Differential Pharmacology and Binding of mGlu₂ Receptor Allosteric Modulators

Abstract

Allosteric modulation of metabotropic glutamate receptor 2 (mGlu) has demonstrated efficacy in preclinical rodent models of several brain disorders, leading to industry and academic drug discovery efforts. Although the pharmacology and binding sites of some mGlu allosteric modulators have been characterized previously, questions remain about the nature of the allosteric mechanism of cooperativity with glutamate and whether structurally diverse allosteric modulators bind in an identical manner to specific allosteric sites. To further investigate the in vitro pharmacology of mGlu allosteric modulators, we developed and characterized a novel mGlu positive allosteric modulator (PAM) radioligand in parallel with functional studies of a structurally diverse set of mGlu PAMs and negative allosteric modulators (NAMs). Using an operational model of allosterism to analyze the functional data, we found that PAMs affect both the affinity and efficacy of glutamate at mGlu, whereas NAMs predominantly affect the efficacy of glutamate in our assay system. More importantly, we found that binding of a novel mGlu PAM radioligand was inhibited by multiple structurally diverse PAMs and NAMs, indicating that they may bind to the mGlu allosteric site labeled with the novel mGlu PAM radioligand; however, further studies suggested that these allosteric modulators do not all interact with the radioligand in an identical manner. Together, these findings provide new insights into the binding sites and modes of efficacy of different structurally and functionally distinct mGlu allosteric modulators and suggest that different ligands either interact with distinct sites or adapt different binding poses to shared allosteric site(s).

Conformational dynamics between transmembrane domains and allosteric modulation of a metabotropic glutamate receptor

“…In contrast, removal of the extracellular domains shifted the dose–response curve to the left, consistent with a model where the LBD provides tonic inhibition of TMD activation by PAMs that is relieved either by LBD closure or by removal of the ECD. Further supporting this model, binding studies have shown up to a 10-fold leftward shift in PAM binding to mGluR2 in the presence of glutamate (Doornbos et al, 2016; O'Brien et al, 2018). Despite the modulatory effects of the LBD on PAM activation, saturating concentrations of LY48 were unable to directly induce LBD closure as assayed using an inter-LBD FRET assay (Figure 1—figure supplement 3).…”

“…Classically, positive allosteric modulators (PAMs) of GPCRs have been defined by their ability to amplify the effects of orthosteric compounds without directly activating the receptor (May et al, 2007) (Lindsley et al, 2016; Wacker et al, 2017). However, some studies have shown both modulation and direct activation of GPCRs by PAMs (May et al, 2007), including mGluRs (Rovira et al, 2015) (O'Brien et al, 2018). Most previous studies, however, relied on end-point assays and thereby failed to provide dynamic information about the onset and reversibility of PAM effects.…”

“…How these microswitches contribute to PAM agonism in mGluRs is not well understood, especially in the context of intersubunit reorientation. Asp735 in helix 5 has been proposed as a central residue within a ‘trigger switch’ (Figure 3—figure supplement 3A) that mediates PAM binding-induced conformational changes for most, but not all, mGluR2 PAMs (O'Brien et al, 2018; Pérez-Benito et al, 2017; Schaffhauser et al, 2003). Consistent with our prediction, mutation of asparagine 735 to aspartate (N735D) resulted in drastically reduced activation by LY48 in the GIRK assay (Figure 3—figure supplement 3B,C).…”

Biased M1positive allosteric modulators reveal novel role of phospholipase D in M1-dependent rodent cortical plasticity

“…While there are multiple examples of allosteric modulators of GPCRs inducing biased signaling, little is known about the structural basis of biased versus non-biased signaling. Previous studies reveal multiple allosteric binding sites for some GPCRs, which could contribute to different responses to distinct classes of allosteric modulators (70)(71)(72). However, recent studies suggest that differences in M1 PAM functionality may not be due to binding to different allosteric binding pockets, but that binding of PAMs to a single allosteric site may stabilize different receptor conformational states (73,74).…”

A case study of foliglurax, the first clinical mGluR4 PAM for symptomatic treatment of Parkinson’s disease: translational gaps or a failing industry innovation model?

“…Assuming that distinct chemotypes interacting allosterically with the mGluRs bind to the same binding site and displace each other is not warranted. Studies have shown that very minor differences in the structure of a drug and a radioligand can lead to unexpected results [74,75]. It has been recommended to de-risk such assumption by conducting the appropriate in vitro tests and confirm competitive behaviors for the pair drug-radioligand to be used in vivo in subsequent studies [74,75].…”