Biased allosteric agonism and modulation of metabotropic glutamate receptor 5: Implications for optimizing preclinical neuroscience drug discovery

Abstract

Allosteric modulators, that exhibit no intrinsic agonist activity, offer the advantage of spatial and temporal fine-tuning of endogenous agonist activity, allowing the potential for increased selectivity, reduced adverse effects and improved clinical outcomes. Some allosteric ligands can differentially activate and/or modulate distinct signaling pathways arising from the same receptor, phenomena referred to as ’biased agonism’ and ’biased modulation’. Emerging evidence for CNS disorders with glutamatergic dysfunction suggests the metabotropic glutamate receptor subtype 5 (mGlu5) is a promising target. Current mGlu5 allosteric modulators have largely been classified based on modulation of intracellular calcium (iCa2+) responses to orthosteric agonists alone. We assessed eight mGlu5 allosteric modulators previously classified as mGlu5 PAMs or PAM-agonists representing four distinct chemotypes across multiple measures of receptor activity, to explore their potential for engendering biased agonism and/or modulation. Relative to the reference orthosteric agonist, DHPG, the eight allosteric ligands exhibited distinct biased agonism fingerprints for iCa2+ mobilization, IP1 accumulation and ERK1/2 phosphorylation in HEK293A cells stably expressing mGlu5 and in cortical neuron cultures. VU0424465, DPFE and VU0409551 displayed the most disparate biased signaling fingerprints in both HEK293A cells and cortical neurons that may account for the marked differences observed previously for these ligands in vivo. Select mGlu5 allosteric ligands also showed ‘probe dependence’ with respect to their cooperativity with different orthosteric agonists, as well as biased modulation for the magnitude of positive cooperativity observed. Unappreciated biased agonism and modulation may contribute to unanticipated effects (both therapeutic and adverse) when translating from recombinant systems to preclinical models.

Constitutive activity of the metabotropic glutamate receptor 2 explored with a whole-cell label-free biosensor

“…For this purpose, glutamate-pyruvate transaminase (GPT; 3 U/ml) was added in the presence of additional pyruvate (3 mM), a procedure commonly used in the field of mGlu receptors, e.g. [37,38]. This treatment led to an almost 4-fold lower concentration of glutamate in the culture medium: 23 ± 0.09 µM.…”

Biased Receptor Signaling in Drug Discovery

“…Induced bias is increasingly seen with synthetic druglike allosteric modulators. For example, the agonist for the metabotropic glutamate receptor 5(S)-3,5-dihydroxyphenylglycine produces activation of calcium, IP1, and phosphor-ERK1/2 signaling in mouse cortical neurons (Sengmany et al, 2017). Values of Log(t/K A ) for activation of these pathways in the absence and presence of a high concentration of PAM agonists for this receptor indicate that the pattern of activation of these three pathways changes significantly after allosteric modification (see Fig.…”

“…In view of the fact that bias measurements made in recombinant systems often differ from those seen in natural systems and also that cell type can change estimates of bias, it is useful to quantitatively compare different functional systems and measurements of bias. Figure 20 shows data for allosteric agonists of the metabotropic glutamate receptor 5 for functional responses (calcium, IP1, ERK) when the receptor is transfected into HEK cells versus that endogenously found in cultured cortical neurons (Sengmany et al, 2017). These data show a trend of greater agonist activity and increased bias [DDLog(max/EC 50 ) values] in HEK cells versus cortical neurons.…”

Control of neuronal excitability by Group I metabotropic glutamate receptors

“…Binding of a negative allosteric modulator to this site results in non-competitive inhibition of the receptor. Positive allosteric modulators do not activate the receptor, but can change the EC50 for an orthosteric agonist or increase its maximum effect (Ellaithy et al 2015;Kinney et al 2005;Knoflach et al 2001;Layton 2005;Sengmany et al 2017). The history of mGlu receptor pharmacology and drug development, as well as comprehensive lists of disclosed compounds with orthosteric or allosteric activity, is well documented (Alexander et al 2011;Ferraguti et al 2008;Pin et al 1999;Schoepp et al 1999).…”