Selective Allosteric Agonist Research Articles

C-X-C Chemokine Receptor Type 4 (CXCR-4) Selective Allosteric Agonist ATI2341 TFA Promotes Uterine Repair in Asherman Syndrome

Our study elucidated the therapeutic effect and mechanism of CXCR4 functional selective allosteric agonist ATI2341 TFA on treating Asherman syndrome (AS). AS was established by damaging mouse uterus and the bone marrow cells from ubiquitin-GFP mice were transplanted into AS mice. After 2 weeks, PBS, CXCR4 agonist ATI2341 TFA, CXCR4 antagonist or combined drugs were administrated into mice followed by analysis of detect pregnancy rate, litter size and pregnancy cycle. In AS model, ATI2341 TFA administration promoted BMSCs recruitment into uterine, which was inhibited by CXCL12 receptor antagonist ADM3100. In addition, ATI2341 TFA administration mitigated the degree of endometrial fibrosis, which was exacerbated by ADM3100. ATI2341 TFA administration also improved the fertility and the number of litter and shorten the pregnancy cycle of mice, while ADM3100 exhibited the opposite impacts. In conclusion, CXCR4 receptor agonist alleviates the infertility or adverse pregnancy outcomes possibly through promoting the recruitment of BMSCs in AS mice.

The unconventional activation of the muscarinic acetylcholine receptor M4R by diverse ligands

Muscarinic acetylcholine receptors (mAChRs) respond to the neurotransmitter acetylcholine and play important roles in human nervous system. Muscarinic receptor 4 (M4R) is a promising drug target for treating neurological and mental disorders, such as Alzheimer’s disease and schizophrenia. However, the lack of understanding on M4R’s activation by subtype selective agonists hinders its therapeutic applications. Here, we report the structural characterization of M4R selective allosteric agonist, compound-110, as well as agonist iperoxo and positive allosteric modulator LY2119620. Our cryo-electron microscopy structures of compound-110, iperoxo or iperoxo-LY2119620 bound M4R-Gi complex reveal their different interaction modes and activation mechanisms of M4R, and the M4R-ip-LY-Gi structure validates the cooperativity between iperoxo and LY2119620 on M4R. Through the comparative structural and pharmacological analysis, compound-110 mostly occupies the allosteric binding pocket with vertical binding pose. Such a binding and activation mode facilitates its allostersic selectivity and agonist profile. In addition, in our schizophrenia-mimic mouse model study, compound-110 shows antipsychotic activity with low extrapyramidal side effects. Thus, this study provides structural insights to develop next-generation antipsychotic drugs selectively targeting on mAChRs subtypes.

Reduced expression of the psychiatric risk gene DLG2 (PSD93) impairs hippocampal synaptic integration and plasticity

Copy number variants indicating loss of function in the DLG2 gene have been associated with markedly increased risk for schizophrenia, autism spectrum disorder, and intellectual disability. DLG2 encodes the postsynaptic scaffolding protein DLG2 (PSD93) that interacts with NMDA receptors, potassium channels, and cytoskeletal regulators but the net impact of these interactions on synaptic plasticity, likely underpinning cognitive impairments associated with these conditions, remains unclear. Here, hippocampal CA1 neuronal excitability and synaptic function were investigated in a novel clinically relevant heterozygous Dlg2+/− rat model using ex vivo patch-clamp electrophysiology, pharmacology, and computational modelling. Dlg2+/− rats had reduced supra-linear dendritic integration of synaptic inputs resulting in impaired associative long-term potentiation. This impairment was not caused by a change in synaptic input since NMDA receptor-mediated synaptic currents were, conversely, increased and AMPA receptor-mediated currents were unaffected. Instead, the impairment in associative long-term potentiation resulted from an increase in potassium channel function leading to a decrease in input resistance, which reduced supra-linear dendritic integration. Enhancement of dendritic excitability by blockade of potassium channels or activation of muscarinic M1 receptors with selective allosteric agonist 77-LH-28-1 reduced the threshold for dendritic integration and 77-LH-28-1 rescued the associative long-term potentiation impairment in the Dlg2+/− rats. These findings demonstrate a biological phenotype that can be reversed by compound classes used clinically, such as muscarinic M1 receptor agonists, and is therefore a potential target for therapeutic intervention.

The regulatory role of GABAA receptor in Actinia equina nervous system and the possible effect of global ocean acidification

Global warming and connected acidification of the world ocean attract a substantial amount of research efforts, in particular in a context of their impact on behaviour and metabolism of marine organisms, such as Cnidaria. Nevertheless, mechanisms underlying Cnidarians’ neural signalling and behaviour and their (possible) alterations due to the world ocean acidification remain poorly understood. Here we researched for the first time modulation of GABAA receptors (GABAARs) in Actinia equina (Cnidaria: Anthozoa) by pH fluctuations within a range predicted by the world ocean acidification scenarios for the next 80–100 years and by selective pharmacological activation. We found that in line with earlier studies on vertebrates, both changes of pH and activation of GABAARs with a selective allosteric agonist (diazepam) modulate electrical charge transfer through GABAAR and the whole-cell excitability. On top of that, diazepam modifies the animal behavioural reaction on startle response. However, despite behavioural reactions displayed by living animals are controlled by GABAARs, changes of pH do not alter them significantly. Possible mechanisms underlying the species resistance to acidification impact are discussed.

