Orthosteric Agonist Research Articles

Elucidating the molecular logic of a metabotropic glutamate receptor heterodimer

Metabotropic glutamate (mGlu) receptor protomers can heterodimerize, leading to different pharmacology compared to their homodimeric counterparts. Here, we use complemented donor-acceptor resonance energy transfer (CODA-RET) technology that distinguishes signaling from defined mGlu heterodimers or homodimers, together with targeted mutagenesis of receptor protomers and computational docking, to elucidate the mechanism of activation and differential pharmacology in mGlu2/4 heteromers. We demonstrate that positive allosteric modulators (PAMs) that bind an upper allosteric pocket in the mGlu4 transmembrane domain are active at both mGlu4/4 homomers and mGlu2/4 heteromers, while those that bind a lower allosteric pocket within the same domain are efficacious in homomers but not heteromers. We further demonstrate that both protomers of mGlu2/4 heteromers are cis-activated by their orthosteric agonists, signaling independently with no trans-activation detected. Intriguingly, however, upper pocket mGlu4 PAMs enable trans-activation in mGlu2/4 heteromers from mGlu4 to the mGlu2 protomer and also enhance cis-activation of the mGlu2 protomer. While mGlu2 PAMs enhanced mGlu2 cis-activation in the heterodimer, we were unable to detect trans-activation in the opposite direction from mGlu2 to the mGlu4 protomer, suggesting an asymmetry of signaling. These insights into the molecular logic of this receptor heteromer are critical to building toward precision targeted therapies for multiple neuropsychiatric disorders.

Positive allosteric modulation of a GPCR ternary complex.

The activation of a G protein-coupled receptor (GPCR) leads to the formation of a ternary complex between agonist, receptor, and G protein that is characterized by high-affinity binding. Allosteric modulators bind to a distinct binding site from the orthosteric agonist and can modulate both the affinity and the efficacy of orthosteric agonists. The influence allosteric modulators have on the high-affinity active state of the GPCR-G protein ternary complex is unknown due to limitations on attempting to characterize this interaction in recombinant whole cell or membrane-based assays. Here, we use the purified M2 muscarinic acetylcholine receptor reconstituted into nanodiscs to show that, once the agonist-bound high-affinity state is promoted by the G protein, positive allosteric modulators stabilize the ternary complex that, in the presence of nucleotides, leads to an enhanced initial rate of signaling. Our results enhance our understanding of how allosteric modulators influence orthosteric ligand signaling and will aid the design of allosteric therapeutics.

A golden age of muscarinic acetylcholine receptor modulation in neurological diseases.

Over the past 40 years, the muscarinic acetylcholine receptor family, particularly the M1-receptor and M4-receptor subtypes, have emerged as validated targets for the symptomatic treatment of neurological diseases such as schizophrenia and Alzheimer disease. However, despite considerable effort and investment, no drugs have yet gained clinical approval. This is largely attributable to cholinergic adverse effects that have halted the majority of programmes and resulted in a waning of interest in these G-protein-coupled receptor targets. Recently, this trend has been reversed. Driven by advances in structure-based drug design and an appreciation of the optimal pharmacological properties necessary to deliver clinical efficacy while minimizing adverse effects, a new generation of M1-receptor and M4-receptor orthosteric agonists and positive allosteric modulators are now entering the clinic. These agents offer the prospect of novel therapeutic solutions for 'hard to treat' neurological diseases, heralding a new era of muscarinic drug discovery.

