Application of an Integrated GPCR SAR-Modeling Platform To Explain

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Application of an Integrated GPCR SAR-Modelling Platform to Explain the Activation Selectivity of Human 5-HT2C over 5-HT2B Alexander Heifetz, R Ian Storer, Gordon McMurray, Tim James, Inaki Morao, Matteo Aldeghi, Mike J. Bodkin, and Philip C. Biggin ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.5b01045 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on March 3, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Chemical Biology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Chemical Biology

Table of Content artwork 297x209mm (300 x 300 DPI)

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Application of an Integrated GPCR SAR-Modelling Platform to Explain the Activation Selectivity of Human 5-HT2C over 5-HT2B Alexander Heifetz1*, R. Ian Storer2*, Gordon McMurray3, Tim James1, Inaki Morao1, Matteo Aldeghi4, Mike J. Bodkin1, and Philip C. Biggin4

1

Evotec (UK) Ltd., 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RZ, UK

2

Worldwide Medicinal Chemistry, 3Discovery Biology, Pfizer Ltd, The Portway, Granta Park,

Cambridge, CB21 6GS, UK 4

Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU,

UK

*Corresponding author. Tel.: +44 (0)1235 83 89 25; Fax: +44 (0)1235 86 31 39 E-Mail addresses: [email protected] and [email protected]

ACS Paragon Plus 1 Environment

Page 2 of 38

Page 3 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ABSTRACT

Agonism of the 5-HT2C serotonin receptor has been associated with the treatment of a number of diseases including obesity, psychiatric disorders, sexual health and urology. However, the development of effective 5-HT2C agonists has been hampered by the difficulty in obtaining selectivity over the closely related 5-HT2B receptor, agonism of which is associated with irreversible cardiac valvulopathy. Understanding how to design selective agonists requires exploration of the structural features governing the functional uniqueness of the target receptor relative to related off targets. X-ray crystallography, the major experimental source of structural information, is a slow and challenging process for integral membrane proteins, and so is currently not feasible for every GPCR or GPCR-ligand complex. Therefore, the integration of existing ligand SAR data with GPCR modelling can be a practical alternative to provide this essential structural insight. To demonstrate this, we integrated SAR data from 39 azepine series 5-HT2C agonists, comprising both selective and unselective examples, with our hierarchical GPCR modelling protocol (HGMP). Through this work we have been able to demonstrate how relatively small differences in the amino-acid sequences of GPCRs can lead to significant differences in secondary structure and function, as supported by experimental data. In particular, this study suggests that conformational differences in the tilt of TM7 between 5-HT2B and 5-HT2C, which result from differences in inter-helical interactions, may be the major source of selectivity in G-protein activation between these two receptors. Our approach also demonstrates how the use of GPCR models in conjunction with SAR data can be used to explain activity cliffs.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION The 5-HT2C receptor belongs to the class A G-protein coupled receptor (GPCR) 5HT2 subfamily and is linked to a range of different physiological functions, including mood regulation, anxiety, reproductive behaviour and feeding. For many years agonism of 5-HT2C receptors has been viewed as an attractive target for drug development, with potential application for the treatment of a number of medical conditions including obesity, psychiatric disorders, sexual dysfunction and urinary incontinence.1 The 5-HT2 subfamily has, however, two other members; 2A and 2B. Incorporating selectivity for 5-HT2C over 5-HT2B has become a major imperative due to the fact that 5-HT2B agonists have been associated with irreversible cardiac valvulopathy and pulmonary hypertension, as illustrated by the market withdrawal of the subtype unselective agonist combination drug Fen-Phen in 1997.2 The difficulty in identifying ligands with good subtype selectivity between 5-HT2B and 5-HT2C can be accounted for by their closely related sequences; >65% sequence identity overall and >82% similarity in the 7 TM region (Figure 1).1 This is a typical case in GPCR drug discovery, where selectivity is required for ligands of highly similar receptors such as the Orexin, Histamine, Muscarinic, Serotonin, Chemokine and other GPCR subfamilies

3-6

. Thus, one would expect that any selectivity that is observed is due to relatively

small differences in the structures of these receptors. The availability of structural information on the target receptor plays a key role in the rationalization, efficiency and cost-effectiveness of the drug design process.7 X-ray crystallography, a traditional source of structural information, is not currently feasible for every GPCR or GPCR-ligand complex. This situation significantly limits the ability of crystallography to impact drug discovery for GPCR targets and hence there remains an urgent need for other practical alternatives. We present here an integrated approach whereby experimental structure activity relationship (SAR) data for 39 azepine class molecules (Table 1) has been combined with a


Page 4 of 38

Page 5 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hierarchical GPCR modelling protocol (HGMP) to explore the structural features of 5-HT2B and 5-HT2C responsible for agonist selectivity. In recent years the discoveries of several azepine-derived subseries that are potent 5-HT2C agonists with varying degrees of selectivity over 5-HT2B have been reported.8, 9 Often SAR data, when combined with GPCR modelling, can provide a powerful approach for receptor structural exploration and structure-based rationalization of efficacy and selectivity with a view to guiding prospective ligand design.8, 10 The HGMP was developed to support structure-based drug discovery programs.

