Structural determinants of 5-HT2B receptor activation and biased agonism

Abstract

Serotonin (5-hydroxytryptamine; 5-HT) receptors modulate a variety of physiological processes ranging from perception, cognition and emotion to vascular and smooth muscle contraction, platelet aggregation, gastrointestinal function and reproduction. Drugs that interact with 5-HT receptors effectively treat diseases as diverse as migraine headaches, depression and obesity. Here we present four structures of a prototypical serotonin receptor-the human 5-HT_2B receptor-in complex with chemically and pharmacologically diverse drugs, including methysergide, methylergonovine, lisuride and LY266097. A detailed analysis of these structures complemented by comprehensive interrogation of signaling illuminated key structural determinants essential for activation. Additional structure-guided mutagenesis experiments revealed binding pocket residues that were essential for agonist-mediated biased signaling and β-arrestin2 translocation. Given the importance of 5-HT receptors for a large number of therapeutic indications, insights derived from these studies should accelerate the design of safer and more effective medications.

Figure 1.. Structural insights into a 5-HT_2B activation mechanism

Identification of an 5-HT_2BR activation mechanism by the 5-HT_2BR-methylergonovine structure a) Structure-activity-relationship of 5-HT_2BR ergoline ligands comparing unsubstituted N(1)-H ligands (red) such as 5-HT, ergotamine, and methylergonovine to N(1)-methyl methysergide (blue) b) 5-HT_2BR Gq calcium flux activity by 5-HT (black, EC₅₀ = 1.4 nM, Emax = 100%), methylergonovine (red, EC₅₀ = 31 nM, Emax = 66%) and lack of agonist activity by methysergide (blue, closed circles). Methysergide instead acts as a competitive antagonist (blue, open circles, IC₅₀ = 2.4 nM) in response to 5-HT. c) Structure of methylergonovine (blue) at 5-HT_2BR (green) with 2D ligand plots of nearby residues (PDB code: 6DRY). d) Close up of methylergonovine binding pose in the binding pocket highlighting D135^3.32 interacting with the charged nitrogen of methysergide, and the indole N(1)-H interacting with both T140^3.37 and A225^5.46 in the orthosteric binding pocket. e) Methylergonovine Gq-mediated calcium flux agonist activity at T140A^3.37 (red), T140V^3.37 (blue), T140S^3.37 (green, EC₅₀ = 18 nM, Emax = 64%) relative to 5-HT_2BR wild-type (WT, black, EC₅₀ = 19 nM, Emax = 66%). f) Methylergonovine Gq-mediated calcium flux agonist activity at A225S^5.46 (red, EC₅₀ = 15 nM, Emax = 58%), A225G^5.46 (blue, EC₅₀ = 12 nM, Emax = 55%) relative to 5-HT_2BR wild-type (WT, black, EC₅₀ = 23 nM, Emax = 64%). Data in panels b, e, and f represent mean and S.E.M from three independent experiments (N=3) performed in triplicate.

Figure 2.. Structure of a 5-HT_2BR-A225G^5.46 mutant designed to be activated by methysergide

Design and structure of a 5-HT_2BR mutant to convert methysergide into an agonist. a) Design of mutation to convert methysergide into an agonist by accommodating the N(1)-methyl (blue) with vdW interaction by T140V^3.37 or with space by A225G^5.46. 5-HT_2BR methysergide Gq-mediated calcium flux activity at 5-HT_2BR wild-type (black), T140V^3.37 (blue) and A225G^5.46 (green, EC₅₀ = 12 nM, Emax = 54%). Data represent mean and S.E.M from three independent experiments (N=3) performed in triplicate. b) Structure of the 5-HT_2BR A225G^5.46 mutant (blue) in complex with methysergide (purple) indicating that the N(1)-methyl is positioned toward residues T140^3.37 and A225^5.46 in the orthosteric binding pocket, which is also illustrated in 2D ligand plot of nearby residues (PDB code: 6DRZ). c) Space-filling comparison of the 5-HT_2BR-methylergonovine and the 5-HT_2BR- A225G^5.46-methysergide structures indicating that the A225G^5.46 mutant creates space to accommodate methysergide’s N(1)-methyl to achieve a similar pose as seen in the 5-HT_2BR-methylergonovine structure. d) Comparison of the binding poses of the 5-HT_2BR- A225G^5.46-methysergide structure to the β2AR (green) in complex with the inverse agonist ICI 118,551 (orange) indicating a 1–2 Å shift difference in TM5 commonly observed in active versus inactive structures.

Figure 3.. Structural basis for a 5-HT_2B activation mechanism via the extended binding pocket