Effect of positive allosteric modulation and orthosteric agonism of dopamine D2-like receptors on respiration in mouse models of Rett syndrome

Rett syndrome (RTT) is an autism spectrum disorder caused by loss-of-function mutations in the methyl-CPG-binding protein 2 (Mecp2) gene. Frequent apneas and irregular breathing are prevalent in RTT, and also occur in rodent models of the disorder, including Mecp2Bird and Mecp2R168X mice. Sarizotan, a serotonin 5-HT1a and dopamine D2-like receptor agonist, reduces the incidence of apneas and irregular breathing in mouse models of RTT (Abdala et al., 2014). Targeting the 5HT1a receptor alone also improves respiration in RTT mice (Levitt et al., 2013). However, the contribution of D2-like receptors in correcting these respiratory disturbances remains untested. PAOPA, a dopamine D2-like receptor positive allosteric modulator, and quinpirole, a dopamine D2-like receptor orthosteric agonist, were used in conjunction with whole-body plethysmography to evaluate whether activation of D2-like receptors is sufficient to improve breathing disturbances in female heterozygous Mecp2Bird/+ and Mecp2R168X/+ mice. PAOPA did not significantly change apnea incidence or irregularity score in RTT mice. PAOPA also had no effect on the ventilatory response to hypercapnia (7 % CO2). In contrast, quinpirole reduced apnea incidence and irregularity scores and improved the hypercapnic ventilatory response in Mecp2R168X/+ and Mecp2Bird/+ mice, while also reducing respiratory rate. These results suggest that D2-like receptors could contribute to the positive effects of sarizotan in the correction of respiratory abnormalities in Rett syndrome. However, positive allosteric modulation of D2-like receptors alone was not sufficient to evoke these effects.

Structural basis of positive allosteric modulation of metabotropic glutamate receptor activation and internalization

The metabotropic glutamate receptors (mGluRs) are neuromodulatory family C G protein coupled receptors which assemble as dimers and allosterically couple extracellular ligand binding domains (LBDs) to transmembrane domains (TMDs) to drive intracellular signaling. Pharmacologically, mGluRs can be targeted at the LBDs by glutamate and synthetic orthosteric compounds or at the TMDs by allosteric modulators. Despite the potential of allosteric compounds as therapeutics, an understanding of the functional and structural basis of their effects is limited. Here we use multiple approaches to dissect the functional and structural effects of orthosteric versus allosteric ligands. We find, using electrophysiological and live cell imaging assays, that both agonists and positive allosteric modulators (PAMs) can drive activation and internalization of group II and III mGluRs. The effects of PAMs are pleiotropic, boosting the maximal response to orthosteric agonists and serving independently as internalization-biased agonists across mGluR subtypes. Motivated by this and intersubunit FRET analyses, we determine cryo-electron microscopy structures of mGluR3 in the presence of either an agonist or antagonist alone or in combination with a PAM. These structures reveal PAM-driven re-shaping of intra- and inter-subunit conformations and provide evidence for a rolling TMD dimer interface activation pathway that controls G protein and beta-arrestin coupling.

Isoquinoline small molecule ligands are agonists and probe-dependent allosteric modulators of the glucagon subfamily of GPCRs

Class B1 G protein-coupled receptors (GPCRs) are peptide hormone receptors and well validated therapeutic targets, however development of non-peptide drugs targeting this class of receptors is challenging. Recently, a series of isoquinoline-based derivates were reported in the patent literature as allosteric ligands for the glucagon receptor subfamily, and two compounds, LSN3451217 and LSN3556672, were used to facilitate structural studies with the glucagon-like peptide-1 receptor (GLP-1R) and glucose dependent insulinotropic peptide receptor (GIPR) bound to orthosteric agonists. Here we pharmacologically characterized stereoisomers of LSN3451217 and LSN3556672, across the class B1 GPCR family. This revealed LSN3556672 isomers are agonists for the glucagon receptor (GCGR), GLP-1R, GIPR and the calcitonin receptor (CTR), albeit the degree of agonism varied at each receptor. In contrast, LSN3451217 isomers were more selective agonists at the GLP-1R, with lower potency at the GCGR and CTR and no activity at the GIPR. All compounds also modulated peptide-mediated cyclic adenosine monophosphate (cAMP) signaling at the GIPR, and to a lesser extent the GLP-1R, in a probe-dependent manner, with modest positive allosteric modulation observed for some peptides, and negligible effects observed with other peptides. In contrast neutral or weak negative/positive allosteric modulation was observed with peptides assessed at the GCGR and CTR. This study expands our knowledge on class B1 GPCR allosteric modulation and may have implications for future structural and drug discovery efforts targeting the class B1 GPCR subfamily.