10, 11

It works by generating a 3D model of a GPCR structure and its complexes with small molecules by applying a set of computational methods. These include homology modelling followed by optimization protocols and flexible ensemble docking, to predict the binding poses and function of ligands bound to GPCRs. HGMP includes a large set of unique plugins to refine the GPCR models and exclusive scoring functions like the GPCR-likeness assessment score (GLAS) to evaluate model quality.

11

It has been successfully applied to a

large number of GPCR drug discovery projects and to support crystallography.10-12

Recently the crystal structures of the 5-HT2B and 5-HT1B receptors with the ligand ergotamine (ERG) were solved by X-ray crystallography.13,

14

Ergotamine predominantly

signals through β-arrestin pathways at 5-HT2B receptors (bias factor of 228), whereas signalling at 5-HT1B receptors appears non-biased. The different signalling patterns are reflected in the crystal structures, which show features of an intermediate activation-state for the 5-HT1B receptor and a β-arrestin-biased activation-state for the 5-HT2B receptor. It was observed that in both 5-HT1B and 5-HT2B, similarly to the β2-adrenergic receptor (β2AR), the residues P5.50, I3.40 and F6.44 (the “P-I-F” motif) form an interface between TM3, TM5 and TM6. However, the crystal structures of 5-HT1B and 5-HT2B exhibit two different conformations of the P-I-F motif. For the 5-HT1B receptor, it was observed that the P-I-F configuration is essentially identical to that of the G-protein activation-state of β2AR. The 5-HT2B receptor adopts similar conformations for residues P2295.50 and I1433.40, but the



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

side-chain geometry of residue F3336.44 was closer to that observed in the β-arrestinbiased activation-state of β2AR.15 The P-I-F motif therefore appears to be in a β-arrestinbiased activation-state in the 5-HT2B receptor. This last observation limited our ability to use the crystal structure of the 5-HT2B receptor in the current investigation, since the azepines compounds for this study have been established to signal via the G-protein pathway as measured via a calcium based FLIPR assay (the activation of the β-arrestin signalling pathway was not tested). Consequently, the β2-adrenergic receptor was selected as the base template for modelling of both active and inactive states of 5-HT2B and 5-HT2C, because it is a related aminergic receptor and has crystal structures available for both G-protein active and inactive conformations.

RESULTS AND DISCUSSION Compounds and bioassay data utilized in the modelling

The discovery of a number of azepine derived subseries of 5-HT2C agonists has been reported in previous publications.8,

16-24

A set of 39 compounds was compiled from across

this class and divided into six subseries as summarized in Table 1. This shortlist of compounds was selected such that each subseries has active and inactive ligands for both 5-HT2C and 5HT2B receptors and where small modifications (matched pairs) in the ligand structures show significant effects on functional potency and efficacy. The compounds were all tested in recombinant FLIPR assays for both receptors to measure agonist activation of the G-protein signalling pathway. In addition, the 5-HT2C binding Ki for the compounds was also measured relative to the radiolabelled 5-HT2C antagonist mesulergine. The translational pharmacology from these assays to physiologically relevant tissues has been evaluated and discussed in previous publications showing good translation to in vivo endpoints.8 The full curves for functional agonist activity of the compounds in both 5-HT2C and 5-HT2B are


Page 6 of 38

Page 7 of 38

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


illustrated in Figure 2A and the 5-HT2C binding assay curves have been provided in Supporting Information Figure S3. As 5-HT2C and 5-HT2B show high sequence homology, one might expect azepine agonists to exhibit very similar activation effects on these two receptors. However, according to the experimental data there was very low correlation observed between the EC50 values measured for 5-HT2C and 5-HT2B (r2 =0.044, Figure 2B). This suggests that the azepines might have different binding poses in the two receptors. A strong correlation of r2 =0.736 (Figure 2B) is observed between the EC50 and Ki values for 5-HT2C, suggesting that the ability of the azepines to activate this receptor is highly dependent on binding.

Building models of the human 5-HT2B and 5-HT2C receptors

The position of each amino acid residue was identified by its unique sequence number as well as by its generic number proposed by Ballesteros and Weinstein

25, 26

(Supporting Information). Sequence alignment between the 5-HT2 receptors and β2AR (Supporting Information Figures S1a and S1b) indicated 57.2% similarity in the 7TM region for 5-HT2B and 59.3% for 5-HT2C. The modelling and refinement of active and inactive states of 5-HT2B and 5-HT2C was performed with the standard HGMP protocol, using potent agonists 1a (for 5-HT2C) and 5a (for 5-HT2B). The model validations were performed with the GPCR-likeness assessment score (GLAS – Supporting Information), which showed values of 0.94 for 5-HT2B and 0.97 for 5-HT2C. These values are comparable with those typically obtained for GPCR crystal structures with resolutions of

Application of an Integrated GPCR SAR-Modeling Platform To Explain

Recommend Documents