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. 2018 Feb 8;172(4):719-730.e14.
doi: 10.1016/j.cell.2018.01.001. Epub 2018 Feb 1.

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology

Yao Peng  1 , John D McCorvy  2 , Kasper Harpsøe  3 , Katherine Lansu  2 , Shuguang Yuan  4 , Petr Popov  5 , Lu Qu  6 , Mengchen Pu  7 , Tao Che  2 , Louise F Nikolajsen  8 , Xi-Ping Huang  9 , Yiran Wu  7 , Ling Shen  10 , Walden E Bjørn-Yoshimoto  3 , Kang Ding  10 , Daniel Wacker  2 , Gye Won Han  11 , Jianjun Cheng  7 , Vsevolod Katritch  5 , Anders A Jensen  3 , Michael A Hanson  12 , Suwen Zhao  10 , David E Gloriam  3 , Bryan L Roth  13 , Raymond C Stevens  14 , Zhi-Jie Liu  15
Affiliations

5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology

Yao Peng et al. Cell. .

Abstract

Drugs frequently require interactions with multiple targets-via a process known as polypharmacology-to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here, we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs.

Keywords: GPCR; crystal structures; ergotamine; polypharmacology; ritanserin; selectivity; serotonin 2C receptor.

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Conflict of interest statement

Declaration of Interests

The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Overall Architecture of 5-HT2C-ERG and -RIT and Their Comparison with 5-HT1B,2B-ERG
(A) ERG (agonist) and RIT (inverse agonist) bound 5-HT2C are shown as orange and green cartoons. ERG and RIT are shown as orange and green balls and sticks, respectively. The helices are indicted with I through VIII. (B) The top panel is the extracellular view. The bottom panel is the intracellular view (loops are omitted for clarity). The arrows show helical shifts as indicated (distance measured by the Cα atoms of I3036.27, A2455.69, R1573.55) between the 5-HT2C-ERG and 5-HT2C-RIT structures. (C) Superposition of the structures: 5-HT2C-ERG (orange), 5-HT2C-RIT (green), 5-HT1B-ERG (light blue; PDB: 4IAR) and 5-HT2B-ERG (yellow; PDB: 4IB4). See also Figures S1 and S2 and Table S1.
Figure 2.
Figure 2.. Different Binding Modes of ERG and RIT in 5-HT2C
(A-D) Superposition of the 5-HT2C-ERG (orange) and RIT (green) ligand binding pockets with an overview and close-up views of the orthosteric and extended binding sites. (E) W3246.48 appears to be a major determinant of RIT’s binding mode as measured by binding affinity loss at mutations, which is recovered partially by the conservative mutation W324F6.48. (F) Mutations of residues F2235.47, F3206.44 and G2185.42 also show RIT affinity loss but less compared to W3246.48. (G and H) Schematic representation of ERG and RIT contacts with the 5-HT2C, respectively. Lines indicate interactions types; orange: polar, salt-bridges and hydrogen bonds, blue: aromatic contacts, and green: dipolar interactions. Data represent mean ± S.E.M. of three independent experiments performed in duplicate (E and F). See also Table S2.
Figure 3.
Figure 3.. Conformational Changes between 5-HT2C-ERG and -RIT Structures and Mutagenesis Validation
(A) ERG (orange sticks) and RIT (green sticks) in the binding pocket of 5-HT2C. Key residues in 5-HT2C-ERG and 5-HT2C-RIT are shown in orange and green sticks, respectively. Hydrogen bonds between D1343.32 and Y3587.43 are shown in dash line. (B) Conformational changes of I1423.40 and F3206.44 in the P-I-F motif and the W3246.48 “toggle switch” in helix VI. (C) Mutations of W3246.48 and F3206.44 completely abolish RIT’s Gαq inverse agonism, yet retain β-arrestin2 inverse agonism. Mutations of I1423.40 selectively abolish RIT’s Gαq inverse agonism and instead show weak agonism. Data represent mean ± S.E.M. from three independent experiments performed in triplicate. See also Figure S3.
Figure 4.
Figure 4.. Polypharmacological Profile of Ergotamine (ERG)
(A) The chemical structure of ERG highlighting the ergoline core (brown), tripeptide (blue) and benzyl portions (purple) in the structures of 5-HT2C (orange), 5-HT1B (light blue; PDB: 4IAR) and 5-HT2B (yellow; PDB: 4IB4). ERG is shown as thick sticks, the protein backbone is represented as a cartoon and the side chains of relevant amino acids as thin sticks. For clarity, the Cα-carbon of G5.42 is depicted as a small sphere. (B) Mean values of ERG affinities (Ki) in nM from radioligand competition binding assays represent the mean from experiments performed in duplicate (see also Table S3). Sequence alignments of the three subsites: Grey background, conservation of positions and residue types critical for ERG binding; SN, short and negative; IH, intermediate length and hydrophobic; SU, small and unbranched; VSP, very small or small and polar; HF, hydrophobic with a maximum size of Phe; HA, hydrophobic and aliphatic. Receptors are listed in the order of decreasing ERG affinity (Ki color scale green over white to orange) but separated into aminergic and non-aminergic receptors. See also Figures S4 and S5 and Tables S3 and S4.
Figure 5.
Figure 5.. Polypharmacology of the 4-benzylidenepiperidine Privileged Structure
(A) The interactions between the 4-benzylidenepiperidine of RIT and its surounding residues in the 5-HT2C-RIT structure. (B) Sequence alignments of the privileged structure subsites with the number of ligands identified in a search for GPCR ligands in ChEMBL containing the privileged structure and Ki < 1μM. Conservation of positions critical for binding is shown in a grey background and conservation is assessed by the residue properties crucial for interaction; 1AR, aromatic residue in at least one of the two positions. (C) Examples of ligands identified in the search with confirmed GPCR targets (Ki < 1 μM) are listed and the privileged structure highlighted in red in the chemical structures. For cinnarizine and clozapine the hits from ChEMBL (Bento et al., 2014) have been supplemented with hits from the PDSP Ki database (Yadav et al., 2011a). See also Figure S6.
Figure 6.
Figure 6.. Ritanserin’s Selectivity on 5-HT2 Receptors
(A) Mean values of RIT affinities (Ki) in nM for radioligand competition binding assays from experiments performed in duplicate. Sequence alignment of the RIT binding site residues in the eleven serotonin receptors for which RIT’s binding affinity was determined. Conservation of residue type according to 5-HT2C is indicated by a grey background and mutated positions are indicated with black boxes. (B) The unique residue types in positions 5.42 and 7.39 of the 5-HT2A-C receptors (see panel A) appear as major determinants of the RIT selectivity for these receptor subtypes as binding affinity decreased when mutated in 5-HT2C to the corresponding residues of 5-HT1A, i.e. G218S5.42 and V354N7.39. Data represent mean ± S.E.M. of three independent experiments performed in duplicate. (C) The interactions between RIT and G2185.42 and V3547.39 in the 5-HT2C-RIT structure. See also Tables S3, S5 and S6 and Figure S6.
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