Identification of an activation mechanism via the extended binding pocket illuminated by the 5-HT_2BR-lisuride structure. a) Comparison of the chemical structures of LSD to lisuride indicating LSD’s (R) diethylamide (purple) and lisuride’s (S)-diethylurea (blue) lead to either 5-HT_2BR agonism or antagonism, respectively. b) 5-HT_2BR Gq-mediated calcium flux activity indicating LSD partial Gq agonist activity (purple, EC₅₀ = 40 nM, Emax = 82%), and lack of lisuride agonist activity (blue, closed circles), and instead demonstrating competitive antagonism by lisuride (blue, open circles, IC₅₀ = 25 nM). c) Structure of lisuride (blue) at the 5-HT_2BR (light blue) comparing LSD (purple) bound to 5-HT_2BR (green) showing lisuride’s (S)-diethylurea wedged between TM3 residues W131^3.28 and L132^3.29 and making no contact with TM7 L362^7.35 (PDB code: 6DRX). d) Lisuride Gq-mediated calcium flux activity at L362N^7.35 (green, EC₅₀ = 395 nM, Emax = 41%), L362Y^7.35 (orange, EC₅₀ = 465 nM, Emax = 69%), and L362F^7.35 (purple, EC₅₀ = 77 nM, Emax = 60%). e) LSD Gq-mediated calcium flux activity at L362A^7.35 (red, EC₅₀ = 340 nM, Emax = 67%) relative to 5-HT_2BR wild-type (WT, purple, EC₅₀ = 37 nM, Emax = 79%). Data in panels b, d, and e represent mean and S.E.M from three independent experiments (N=3) performed in triplicate. f) Schematic illustrating ligand contact with residue L362^7.35 in TM7 in the extended binding pocket leads to 5-HT_2BR activation.

Figure 4.. Divergent actions on β-arrestin2 recruitment by OBP versus EBP mutations

Examination of β-arrestin2 recruitment activity at OBP T140A^3.37 and A225G^5.46 mutations versus EBP mutation L362F^7.35. a) Methylergonovine Gq-mediated calcium flux (left panel) comparing T140A^3.37 (red) to 5-HT_2BR WT (black, EC₅₀ = 21 nM). β-arrestin2 recruitment (right panel) comparing T140A^3.37 (red) to 5-HT_2BR WT (black, EC₅₀ = 1.2 nM). b) Methysergide Gq-mediated calcium flux (left panel) comparing A225G^5.46 (green, EC₅₀ = 33 nM) to 5-HT_2BR WT (black). β-arrestin2 recruitment (right panel) comparing A225G^5.46 (green, EC₅₀ = 1.7 nM) to 5-HT_2BR WT (black). c) Lisuride Gq-mediated calcium flux (left panel) comparing L362F^7.35 (purple, EC₅₀ = 65 nM) to 5-HT_2BR WT (black). β-arrestin2 recruitment (right panel) comparing L362F^7.35 (purple) to 5-HT_2BR WT (black). d) LSD Gq-mediated calcium flux (left panel) comparing L362F^7.35 (purple, EC₅₀ = 40 nM) to 5-HT_2BR WT (black, EC₅₀ = 42 nM). β-arrestin2 recruitment (right panel) comparing L362F^7.35 (purple) to 5-HT_2BR WT (black, EC₅₀ = 0.97 nM). Data in panels a-d are expressed as fold-over-basal and represent mean and S.E.M from three independent experiments (N=3) performed in triplicate. e) LSD dissociation comparing 5-HT_2BR wild-type (black, k_off = 0.015 min⁻¹) to the L362F^7.35 mutant (purple, k_off = 0.240 min⁻¹). Data represent percent specific binding indicating mean and S.E.M from three independent experiments (N=3) performed in duplicate. f) Schematic comparing the location of the EBP residues L209^EL2 and L362^7.35 (purple), which can result in either Gq or β-arrestin2 recruitment preference, to the location of OBP residues T140^3.37 and A225^5.46 (green), which result in equal contributions to Gq activity and β-arrestin2 recruitment.

Figure 5.. Structure of 5-HT_2BR-LY266097 reveals TM7 as a trigger for biased signaling

Structure of 5-HT_2BR in complex with LY266097 reveals determinants of ligand bias via TM7 a) 5-HT_2BR (orange) in complex with LY266097 (green) with 2D ligand plot of nearby residues (PDB code: 6DS0). b) View from the top of the receptor showing 2-chloro-3,4-dimethoxybenzyl substitution of LY266097 is oriented in close proximity to residue L362^7.35 in TM7. c) Alignment of the 5-HT_2BR-LY266097 and 5-HT_2BR-lisuride structures showing that LY266097’s 2-chloro-3,4-dimethoxybenzyl substitution is within 3.3 Å from L362^7.35, whereas lisuride’s (S)-diethylurea is further away at 6.3 Å from L362^7.35. d) Profiling of LY266097 for ligand bias showing partial Gq agonist activity (red closed circles, EC₅₀ = 37 nM, Emax = 62%) and partial antagonist activity (red open circles, IC₅₀ = 78 nM), but no β-arrestin2 recruitment activity (blue). e) LY266097 Gq-mediated calcium flux activity comparing L362^7.35 (red) to 5-HT_2BR wild-type (WT, black, EC₅₀ = 41 nM, Emax = 54%). f) Gq-mediated calcium flux activity of benzyl substituted LY266097 analogs, 3,4-diMeOBenzyl (blue, EC₅₀ = 24 nM, Emax = 46%), 2-ClBenzyl (orange, EC₅₀ = 35 nM, Emax = 28%), and unsubstituted Benzyl (red). g) 5-HT_2BR-LY266097 structure shows methyl substitution on the tetrahydro-beta-carboline scaffold interacts with the 5-HT₂-specific residue G221^5.42. h) Gq-mediated calcium flux activity of des-methyl LY266097 analog (purple, EC₅₀ = 20 nM, Emax = 93%) results in near full agonist activity compared to 5-HT (black, EC₅₀ = 0.7 nM) and LY266097 (red, EC₅₀ = 41 nM, Emax = 54%). Data in panels d, e, f, and h represent mean and S.E.M from three independent experiments (N=3) performed in triplicate.