Commentary

An allosteric model for benzodiazepine receptor function

I interpret some recent data to indicate that co-operative effects take place between the (identical) orthosteric binding sites in a G-protein-coupled receptor dimer. In the current study, the reasonability of this concept was tested by creating a mathematical model. The model is composed of a symmetrical constitutive receptor dimer in which the protomers are able to affect each other allosterically, and it includes binding, receptor activation and signal amplification steps. The model was utilized for analyses of previous data as well as simulations of predicted behaviour. The model demonstrates the behaviour stated in the hypotheses, i.e. even an apparently neutral receptor ligand can allosterically affect agonist binding or receptor activation by binding to the normal orthosteric ligand binding site. Therewith the speculated allosteric action originating from the orthosteric binding site of the dimeric receptor is a realistic possibility. The results of the simulations and curve fitting constitute a reasonable starting point for further studies, and the model can be utilized to design meaningful experiments to investigate these questions.

Preclinical studies have shown that anesthesia might accelerate the clinical progression of Alzheimer's disease (AD) and can have an impact on tau pathology, a hallmark of AD. Although benzodiazepines have been suggested to increase the risk of incident dementia, their impact on tau pathology in vivo is unknown. We thus examined the impact of midazolam, a benzodiazepine that is often administered perioperatively as an anxiolytic, on tau hyperphosphorylation in nontransgenic and in hTau mice, the latter a model of AD-like tau pathology. The acute administration of midazolam in C57BL/6 mice was associated with downregulation of protein phosphatase-1 and a significant and persistent increase in brain tau phosphorylation. In hTau mice, tau hyperphosphorylation was also observed; however, midazolam was neither associated with proaggregant changes nor spatial reference memory impairment. In C57BL/6 mice, chronic midazolam administration immediately increased hippocampal tau phosphorylation, and this effect was more pronounced in older mice. Interestingly, in young C57BL/6 mice, chronic midazolam administration induced hippocampal tau hyperphosphorylation, which persisted for 1 week. In hTau mice, chronic midazolam administration increased hippocampal tau phosphorylation and, although this was not associated with proaggregant changes, this correlated with a decreased capacity of tau to bind to preassembled microtubules. These findings suggest that midazolam can induce significant tau hyperphosphorylation in vivo, which persists well beyond recovery from its sedative effects. Moreover, it can disrupt one of tau's critical functions. Hence, future studies should focus on the impact of more prolonged or repeated benzodiazepine exposure on tau pathology and cognitive decline.

Drugs and natural ligands induce conformational changes in receptors by selecting for natural active and inactive states of receptors. This process can be modeled by a Monod-Wyman-Changeux (MWC) model, which includes in its scheme of equilibriums an isomerization constant for the unoccupied receptor. The isomerization constant typically has a small value that represents the spontaneous activation of the receptor in the absence of ligands (constitutive activity). Allosteric ligands have an effect on the response to an agonist that is indistinguishable from an increase or decrease in the isomerization constant. The nature of allosteric interactions at the GABA_A receptor and other ligand-gated ion channels is consistent with the consequences of a MWC model. Many of the allosteric ligands for these receptors act on sites located on interfaces between adjacent subunits or on the transmembrane helices that are distinct from those of the orthosteric site. By contrast, the M₂ muscarinic receptor has an allosteric site in the extracellular vestibule that acetylcholine must pass through before entering the orthosteric site. Allosteric interactions at the M₂ muscarinic receptor are more complicated than that predicted by the MWC model probably because of the close proximity of the orthosteric and allosteric sites. The affinities of ligands for the active and inactive receptor states provide an unequivocal estimate of ligand bias because these parameters provide the most fundamental measure of how well a ligand induces receptor activation or inhibition. These estimates can be made for ligands interacting with GPCRs by measuring agonist-induced receptor function in the presence of an allosteric agonist or by using both wild type and constitutively active mutant receptors.

Cannabinoid pharmacology has proven nettlesome with issues of promiscuity a common theme among both agonists and antagonists. One recourse is to develop allosteric ligands to modulate cannabinoid receptor signaling. Cannabinoids have come late to the allosteric table. The ‘first-generation’ negative and positive allosteric modulators (NAMs and PAMs) represent an important first effort. However, most studies have relied on synthetic agonists, often tested in over-expression systems rather than a defined neuronal model system that utilizes endogenously synthesized and released cannabinoids.

We have systematically examined first-generation NAMs and a PAM on endocannabinoid modulation of synaptic transmission in cultured autaptic hippocampal neurons. These neurons exhibit CB₁ and 2-arachidonoyl glycerol (2-AG)-mediated depolarization induced suppression of excitation (DSE) and therefore serve as a model to test CB₁ modulators in a neuronal model of endogenous cannabinoid signaling.

We find ORG27569, PSNCBAM-1, and PEPCAN12 attenuate DSE and do not directly inhibit CB₁ receptors. Of these PSNCBAM-1 is the most efficacious while PEPCAN12 has the distinction of being an endogenous NAM. The reported NAMs pregnenolone and hemopressin as well as the reported PAM lipoxin A4 are without effect in this model of endocannabinoid signaling.

In summary, three of the allosteric modulators evaluated function in a manner consistent with allosterism in a neuronal 2-AG-based model of endogenous cannabinoid signaling.

The GABA_A receptor is the target for a number of important allosteric drugs used in medicine, including benzodiazepines and anesthetics. These modulators have variable effects on the potency and maximal response of macroscopic currents elicited by different GABA_A receptor agonists, yet this modulation is consistent with a two-state model in which the allosteric ligand has invariant affinity constants for the active and inactive states. Analysis of the effects of an allosteric agonist, like etomidate, on the population current provides a means of estimating the gating constant of the unliganded GABA_A receptor (∼ 10^− 4). In contrast, allosteric interactions at the M₂ muscarinic receptor are often inconsistent with a two-state model. Analyzing allosterism within the constraints of a two-state model, nonetheless, provides an unbiased measure of probe dependence as well as clues to the mechanism of allosteric modulation. The rather simple allosteric effect of affinity-only modulation is difficult to explain and suggests modulation of a peripheral orthosteric ligand-docking site on the M₂ muscarinic receptor.

The ghrelin receptor is a G protein-coupled receptor (GPCR) mainly distributed in the brain, and also expressed in peripheral tissues. Remarkably, the ghrelin receptor possesses a naturally high constitutive activity representing 50% of its maximal activity. Its endogenous ligand ghrelin is the only known orexigenic gastrointestinal peptide and plays a central role in the regulation of appetite, food intake, and energy homeostasis.

Reducing the constitutive activity of the ghrelin receptor by inverse agonists is the strategy adopted by our group to develop anti-obesity drugs. Therefore, short peptides were synthesized and showed high inverse agonist potency toward the ghrelin receptor.

This review describes the methods used to synthesize the peptides and to evaluate their biological activity. Peptide synthesis was performed on solid phase using a Fmoc/tBu-strategy. Peptide potency was measured with a signal transduction assay, the inositol trisphosphate turnover assay, adapted to a receptor expressing constitutive activity.