Biased Ligands Differentially Shape the Conformation of The
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International Journal of Molecular Sciences Article Biased Ligands Differentially Shape the Conformation of the Extracellular Loop Region in 5-HT2B Receptors Katrin Denzinger, Trung Ngoc Nguyen, Theresa Noonan, Gerhard Wolber and Marcel Bermudez * Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Strasse 2-4, 14195 Berlin, Germany; [email protected] (K.D.); [email protected] (T.N.N.); [email protected] (T.N.); [email protected] (G.W.) * Correspondence: [email protected] Received: 22 November 2020; Accepted: 18 December 2020; Published: 20 December 2020 Abstract: G protein-coupled receptors are linked to various intracellular transducers, each pathway associated with different physiological effects. Biased ligands, capable of activating one pathway over another, are gaining attention for their therapeutic potential, as they could selectively activate beneficial pathways whilst avoiding those responsible for adverse effects. We performed molecular dynamics simulations with known β-arrestin-biased ligands like lysergic acid diethylamide and ergotamine in complex with the 5-HT2B receptor and discovered that the extent of ligand bias is directly connected with the degree of closure of the extracellular loop region. Given a loose allosteric coupling of extracellular and intracellular receptor regions, we delineate a concept for biased signaling at serotonin receptors, by which conformational interference with binding pocket closure restricts the signaling repertoire of the receptor. Molecular docking studies of biased ligands gathered from the BiasDB demonstrate that larger ligands only show plausible docking poses in the ergotamine-bound structure, highlighting the conformational constraints associated with bias. This emphasizes the importance of selecting the appropriate receptor conformation on which to base virtual screening workflows in structure-based drug design of biased ligands. As this mechanism of ligand bias has also been observed for muscarinic receptors, our studies provide a general mechanism of signaling bias transferable between aminergic receptors. Keywords: GPCR; biased signaling; molecular dynamics; serotonin receptors; virtual screening; conformational descriptor; pharmacophore; drug design 1. Introduction G protein-coupled receptors (GPCRs) are involved in the regulation of a plethora of physiological processes, and about 35% of currently marketed therapeutics directly target GPCRs [1]. In the last decade, structural and biophysical data helped to revisit our understanding of GPCR activation and revealed a surprisingly complex pharmacology [2] including several dimensions of spatiotemporal signaling [3–8]. One important aspect of this complexity is the preference of a certain ligand–receptor complex for one intracellular transducer protein over another, commonly referred to as biased signaling or functional selectivity [9]. Signaling bias can contribute to a desired effect or trigger adverse reactions [10–12]. Its rational design remains challenging, but without any doubt, biased ligands bear a vast therapeutic potential to be uncovered [13–15]. Mechanistic understanding of ligand-triggered receptor activation is key to develop drugs with biased signaling properties. Current activation models are based on allosteric coupling of the ligand binding site with intracellular receptor regions allowing for binding of different intracellular binding partners like G Int. J. Mol. Sci. 2020, 21, 9728; doi:10.3390/ijms21249728 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2020, 21, x FOR PEER REVIEW 2 of 14 Int. J. Mol. Sci. 2020, 21, 9728 2 of 14 Current activation models are based on allosteric coupling of the ligand binding site with intracellular receptor regions allowing for binding of different intracellular binding partners like G proteinsproteins oror ββ-arrestin.-arrestin. ActivationActivation uponupon ligandligand bindingbinding causescauses long-rangelong-range conformationalconformational changeschanges withinwithin thethe receptor,receptor, resultingresulting inin anan outwardoutward movementmovement ofof thethe intracellularintracellular partpart ofof transmembranetransmembrane domaindomain 66 (TM6)(TM6) toto enableenable bindingbinding ofof intracellularintracellular transducerstransducers andand subsequentsubsequent releaserelease ofof secondsecond messengermessenger molecules [16]. [16]. Reciprocally, Reciprocally, G-protein G-protein binding binding causes causes a aclosure closure of ofthe the binding binding pocket pocket by bydrawing drawing together together extracellular extracellular loops loops 2 2and and 3 3(ECL (ECL22 and and ECL3, ECL3, respectively), trapping the ligandligand withinwithin thethe