Sengmany, K. | bioRxiv Science

bioRxiv2024

Pharmacological characterisation of allosteric modulators at human mGlu5

The metabotropic glutamate receptor 5 (mGlu5) is a Class C G protein-coupled receptor, ubiquitously expressed throughout the CNS. With major roles in cognition, learning and memory, mGlu5 dysfunction is linked with numerous neurodegenerative and neuropsychiatric disorders, presenting a viable therapeutic target. Allosteric modulators bind topographically distinct sites from glutamate and other orthosteric agonists and enhance (positive allosteric modulators, PAMs), inhibit (negative allosteric modulators, NAMs) or do not effect (neutral allosteric ligands, NALs) mGlu5 function. While mGlu5 modulators have efficacy in in vivo rodent models of CNS disorders, none have made it to the clinic. We hypothesise this lack of translatability is arises from preclinical optimisation using on non-human pharmacological data, as functional studies are predominantly performed using rat mGlu5 and non-human brain neuronal cultures. Here we assess and quantify the impact of eleven chemically and pharmacologically diverse mGlu5 PAMs, NAMs and NALs on human mGlu5 activity using radioligand binding, iCa2+ mobilisation and IP1 accumulation assays. By comparing to published and newly generated data for rat mGlu5 we show that while modulator pharmacology is relatively consistent across species, ligand dependent species differences in allosteric modulator affinity, cooperativity and probe dependence are evident. Additionally, we report PAM-dependent effects on orthosteric agonist kinetic profiles at human mGlu5. Together, these data highlight the importance of systematic evaluation of mGlu5 allosteric ligand activity at human mGlu5 to improve drug design and overcome potential barriers to translatability to clinical settings.

pharmacology and toxicology↗

bioRxiv2024

Ligand-induced conformational changes in the β1-Adrenergic Receptor Revealed by Hydrogen-Deuterium Exchange Mass Spectrometry

G-Protein Coupled Receptors (GPCRs) constitute the largest family of signalling proteins responsible for translating extracellular stimuli into intracellular functions. When dysregulated, GPCRs drive numerous diseases and are the most targeted proteins in drug discovery. GPCR structural dynamics and activity can be modulated by a wide range of drugs, including full/partial agonists and antagonists. While crucial for developing novel therapeutics targeting GPCRs, the structural dynamics of the receptors associated with their activity upon drug interactions are not yet fully understood. Here, we employ Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS), to characterise the structural dynamics of turkey {beta}1-adrenergic receptor (t{beta}1AR) in complex with nine ligands, including agonists, partial agonists and antagonists. We show that dynamic signatures across the GPCR structure can be grouped by compound modality. Surprisingly, we discovered repeated destabilisation of the intracellular loop 1 (ICL1) upon full agonist binding and stabilisation upon antagonist binding, suggesting that increased dynamics in this region are an essential component for G-protein recruitment. Multiple sequence alignments and molecular dynamics simulations indicate that L72 in ICL1 plays important structural role. Differential HDX-MS experiment of t{beta}1AR and t{beta}1AR L72A construct in complex with miniGs, in response to various ligands, suggests involvement of ICL1 in stabilising the GDP bound state by influencing the stability of HG helix of miniGs. Overall, our results provide a platform for determining drug modality and highlight how HDX-MS can be used to dissect receptor ligand interaction properties and GPCR mechanism. Significance statementRecent advances in hydrogen-deuterium exchange mass spectrometry have allowed probing conformational signatures of challenging membrane protein assemblies. We studied the structural dynamics of a class A GPCR, namely t{beta}1AR, in response to diverse ligands including agonists, antagonists and partial agonists. We demonstrate that the functional effect of compounds can be discerned by simply profiling the dynamics induced across the receptor, without the need for downstream interaction partners. We showed that ICL1 undergoes a significant change in dynamics between activated and inhibited states consistent with a role in downstream signaling pathways in class A GPCRs.

biophysics↗