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Article Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX

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Covalent Allosteric Probe for the Metabotropic Glutamate Receptor 2: Design, Synthesis, and Pharmacological Characterization Maarten L. J. Doornbos,† Xuesong Wang,† Sophie C. Vermond,† Luc Peeters,‡ Laura Pérez-Benito,§,∥ Andrés A. Trabanco,§ Hilde Lavreysen,‡ José María Cid,§ Laura H. Heitman,† Gary Tresadern,*,§ and Adriaan P. IJzerman*,† †

Division of Drug Discovery and Safety, Leiden Academic Centre for Drug Research (LACDR), Leiden University, P.O. Box 9502, 2300RA Leiden, The Netherlands ‡ Janssen Research and Development, Turnhoutseweg 30, 2340 Beerse, Belgium § Janssen Research and Development, Calle Jarama 75A, 45007 Toledo, Spain ∥ Laboratori de Medicina Computacional Unitat de Bioestadistica, Facultat de Medicina, Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain S Supporting Information *

ABSTRACT: Covalent labeling of G protein-coupled receptors (GPCRs) by small molecules is a powerful approach to understand binding modes, mechanism of action, pharmacology, and even facilitate structure elucidation. We report the first covalent positive allosteric modulator (PAM) for a class C GPCR, the mGlu2 receptor. Three putatively covalent mGlu2 PAMs were designed and synthesized. Pharmacological characterization identified 2 to bind the receptor covalently. Computational modeling combined with receptor mutagenesis revealed T7917.29×30 as the likely position of covalent interaction. We show how this covalent ligand can be used to characterize the PAM binding mode and that it is a valuable tool compound in studying receptor function and binding kinetics. Our findings advance the understanding of the mGlu2 PAM interaction and suggest that 2 is a valuable probe for further structural and chemical biology approaches.

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INTRODUCTION Over the past years covalent ligands for G protein-coupled receptors (GPCRs) have re-emerged as valuable tool compounds to characterize the structure, expression pattern, and function of these proteins.1 A major obstacle in GPCR structure elucidation using crystallization is the dynamic behavior of their seven-transmembrane (7TM) domain, especially when in the active state.2 Covalent ligands can stabilize the 7TM domain of the receptor without the likelihood of dissociation from the binding site. The use of covalent ligands for structure elucidation has been taken as an approach to facilitate receptor crystallization, as was shown recently for the crystal structures of the adenosine A1 and multiple β2 adrenergic receptors among others.3,4 Beyond structural considerations, covalent molecules are valuable pharmacological tools useful for further understanding of binding modes and other chemical biology and proteomics applications. The metabotropic glutamate (mGlu) receptors belong to the class C GPCRs and are activated by glutamate, the most abundant neurotransmitter.5 The mGlu receptors are obligatory dimers and are characterized by their large extracellular Venus flytrap (VFT) domain (binding endogenous glutamate) which is connected to the 7TM domain via a cysteine rich domain.6 © XXXX American Chemical Society

For mGlu receptors, allosteric modulators that bind in the 7TM domain are pursued widely for drug discovery as they are typically more subtype-selective than orthosteric ligands and only function in the presence of endogenous agonist.7 Positive allosteric modulation of the mGlu2 receptor has been shown to be a potential strategy for the treatment of neurological disorders such as schizophrenia and anxiety.8 Although the structure of the extracellular domain of the mGlu2 receptor is known,9 the current understanding of the structure of the 7TM domain is based on the crystal structures of the mGlu1 and mGlu5 7TM domains, which were crystallized in an inactive state with a negative allosteric modulator (NAM) bound in the allosteric binding pocket.10−12 We have had a long-standing interest in mGlu2 receptor PAMs leading to characterization of multiple medicinal chemistry series13,14 that were also studied with site-directed mutagenesis.15,16 We have further characterized the binding kinetics and pharmacology of selected leads.17−19 Robust in vivo pharmacodynamic effects were observed in several animal Special Issue: Allosteric Modulators Received: January 12, 2018 Published: March 1, 2018 A

DOI: 10.1021/acs.jmedchem.8b00051 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

Figure 1. Structures of 1 and novel mGlu2 PAMs 2−4. The position of the tritium label of [3H]-1 is denoted by ∗.

Scheme 1. Synthesis of Compounds 2−4a

a Reagents and conditions: (i) Pd(PPh3)4, NaHCO3, H2O/1,4-dioxane, 150 °C, 10−15 min, microwave, 61−68% for 7a−c. (ii) (a) 8a, HATU, DIPEA, DMF, rt, 3h, 68−75% for 2 and 3, (b) 8b, 1,4-dioxane, 90 °C, 30 min, 33% for 4.

Table 1. Functional Activity (pEC50), Affinity (pKi), and Kinetic Parameters (kon, koff, RT) for mGlu2 PAMs 1−4 compd

pEC50a

pKia

pKi (3 h pre-incubation)a

1 2 3 4

± ± ± ±

± ± ± ±

± ± ± ±

7.74 6.80 6.80 7.82

0.03 0.06 0.04 0.06

8.12 7.21 6.95 8.24

0.13 0.11 0.11 0.08

8.12 8.21 6.78 8.38

kon (M−1 s−1)a

koff (s−1)a

(1.2 ± 0.072) × 10 “(3.2 ± 1.2) × 103” (2.1 ± 0.77) × 104 (2.2 ± 0.17) × 105

6c

0.19 0.14* 0.09 0.10

RT (min)a,b

0.0013 ± 0.0002 “(3.2 ± 3.1) × 10−13” 0.00091 ± 0.00033 0.00057 ± 0.00016 c

12 ± 2.3c “(5.2 ± 4.9) × 1010” 18 ± 6.7 29 ± 8.3

a Values represent the mean ± SEM of at least three individual experiments, performed in duplicate. bRT (min) = 1/(60 × koff). cAs described previously.17 *