bindingbinding pocketpocket andand stabilizingstabilizing thethe ternaryternary GPCRGPCR complexcomplex [[17].17]. DueDue toto thethe dynamicdynamic naturenature ofof GPCRs,GPCRs, multiplemultiple receptorreceptor conformationsconformations existexist betweenbetween thethe fullyfully activeactive andand inactiveinactive state,state, andand eacheach ligandligand stabilizes a a distinct distinct ensemble ensemble of of receptor receptor co conformationsnformations [18,19]. [18,19 Thus,]. Thus, ligands ligands that that extend extend into intothe extracellular the extracellular vestibule vestibule can affect can atheffect degree the degree of binding of binding pocket pocketclosure closurevia their via interaction their interaction patterns patternswith extracellular with extracellular epitopes. epitopes. This in turn This might in turn influence might influence the degree the of degree intracellular of intracellular opening resulting opening resultingin a differential in a diff recruitmenterential recruitment of signal of transducer signal transducerss (Figure (Figure1) [20].1 The)[ 20 relationship]. The relationship between between ligand ligandinteraction interaction patterns patterns at the at extracellular the extracellular loop loop region, region, ECL ECL closure, closure, and and resulting resulting pharmacological functionsfunctions hashas recentlyrecently beenbeen studiedstudied withwith bitopicbitopic ligandsligands at muscarinic receptors (SI Figure S1) [21,22]. [21,22]. TheseThese findingsfindings show show that that ligand-dependent ligand-dependent conformational conformational constraints constraints at theat the extracellular extracellular binding binding site cansite becan linked be linked to a restrictionto a restriction of the of muscarinic the muscarinic receptor’s receptor’s signaling signaling repertoire. repertoire. Figure 1. A schematic representation of the general activation mechanism illustrates the full signaling Figure 1. A schematic representation of the general activation mechanism illustrates the full signaling repertoire of a classical orthosteric agonist (left, green circle) and the restricted conformational freedom repertoire of a classical orthosteric agonist (left, green circle) and the restricted conformational by extended molecular motives (middle, salmon shape) resulting in biased signaling. One example of freedom by extended molecular motives (middle, salmon shape) resulting in biased signaling. One such extended (bitopic) ligands is iper-8-phth, which is shown on the right in its proposed binding example of such extended (bitopic) ligands is iper-8-phth, which is shown on the right in its proposed mode at the M1 receptor [22]. While the iperoxo building block (blue surface) binds to the orthosteric binding mode at the M1 receptor [22]. While the iperoxo building block (blue surface) binds to the binding pocket, the extended molecular structure (grey shape) spatially interferes with extracellular orthosteric binding pocket, the extended molecular structure (grey shape) spatially interferes with loop regions. extracellular loop regions. The present study follows the hypothesis that this mechanism for biased signaling is transferable to otherThe aminergicpresent study receptors. follows the Given hypothesis the availability that this mechanism of structural for and biased functional signaling data, is transferable we focus to other aminergic receptors. Given the availability of structural and functional data, we focus on the on the 5-HT2BR and its co-crystallized, nature-derived, β–arrestin-biased ligands lysergic acid diethylamide5-HT2BR and its (LSD) co-crystallized, and ergotamine nature-derived, [23,24]. Weβ–arrestin-biased compare their ligands influence lysergic on extracellularacid diethylamide loop dynamics(LSD) and with ergotamine the unbiased [23,24]. endogenous We compare ligand theirserotonin influence (5-HT)on extracellular by means loop of molecular dynamics dynamics with the unbiased endogenous ligand serotonin (5-HT) by means of molecular dynamics (MD) simulations (MD) simulations and dynamic pharmacophores. The analysis of additional biased 5-HT2BR ligands fromand dynamic the BiasDB pharmacophores., a publicly available The analysis database of additional for biased biased GPCR 5-HT ligands2BR [ligands25], and from recent the structural BiasDB, a publicly available database for biased GPCR ligands [25], and recent structural data of the 5-HT1AR, data of the 5-HT1AR, 5-HT1BR, and dopamine D2 receptor support our findings [23,26–28]. Given the existence5-HT1BR, ofand pathway-specific dopamine D2 receptor binding support site conformations, our findings we[23,26 discuss–28]. implicationsGiven the existence for structure-based of pathway- approachesspecific binding in drug site design, conformations, providing