Effect of S–Se Bioisosteric Exchange on Affinity and Intrinsic Efficacy of Novel N-acylhydrazone Derivatives at the Adenosine A2A Receptor

In this work, we evaluated the conformational effect promoted by the isosteric exchange of sulfur by selenium in the heteroaromatic ring of new N-acylhydrazone (NAH) derivatives (3–8, 13, 14), analogues of the cardioactive compounds LASSBio-294 (1) and LASSBio-785 (2). NMR spectra analysis demonstrated a chemical shift variation of the iminic Csp2 of NAH S/Se-isosters, suggesting a stronger intramolecular chalcogen interaction for Se-derivatives. To investigate the pharmacological profile of these compounds at the adenosine A2A receptor (A2AR), we performed a previously validated functional binding assay. As expected for bioisosteres, the isosteric-S/Se replacement affected neither the affinity nor the intrinsic efficacy of our NAH derivatives (1–8). However, the N-methylated compounds (2, 6–8) presented a weak partial agonist profile at A2AR, contrary to the non-methylated counterparts (1, 3–5), which appeared as weak inverse agonists. Additionally, retroisosterism between aromatic rings of NAH on S/Se-isosters mimicked the effect of the N-methylation on intrinsic efficacy at A2AR, while meta-substitution in the phenyl ring of the acyl moiety did not. This study showed that the conformational effect of NAH-N-methylation and aromatic rings retroisosterism changed the intrinsic efficacy on A2AR, indicating the S/Se-chalcogen effect to drive the conformational behavior of this series of NAH.

Evaluation of G protein bias and β-arrestin 2 signaling in opioid-induced respiratory depression.

Respiratory depression is a potentially fatal side effect of opioid analgesics and major limitation to their use. G-protein-biased opioid agonists have been proposed as "safer" analgesics with less respiratory depression. These agonists are biased to activate G proteins rather than β-arrestin signaling. Respiratory depression has been shown to correlate with both G-protein bias and intrinsic efficacy, and recent work has refuted the role of β-arrestin signaling in opioid-induced respiratory depression. In addition, there is substantial evidence that G-proteins do, in fact, mediate respiratory depression by actions in respiratory-controlling brainstem neurons. Based on these studies, we provide the perspective that protection from respiratory depression displayed by newly developed G-protein biased agonists is due to factors other than G-protein versus arrestin bias.

β2-adrenoceptor ligand efficacy is tuned by a two-stage interaction with the Gαs C terminus

Classical pharmacological models have incorporated an “intrinsic efficacy” parameter to capture system-independent effects of G protein–coupled receptor (GPCR) ligands. However, the nonlinear serial amplification of downstream signaling limits quantitation of ligand intrinsic efficacy. A recent biophysical study has characterized a ligand “molecular efficacy” that quantifies the influence of ligand-dependent receptor conformation on G protein activation. Nonetheless, the structural translation of ligand molecular efficacy into G protein activation remains unclear and forms the focus of this study. We first establish a robust, accessible, and sensitive assay to probe GPCR interaction with G protein and the Gα C terminus (G-peptide), an established structural determinant of G protein selectivity. We circumvent the need for extensive purification protocols by the single-step incorporation of receptor and G protein elements into giant plasma membrane vesicles (GPMVs). We use previously established SPASM FRET sensors to control the stoichiometry and effective concentration of receptor–G protein interactions. We demonstrate that GPMV-incorporated sensors (v-SPASM sensors) provide enhanced dynamic range, expression-insensitive readout, and a reagent level assay that yields single point measurements of ligand molecular efficacy. Leveraging this technology, we establish the receptor–G-peptide interaction as a sufficient structural determinant of this receptor-level parameter. Combining v-SPASM measurements with molecular dynamics (MD) simulations, we elucidate a two-stage receptor activation mechanism, wherein receptor–G-peptide interactions in an intermediate orientation alter the receptor conformational landscape to facilitate engagement of a fully coupled orientation that tunes G protein activation.

Re-evaluating how low intrinsic efficacy and apparent bias for G protein activation relates to the improved side effect profiles of new opioid agonists

In a recent report by Gillis et al., 2020 (1), it was suggested that low intrinsic agonism, and not biased agonism, leads to an improvement in the separation of potency in opioid-induced respiratory suppression versus antinociception. Although the compounds that were tested have been shown to display G protein signaling bias in prior publications, the authors conclude that since they cannot detect biased agonism in their cellular signaling studies the compounds are therefore not biased agonists. Rather, they conclude that it is low intrinsic efficacy that leads to the therapeutic window improvement. Intrinsic efficacy is the extent to which an agonist can stimulate a G protein-coupled receptor (GPCR) response in a system. The designation of full agonist is made to compounds that produce the highest observable activation in a system (maximum intrinsic efficacy); agonists producing some fraction of that response are considered partial agonists. The maximum response window is determined by the cellular environment, receptor and effector expression levels, and the amplification readout of the system. Biased agonism takes into consideration not only intrinsic efficacy, but also potency (concentration required to reach half maximal efficacy) of an agonist in an assay. Herein, the data published in the aforementioned manuscript was used to rederive the intrinsic efficacy and bias factors as ΔΔlog(τ/KA) and ΔΔlog(Emax/EC50). Based on this reanalysis, the data does not support the conclusion that biased agonism, favoring G protein signaling, was not present. Further, these observations agree with prior studies wherein oliceridine, PZM21 and SR-17018 were first described as biased agonists with improvement in antinociception over respiratory suppression in mice. Therefore, introducing G protein signaling bias may be a means to improve opioid analgesia while avoiding certain undesirable side effects.