The human-specific nicotinic receptor subunit CHRFAM7A reduces α7 receptor function in human induced pluripotent stem cells-derived and transgenic mouse neurons.

We investigated the impact of the human-specific gene CHRFAM7A on the function of α7 nicotinic acetylcholine receptors (α7 nAChRs) in two different types of neurons: human-induced pluripotent stem cell (hiPSC)-derived cortical neurons, and superior cervical ganglion (SCG) neurons, taken from transgenic mice expressing CHRFAM7A. dupα7, the gene product of CHRFAM7A, which lacks a major part of the extracellular N-terminal ligand-binding domain, co-assembles with α7, the gene product of CHRNA7. We assessed the receptor function in hiPSC-derived cortical and SCG neurons with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists PNU282987 and choline with the type-2 positive allosteric modulator (PAM II) PNU120596. In line with different cellular models used previously, we demonstrate that CHRFAM7A has a major impact on nicotinic α7 nAChRs by reducing calcium transients in response to all three agonists.

Activation of μ receptors by SR-17018 through a distinctive mechanism

Agonists at μ opioid receptors relieve acute pain, however, their long-term use is limited by side effects, which may involve β-arrestin2. Agonists biased against β-arrestin2 recruitment may be advantageous. However, the classification of bias may be compromised by assays utilising overexpressed μ receptors which overestimate efficacy for G-protein activation. There is a need for re-evaluation with restricted receptor availability to determine accurate agonist efficacies. We depleted μ receptor availability in PathHunter CHO cells using the irreversible antagonist, β-funaltrexamine (β-FNA), and compared efficacies and apparent potencies of twelve agonists, including several previously reported as biased, in β-arrestin2 recruitment and cAMP assays. With full receptor availability all agonists had partial efficacy for stimulating β-arrestin2 recruitment relative to DAMGO, while only TRV130 and buprenorphine were partial agonists as inhibitors of cAMP accumulation. Limiting receptor availability by prior exposure to β-FNA (100 nM) revealed morphine, oxycodone, PZM21, herkinorin, U47700, tianeptine and U47931e are also partial agonists in the cAMP assay. The efficacies of all agonists, except SR-17018, correlated between β-arrestin2 recruitment and cAMP assays, with depleted receptor availability in the latter. Furthermore, naloxone and cyprodime exhibited non-competitive antagonism of SR-17018 in the β-arrestin2 recruitment assay. Limited antagonism by naloxone was also non-competitive in the cAMP assay, while cyprodime was competitive. Furthermore, SR-17018 only negligibly diminished β-arrestin2 recruitment stimulated by DAMGO (1 μM), whereas fentanyl, morphine and TRV130 all exhibited the anticipated competitive inhibition. The data suggest that SR-17018 achieves bias against β-arrestin2 recruitment through interactions with μ receptors outside the orthosteric agonist site.This article is part of the Special Issue on “Ligand Bias”.

SCA44- and SCAR13-associated GRM1 mutations affect metabotropic glutamate receptor 1 function through distinct mechanisms.

