Practical Strategies and Concepts in GPCR Allosteric Modulator Discovery: Recent Advances with Metabotropic Glutamate Receptors

Abstract

Allosteric modulation of GPCRs has initiated a new era of basic and translational discovery, filled with therapeutic promise yet fraught with caveats. Allosteric ligands stabilize unique conformations of the GPCR that afford fundamentally new receptors, capable of novel pharmacology, unprecedented subtype selectivity, and unique signal bias. This review provides a comprehensive overview of the basics of GPCR allosteric pharmacology, medicinal chemistry, drug metabolism, and validated approaches to address each of the major challenges and caveats. Then, the review narrows focus to highlight recent advances in the discovery of allosteric ligands for metabotropic glutamate receptor subtypes 1-5 and 7 (mGlu1-5,7) highlighting key concepts ("molecular switches", signal bias, heterodimers) and practical solutions to enable the development of tool compounds and clinical candidates. The review closes with a section on late-breaking new advances with allosteric ligands for other GPCRs and emerging data for endogenous allosteric modulators.

Figure 1

Structures of compounds 1–25 discussed in the Introduction.

Figure 2

Allosteric interactions can manifest as altered affinity and/or efficacy. (a) Simulations of the effect of an allosteric modulator on receptor occupancy by an orthosteric ligand, as described by the allosteric ternary complex model (center). In the absence of an allosteric ligand (black curve), relative receptor occupancy is determined by the concentration of orthosteric ligand (A) and the equilibrium dissociation constant (K_A), which is the concentration of A that occupies 50% of receptors. Increasing concentrations (red, K_B; orange, 3K_B; yellow, 10K_B; green, 30K_B; blue, 100K_B) of a negative allosteric modulator (left, α = 0.1) or a positive allosteric modulator (right, α = 10) alter the apparent affinity of the orthosteric ligand 10fold. (b) Simulations of allosteric interactions in a functional assay, as described by the operational model of allosterism (center). Top left, an allosteric ligand that negatively modulates both affinity and efficacy. Top right, an allosteric modulator that potentiates both affinity and efficacy. Bottom left and right, allosteric ligands with opposing effects on affinity versus efficacy.

Figure 3

Comparison of mGlu₅ modulator potency and affinity estimates. Potency values were pooled for (a) inhibition or (b) potentiation of orthosteric agonist activity (glutamate or quisqualate) in multiple paradigms including recombinant cell lines expressing either human or rat mGlu₅, or primary cultures, and using intracellular Ca²⁺ mobilization and inositol phosphate accumulation. Affinity estimates were pooled from inhibition binding studies using radiolabeled allosteric ligands using membranes or whole cells from recombinant cells lines expressing either human or rat mGlu₅, primary cultures, or tissue homogenates.

Figure 4

Heteromerization of mGlu₄ and mGlu₂ permits an mGlu₂ NAM to block mGlu₄ agonist-mediated responses. (A) When mGlu₄ (gray) is expressed alone, both protomers respond to L-AP4 (mGlu₄-selective agonist, green circles), which induces responses with predicted potency and full efficacy (lower panel, black circles in graph). When mGlu₂ (maroon) is coexpressed with mGlu₄, the L-AP4 response is more shallow, and the response is approximately 75% (white circles) that of L-AP4 when mGlu₄ is expressed alone. (B) Incubation of increasing concentrations of an mGlu₂ NAM with mGlu₄ homomers results in no blockade of response. Incubation of NAM (yellow X) with mGlu_2/4 heteromers results in a concentration-dependent, noncompetitive blockage of L-AP4 responses, indicating transactivation between the two protomer subunits. Graphs are simulated based on data presented in ref .

Figure 5

Iterative, multidimensional parallel synthesis approach, coupled with matrix libraries for the chemical lead optimization of GPCR allosteric modulators.

Figure 6

Matrix library strategy for the chemical lead optimization of a series of M₅ negative allosteric modulators with steep SARs.

Figure 7

Molecular switches were apparent in the first reported series of mGlu₅ PAMs, wherein small modifications afforded PAMs, NAMs, and NALs.

Figure 8

Biotransformation of a potent mGlu₅ agonist-PAM, VU0403602 (32), through cytochrome P450-mediated metabolism to a major circulating and brain-penetrant active metabolite (M1, 33) displaying similar PAM pharmacology with higher efficacy and lower potency intrinsic agonist activity in rat. Values represent means of at least three independent determinations in fluorometric calcium mobilization assays using rat mGlu₅-expressing HEK cells.

Figure 9

Historical (34–38) and recent (39–43) mGlu1 PAMs, the latter of which were developed by exploiting molecular switches.

Figure 10

mGlu_2/3 orthosteric agonists 45 (LY354640), 46 (LY379268), and 47 (LY404039); orally available clinical prodrug 48 (LY2140023); and mGlu_2/3 orthosteric antagonists 49 (LY341495) and 50 (MGS0039).

Figure 11

Prototypical mGlu₂ PAM tools 51 (LY487379) and 19 (BINA).

Figure 12

mGlu₂ PAMs 52, 53 and 19 (BINA) and mGlu_2/3 PAM 54.

Figure 13

2,3-Dihydroimidazo[2,1-b]oxazole-based mGlu₂ PAMs 55 (TASP0433864) and 56.

Figure 14

Imidazopyridine mGlu₂ PAMs 57–62, triazolopyridine mGlu₂ PAM 61 (JNJ-42153605), and [¹¹C]-labeled triazolopyridine mGlu₂ PAM 62.

Figure 15

Pyridone mGlu₂ PAMs: HTS hit 63, in vivo tools 64 and 65 (JNJ-40068782), and clinical compound 11 (JNJ-40411813).

Figure 16

mGlu₂ PAM clinical compound 11 (AZD8529) and its synthetic precursor 57.

Figure 17

mGlu_2/3 NAM tools 58 (RO4491533) and 59 (RO4432717) and clinical compound 60 (decoglurant).

Figure 18

Screening hit 61 and pyrazolo[1,5-a]quinazolin-5-one mGlu_2/3 NAMs 62 and 63.

Figure 19

Cross-screening hit 64 and 1,2-diphenylethyne mGlu₃ NAMs 65 (VU0463597, ML289) and 66 (VU0469942, ML337).

Figure 20

Cross-screening hit 67 and optimized mGlu3 NAM in vivo tool 68 (VU0650786).

Figure 21

Markush structures of 69 and mGlu₂ NAM 70 (MRK-8–29). The similarity between 70 and the prototypical M₁ PAM BQCA (71) led to a scaffold-hopping exercise that identified the novel mGlu₂ NAM VU6001192 (72).

Figure 22

Structures of mGlu₄ PAMs with reported in vivo activity in preclinical models of Parkinson’s disease.

Figure 23

Structures of mGlu₅ full (75 and 76) and partial (77–79) NAMs with reported in vivo activity in preclinical models of drug abuse and depression.

Figure 24

Structures 80–86 of mGlu₅ PAMs and ago-PAMs displaying signal bias, including two (85 and 86) that have advanced to safety assessment.

Figure 25

Structures of mGlu₇ allosteric agonist (87, AMN082), pan-group III PAMs (88, VU0422288; 89, VU0155094), and antagonist/NAMs (90, XAP044; 91 MMPIP; and 92 ADX71743).

Figure 26

Structures of the first GABA_B NAM (93) and the GABA_B PAM CGP7930 (94), from which 93 was derived by scaffold hopping.

Figure 27

Structures of the first GPR4 NAM (95) and the orhtosteric antagonist psychosine (96). Gal is galactosyl.