, Herestraat 49, PB601, Leuven, B-3000, Belgium.

Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116369PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4308110PMC
January 2016

The prokaryote ligand-gated ion channel ELIC captured in a pore blocker-bound conformation by the Alzheimer's disease drug memantine.

Structure 2014 Oct 4;22(10):1399-407. Epub 2014 Sep 4.

Department of Biochemistry, University of Cambridge, Cambridge CB2 1QW, UK.

Pentameric ligand-gated ion channels (pLGIC) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules, including the Alzheimer's drug memantine, block ion conduction at vertebrate pLGICs by plugging the channel pore. We show that memantine has similar potency in ELIC, a prokaryotic pLGIC, when it contains an F16'S pore mutation. X-ray crystal structures, using both memantine and its derivative, Br-memantine, reveal that the ligand is localized at the extracellular entryway of the channel pore, and the pore is in a more closed conformation than wild-type ELIC in both the presence and absence of memantine. However, using voltage clamp fluorometry we observe fluorescence changes in opposite directions during channel activation and pore block, revealing an additional conformational transition not apparent from the crystal structures. These results have important implications for drugs such as memantine, which block channel pores.
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http://dx.doi.org/10.1016/j.str.2014.07.013DOI Listing
October 2014

Family of prokaryote cyclic nucleotide-modulated ion channels.

Proc Natl Acad Sci U S A 2014 May 12;111(21):7855-60. Epub 2014 May 12.

Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and

Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24-32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K(+) ions over Na(+) ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.
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http://dx.doi.org/10.1073/pnas.1401917111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040583PMC
May 2014

Structure-function elucidation of a new α-conotoxin, Lo1a, from Conus longurionis.

J Biol Chem 2014 Apr 24;289(14):9573-83. Epub 2014 Feb 24.

From Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg, O&N2, 3000 Leuven, Belgium.

α-Conotoxins are peptide toxins found in the venom of marine cone snails and potent antagonists of various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs are cholinergic receptors forming ligand-gated ion channels in the plasma membranes of certain neurons and the neuromuscular junction. Because nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, nAChR dysfunctions have been implicated in a variety of severe pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson disease, and Alzheimer disease. To expand the knowledge concerning cone snail toxins, we examined the venom of Conus longurionis. We isolated an 18-amino acid peptide named α-conotoxin Lo1a, which is active on nAChRs. To the best of our knowledge, this is the first characterization of a conotoxin from this species. The peptide was characterized by electrophysiological screening against several types of cloned nAChRs expressed in Xenopus laevis oocytes. The three-dimensional solution structure of the α-conotoxin Lo1a was determined by NMR spectroscopy. Lo1a, a member of the α4/7 family, blocks the response to acetylcholine in oocytes expressing α7 nAChRs with an IC50 of 3.24 ± 0.7 μM. Furthermore, Lo1a shows a high selectivity for neuronal versus muscle subtype nAChRs. Because Lo1a has an unusual C terminus, we designed two mutants, Lo1a-ΔD and Lo1a-RRR, to investigate the influence of the C-terminal residue. Lo1a-ΔD has a C-terminal Asp deletion, whereas in Lo1a-RRR, a triple-Arg tail replaces the Asp. They blocked the neuronal nAChR α7 with a lower IC50 value, but remarkably, both adopted affinity for the muscle subtype α1β1δε.
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http://dx.doi.org/10.1074/jbc.M114.556175DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3975008PMC
April 2014

Affinity comparison of p3 and p8 peptide displaying bacteriophages using surface plasmon resonance.

Anal Chem 2013 Nov 16;85(21):10075-82. Epub 2013 Oct 16.

BIOSYST-MeBioS, KU Leuven , Willem De Croylaan 42, P.O. Box 2428, B-3001 Leuven, Belgium.

Ever increasing demands in sensitivity and specificity of biosensors have recently established a trend toward the use of multivalent bioreceptors. This trend has also been introduced in the field of bacteriophage affinity peptides, where the entire phage is used as a receptor rather than the individual peptides. Although this approach is gaining in popularity due to the numerous advantages, binding kinetics of complete phage particles have never been studied in detail, notwithstanding being essential for the efficient design of such applications. In this paper we used an in house developed fiber-optic surface plasmon resonance (FO-SPR) biosensor to study the affinity and binding kinetics of phages, displaying peptide libraries. By using either peptide expression on the p3 or on the p8 coat proteins, a corresponding density of 5 up to more than 2000 peptides on a single virus particle was obtained. Binding parameters of 26 different filamentous phages, displaying peptides selective for enhanced Green Fluorescent Protein (eGFP), were characterized. This study revealed a broad affinity range of phages for the target eGFP, indicating their potential to be used for applications with different requirements in binding kinetics. Moreover, detailed analysis of koff and kon values of several selected p3 and p8 phages, using the FO-SPR biosensor, clearly showed the correlation between the binding parameters and the density at which eGFP-peptides are being expressed. Consequently, although p3 and p8-based phages both revealed exceptionally high affinities for eGFP, two p8 phages were found to have the highest affinity with dissociation constants (Kd) in the femtomolar range.
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http://dx.doi.org/10.1021/ac402192kDOI Listing
November 2013

Structural insights into Cys-loop receptor function and ligand recognition.

Biochem Pharmacol 2013 Oct 10;86(8):1042-53. Epub 2013 Jul 10.

Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, KU Leuven, Herestraat 49, PB 601, B-3000 Leuven, Belgium. Electronic address:

This review outlines recent insights into ligand recognition, channel gating and ion permeation for the family of pentameric ligand-gated ion channels (pLGICs). These receptors are involved in the fast inhibitory and excitatory neurotransmission. Prototypical anion-selective members are the γ-amino butyric acid type A (GABA(A)), γ-amino butyric acid type C (GABA(C)) and glycine receptor. The cation-selective members are the 5-HT3 serotonin and nicotinic acetylcholine (nACh) receptors. They are the target for a wide variety of drugs and dysfunction in these receptors is associated with several diseases. We summarize recent structural knowledge in combination with electrophysiological data and molecular dynamic simulations, thereby describing key features of ligand binding, channel gating and ion permeation. A conserved cation-π interaction between ligand and aromatic residues of the ligand binding site critically contributes to ligand recognition, as revealed by X-ray crystal structures of acetylcholine binding proteins, as well as the integral pLGICs, ELIC and GluCl. In addition, we summarize the possible downstream effects on gating of structural rearrangements in the extracellular ligand-binding domain, which mainly occur in loop C and loop F. These data are discussed in the context of different conformational states of the pore-forming domain observed in crystal structures of GLIC and GluCl, which likely represent the open pore conformation, and ELIC, which likely corresponds to a closed pore conformation. We conclude with a current structural view on the determinants of ion selection and permeation.
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http://dx.doi.org/10.1016/j.bcp.2013.07.001DOI Listing
October 2013

Multisite binding of a general anesthetic to the prokaryotic pentameric Erwinia chrysanthemi ligand-gated ion channel (ELIC).

J Biol Chem 2013 Mar 30;288(12):8355-64. Epub 2013 Jan 30.

Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, KU Leuven, Herestraat 49, PB 601, B-3000 Leuven, Belgium.

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABA(A/C) receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7-β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.
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http://dx.doi.org/10.1074/jbc.M112.424507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3605653PMC
March 2013

Structural basis of ligand recognition in 5-HT3 receptors.

EMBO Rep 2013 Jan 30;14(1):49-56. Epub 2012 Nov 30.

Laboratory of Structural Neurobiology, KULeuven, Leuven, Belgium.

The 5-HT(3) receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT(3) receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation-π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT(3) receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT(3) receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT(3) receptor.
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http://dx.doi.org/10.1038/embor.2012.189DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3537142PMC
January 2013

Structure of NPP1, an ectonucleotide pyrophosphatase/phosphodiesterase involved in tissue calcification.

Structure 2012 Nov 4;20(11):1948-59. Epub 2012 Oct 4.

Laboratory of Biosignaling and Therapeutics, Department of Cellular and Molecular Medicine, University of Leuven, 3000 Leuven, Belgium.

Ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) converts extracellular nucleotides into inorganic pyrophosphate, whereas its close relative NPP2/autotaxin hydrolyzes lysophospholipids. NPP1 regulates calcification in mineralization-competent tissues, and a lack of NPP1 function underlies calcification disorders. Here, we show that NPP1 forms homodimers via intramembrane disulfide bonding, but is also processed intracellularly to a secreted monomer. The structure of secreted NPP1 reveals a characteristic bimetallic active site and a nucleotide-binding groove, but it lacks the lipid-binding pocket and open tunnel present in NPP2. A loop adjacent to the nucleotide-binding site, which is disordered in NPP2, is well ordered in NPP1 and might promote nucleotide binding. Remarkably, the N-terminal somatomedin B-like domains of NPP1, unlike those in NPP2, are flexible and do not contact the catalytic domain. Our results provide a structural basis for the nucleotide pyrophosphatase activity of NPP1 and help to understand how disease-causing mutations may affect NPP1 structure and function.
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http://dx.doi.org/10.1016/j.str.2012.09.001DOI Listing
November 2012

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines.

Proc Natl Acad Sci U S A 2012 Oct 3;109(44):E3028-34. Epub 2012 Oct 3.

Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, Catholic University of Leuven, 3000 Leuven, Belgium.

GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA(A) receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.
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http://dx.doi.org/10.1073/pnas.1208208109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497736PMC
October 2012

Insights into the structural determinants required for high-affinity binding of chiral cyclopropane-containing ligands to α4β2-nicotinic acetylcholine receptors: an integrated approach to behaviorally active nicotinic ligands.

J Med Chem 2012 Sep 7;55(18):8028-37. Epub 2012 Sep 7.

Drug Discovery Program, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612, USA.

Structure-based drug design can potentially accelerate the development of new therapeutics. In this study, a cocrystal structure of the acetylcholine binding protein (AChBP) from Capitella teleta (Ct) in complex with a cyclopropane-containing selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for ligand binding obtained from this AChBP X-ray structure were used to refine a previous model of the human α4β2-nAChR, thus possibly providing a better understanding of the structure of the human receptor. To validate the potential application of the structure of the Ct-AChBP in the engineering of new α4β2-nAChR ligands, homology modeling methods, combined with in silico ADME calculations, were used to design analogues of compound 5. The most promising compound, 12, exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining favorable pharmacological parameters together with appropriate behavioral end points in the rodent studies.
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http://dx.doi.org/10.1021/jm3008739DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3464052PMC
September 2012