Metabotropic glutamate receptor 1 (mGlu1) is a promising therapeutic target for neurodegenerative CNS disorders including spinocerebellar ataxias (SCAs). Clinical reports have identified naturally-occurring mGlu1 mutations in rare SCA subtypes and linked symptoms to mGlu1 mutations. However, how mutations alter mGlu1 function remains unknown, as does amenability of receptor function to pharmacological rescue. Here, we explored SCA-associated mutation effects on mGlu1 cell surface expression, canonical signal transduction and allosteric ligand pharmacology. Orthosteric agonists, positive allosteric modulators (PAMs) and negative allosteric modulators (NAMs) were assessed at two functional endpoints (iCa2+ mobilisation and inositol 1-phosphate [IP1] accumulation) in FlpIn Trex HEK293A cell lines expressing five mutant mGlu1 subtypes. Key pharmacological parameters including ligand potency, affinity and cooperativity were derived using operational models of agonism and allostery. mGlu1 mutants exhibited differential impacts on mGlu1 expression, with a C-terminus truncation significantly reducing surface expression. Mutations differentially influenced orthosteric ligand affinity, efficacy and functional cooperativity between allosteric and orthosteric ligands. Loss-of-function mutations L454F and N885del reduced orthosteric affinity and efficacy, respectively. A gain-of-function Y792C mutant mGlu1 displayed enhanced constitutive activity in IP1 assays, which manifested as reduced orthosteric agonist activity. The mGlu1 PAMs restored glutamate potency in iCa2+ mobilisation for loss-of-function mutations and mGlu1 NAMs displayed enhanced inverse agonist activity at Y792C relative to wild-type mGlu1. Collectively, these data highlight distinct mechanisms by which mGlu1 mutations affect receptor function and show allosteric modulators may present a therapeutic strategy to restore aberrant mGlu1 function in rare SCA subtypes.

Effect of mGluR2 and mGluR2/3 activators on parkinsonism in the MPTP-lesioned non-human primate.

We have previously discovered that the selective activation of metabotropic glutamate type 2 receptors (mGluR2) and concurrent stimulation of metabotropic glutamate types 2 and 3 receptors (mGluR2/3) enhance the anti-parkinsonian action of L-3,4-dihydroxyphenylalanine (L-DOPA). Here, we sought to determine the effects of the mGluR2/3 orthosteric agonists LY-354,740 and LY-404,039, as well as the effects of the mGluR2 positive allosteric modulators LY-487,379 and CBiPES on the range of movement, bradykinesia, posture and alertness as adjuncts to L-DOPA. Ten 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned marmosets entered 4 experimental streams: L-DOPA + LY-354,740 (vehicle, 0.1, 0.3 and 1 mg/kg), L-DOPA + LY-404,039 (vehicle, 0.1, 1 and 10 mg/kg), L-DOPA + LY-487,379 (vehicle, 0.1, 1 and 10 mg/kg), L-DOPA + CBiPES (vehicle, 0.1, 1 and 10 mg/kg). For each molecule, treatments were randomised, and the range of movement, bradykinesia, posture and alertness were assessed by a blinded rater. None of the tested compounds significantly altered the global range of movement. LY-404,039 and CBiPES both reduced global bradykinesia, by up to 46% (both P < 0.05). LY-354,740, LY-404,039 and CBiPES each improved global posture by 35%, 44% and 39% (each P < 0.05), respectively. LY-404,039 and CBiPES both enhanced alertness by 54% (P < 0.05) and 79% (P < 0.01), respectively. LY-487,379 did not improve any of the parameters. Our results suggest that selective mGluR2 positive allosteric modulation and combined mGluR2/3 orthosteric stimulation might benefit bradykinesia, posture and alertness in PD when added to L-DOPA, which potentially represent novel therapeutic indications for molecules acting via these mechanisms.

Comparative Analysis of Orthosteric and Allosteric GLP-1R Agonists' Effects on Insulin Secretion from Healthy, Diabetic, and Recovered INS-1E Pancreatic Beta Cells.

Despite the availability of different treatments for type 2 diabetes (T2D), post-diagnosis complications remain prevalent; therefore, more effective treatments are desired. Glucagon-like peptide (GLP)-1-based drugs are currently used for T2D treatment. They act as orthosteric agonists for the GLP-1 receptor (GLP-1R). In this study, we analyzed in vitro how the GLP-1R orthosteric and allosteric agonists augment glucose-stimulated insulin secretion (GSIS) and intracellular cAMP production (GSICP) in INS-1E pancreatic beta cells under healthy, diabetic, and recovered states. The findings from this study suggest that allosteric agonists have a longer duration of action than orthosteric agonists. They also suggest that the GLP-1R agonists do not deplete intracellular insulin, indicating they can be a sustainable and safe treatment option for T2D. Importantly, this study demonstrates that the GLP-1R agonists variably augment GSIS through GSICP in healthy, diabetic, and recovered INS-1E cells. Furthermore, we find that INS-1E cells respond differentially to the GLP-1R agonists depending on both glucose concentration during and before treatment and/or whether the cells have been previously exposed to these drugs. In conclusion, the findings described in this manuscript will be useful in determining in vitro how pancreatic beta cells respond to T2D drug treatments in healthy, diabetic, and recovered states.