Dronedarone Modulates M1 and M3 Muscarinic Receptors with Subtype Selectivity, Functional Selectivity, and Probe Dependence

We have previously reported that amiodarone interacts with a novel allosteric site on muscarinic receptors. Amiodarone's most striking effect is to enhance the maximal response elicited by muscarinic agonists at the M₁, M₃, and M₅ receptors. Furthermore, the quaternary analog N-ethylamiodarone (NEA) is inhibitory at these receptors and appears to compete with amiodarone at that allosteric site. In the present studies, we show that dronedarone also modulates Gq-mediated responses at M₁ and M₃, although in a more discriminating manner. For example, dronedarone markedly enhances pilocarpine-stimulated release of arachidonic acid from CHO cells, via the M₃ receptor subtype, but does not affect the acetylcholine-stimulated response. Such probe-dependent effects are diagnostic of an allosteric interaction. In comparison to these effects at M₃, dronedarone is strongly inhibitory toward both pilocarpine and acetylcholine at the M₁ subtype. The effects of dronedarone are consistent with an interaction at the amiodarone site: dronedarone inhibits the enhancement of acetylcholine's response produced by amiodarone at the M₃ subtype; also, NEA reverses the enhancement of pilocarpine's response at M₃ produced by either dronedarone or amiodarone. In studies with the M₁-selective allosteric agonist 4-[3-(4-butylpiperidin-1-yl)-propyl]-7-fluoro-4H-benzo[1,4]oxazin-3-one (AC-260584), amiodarone enhanced the maximal response observed, whereas dronedarone was inhibitory. On the other hand, benzyl quinolone carboxylic acid, the well-known allosteric ligand that dramatically enhances the potency of acetylcholine at the M₁ subtype, had no effect on the response profile of AC-260584. In summary, dronedarone acts at M₁ and M₃ muscarinic receptors in a manner that complements amiodarone and provides an additional tool with which to investigate this novel allosteric site.

Effects of extrasynaptic GABA‐A signaling in the PAG on morphine antinociception.

Mu‐opioid receptors (MOPrs) reduce GABA‐mediated inhibition of ventrolateral periaqueductal gray (vlPAG) output neurons to activate the descending pain modulatory system and induce antinociception. Both tonic and phasic GABAA‐mediated currents are observed in the vlPAG. Tonic GABAA currents are reduced in vlPAG neurons from female rats pretreated with Complete Freud's Adjuvant (CFA) for 5‐7 days to induce chronic inflammatory pain (2‐way ANOVA; F (1, 45) = 15.24, p = 0.0003). The mRNA and protein levels of the GABAA delta subunit, known to mediate tonic currents in other brain areas, are increased in the vlPAG of female, but not male rats, pretreated with CFA. The decrease in current in females may be due to the rapid clearance of GABA because of upregulation of GABA transporters. Neuronal GAT1 transcripts were increased in female vlPAG pretreated with CFA (Two‐way ANOVA, F (1, 16) = 25.63, p < 0.0001). Glial GAT3 transcripts were decreased in males and increased in female vlPAG pretreated with CFA (Two way ANOVA, F (1, 16) = 32.93). Finally, we explored the role of the GABAA delta subunit in morphine‐induced antinociception with the novel selective allosteric agonist (DS2). DS2 (3 µg) was injected in to the vlPAG of male and female rats prior to cumulative morphine doses and testing of thermal nociceptive thresholds using the hot plate test. DS2 significantly decreased the ED50 of morphine in male rats (vehicle = 4.8 µg vs. DS2 = 10.8 µg, F(1,61) = 6.57, p = 0.013). Interestingly, female rats exhibited a leftward shift of the morphine dose response following DS2 microinjection (vehicle = 15.5 µg vs. DS2 = 5.1 µg, p < 0.05). These data support a role for the GABAA receptors containing the delta subunit in pain modulation in vivo.

Rational design of a selective antagonist of ε protein kinase C derived from the selective allosteric agonist, pseudo-RACK peptide

We have previously shown that domains involved in binding of protein kinase C (PKC) isozymes to their respective anchoring proteins (RACKs) and short peptides derived from these domains are PKC isozyme-selective antagonists. We also identified PKC isozyme-selective agonists, named ψRACK peptides, derived from a sequence within each PKC with high homology to its respective RACK. We noted that all the ψRACK sequences within each PKC isozyme have at least one non-homologous amino acid difference from their corresponding RACK that constitutes a charge change. Based on this information, we have devised here a new approach to design an isozyme-selective PKC antagonist, derived from the ψRACK sequence. We focused on εPKC ψRACK peptide, where the pseudo-εRACK sequence (ψεRACK; H D APIGYD; corresponding to εPKC85-92) is different in charge from the homologous RACK-derived sequence (N N VALGYD; corresponding to εRACK285-292) in the second amino acid. Here we show that changing the charge of the ψεRACK peptide through a substitution of only one amino acid (aspartate to asparagine) resulted in a peptide with an opposite activity on the same cell function and a substitution for aspartate with an alanine resulted in an inactive peptide. These data support our hypothesis regarding the mechanism by which pseudo-RACK peptide activates PKC in heart cells and suggest that this approach is applicable to other signaling proteins with inducible protein–protein interactions.