G protein–coupled receptors (GPCRs) use a series of conserved microswitches to transmit signals across the cell membrane via an allosteric network encompassing the ligand-binding site and the G protein–binding site. Crystal structures of GPCRs provide snapshots of their inactive and active states, but poorly describe the conformational dynamics of the allosteric network that underlies GPCR activation. Here, we analyzed the correlation between ligand binding and receptor conformation of the α1_A-adrenoceptor, a GPCR that stimulates smooth muscle contraction in response to binding noradrenaline. NMR of ¹³C^εH₃-methionine–labeled α_1A-adrenoreceptor variants, each exhibiting differing signaling capacities, revealed how different classes of ligands modulate the conformational equilibria of this receptor. ¹³C^εH₃-methionine residues near the microswitches exhibited distinct states that correlated with ligand efficacies, supporting a conformational selection mechanism. We propose that allosteric coupling among the microswitches controls the conformation of the α_1A-adrenoceptor and underlies the mechanism of ligand modulation of GPCR signaling in cells.

α_1A‐ and α_1B‐adrenoceptors (ARs) are G protein‐coupled receptors (GPCRs) that are activated by adrenaline and noradrenaline to modulate smooth muscle contraction in the periphery, and neuronal outputs in the central nervous system (CNS). α_1A‐ and α_1B‐AR are clinically targeted with antagonists for hypertension and benign prostatic hyperplasia and are emerging CNS targets for treating neurodegenerative diseases. The benzodiazepines midazolam, diazepam, and lorazepam are proposed to be positive allosteric modulators (PAMs) of α₁‐ARs. Here, using thermostabilized, purified, α_1A‐ and α_1B‐ARs, we sought to identify the benzodiazepine binding site and modulatory mechanism to inform the design of selective PAMs. However, using a combination of biophysical approaches no evidence was found for direct binding of several benzodiazepines to purified, stabilized α_1A‐ and α_1B‐ARs. Similarly, in cell‐based assays expressing unmodified α_1A‐ and α_1B‐ARs, benzodiazepine treatment had no effect on fluorescent ligand binding, agonist‐stimulated Ca²⁺ release, or G protein activation. In contrast, several benzodiazepines positively modulated phenylephrine stimulation of a cAMP response element pathway by α_1A‐ and α_1B‐ARs; however, this was shown to be caused by off‐target inhibition of phosphodiesterases, known targets of diazepam. This study highlights how purified, stabilized GPCRs are useful for validating allosteric ligand binding and that care needs to be taken before assigning new targets to benzodiazepines.

The colony-stimulating factor-1 receptor (CSF1R) is atyrosine-proteinkinase that is a potential target for asthma therapeutics. We haveapplied a fragment-lead combination approach to identify small fragmentsthat act synergistically with GW2580, a known inhibitor of CSF1R.Two fragment libraries were screened in combination with GW2580 bysurface plasmon resonance (SPR). Binding affinity measurements confirmedthat thirteen fragments bind specifically to the CSF1R, and a kinaseactivity assay further validated the inhibitory effect of these fragments.Several fragment compounds enhanced the inhibitory activity of thelead inhibitor. Computational solvent mapping, molecular docking,and modeling studies suggest that some of these fragments bind adjacentto the binding site of the lead inhibitor and further stabilize theinhibitor-bound state. Modeling results guided the computational fragment-linkingapproach to design potential next-generation compounds. The inhalabilityof these proposed compounds was predicted using quantitative structure-propertyrelationships (QSPR) modeling based on an analysis of 71 drugs currentlyon the market. This work provides new insights into the developmentof inhalable small molecule therapeutics for asthma.