Positive allosteric modulation of the cannabinoid CB1 receptor potentiates endocannabinoid signalling and changes ERK1/2 phosphorylation kinetics.

Activation of CB1 by exogenous agonists causes adverse effects in vivo. Positive allosteric modulation may offer improved therapeutic potential and a reduced on-target adverse effect profile compared with orthosteric agonists, due to reduced desensitisation/tolerance, but this has not been directly tested. This study investigated the ability of PAMs/ago-PAMs to induce receptor regulation pathways, including desensitisation and receptor internalisation. Bioluminescence resonance energy transfer (BRET) assays in HEK293 cells were performed to investigate G protein dissociation, ERK1/2 phosphorylation and β-arrestin 2 translocation, while immunocytochemistry was performed to measure internalisation of CB1 in response to the PAMs ZCZ011, GAT229 and ABD1236 alone and in combination with the orthosteric agonists AEA, 2-AG, and AMB-FUBINACA. ZCZ011, GAT229 and ABD1236 were allosteric agonists in all pathways tested. The ago-PAM ZCZ011 induced a biphasic ERK1/2 phosphorylation time course compared to transient activation by orthosteric agonists. In combination with 2-AG but not AEA or AMB-FUBINACA, ZCZ011 and ABD1236 caused the transient peak of ERK1/2 phosphorylation to become sustained. All PAMs increased the potency and efficacy of AEA-induced signalling in all pathways tested; however, no notable potentiation of 2-AG or AMB-FUBINACA was observed. Ago-PAMs can potentiate endocannabinoid CB1 agonism by AEA to a larger extent compared with 2-AG. However, all compounds were found to be allosteric agonists and induce activation of CB1 in the absence of endocannabinoid, including β-arrestin 2 recruitment and internalisation. Thus, the spatiotemporal signalling of endogenous cannabinoids will not be retained in vivo.

Allosteric Modulators of Serotonin Receptors: A Medicinal Chemistry Survey.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter regulating numerous physiological functions, and its dysregulation is a crucial component of the pathological processes of schizophrenia, depression, migraines, and obesity. 5-HT interacts with 14 different receptors, of which 5-HT1A-1FRs, 5-HT2A-CRs, and 5-HT4-7Rs are G protein-coupled receptors (GPCRs), while 5-HT3R is a ligand-gated ion channel. Over the years, selective orthosteric ligands have been identified for almost all serotonin receptors, yielding several clinically relevant drugs. However, the high degree of homology between 5-HTRs and other GPCRs means that orthosteric ligands can have severe side effects. Thus, there has recently been increased interest in developing safer ligands of GPCRs, which bind to less conserved, more specific sites, distinct from that of the receptor's natural ligand. The present review describes the identification of allosteric ligands of serotonin receptors, which are largely natural compounds (oleamide, cannabidiol, THC, and aporphine alkaloids), complemented by synthetic modulators developed in large part for the 5-HT2C receptor. The latter are positive allosteric modulators sought after for their potential as drugs preferable over the orthosteric agonists as antiobesity agents for their potentially safer profile. When available, details on the interactions between the ligand and allosteric binding site will be provided. An outlook on future research in the field will also be provided.

S-3,4-DCPG, a potent orthosteric agonist for the mGlu8 receptor, facilitates extinction and inhibits the reinstatement of morphine-induced conditioned place preference in male rats

The limbic system, particularly the NAc, shows a high concentration of metabotropic glutamate receptors (mGluRs). Recent evidence suggests the significant involvement of mGluRs in mental disorders, including substance abuse and addiction. The objective of this study was to examine the involvement of mGlu8 receptors in the NAc in the mechanisms underlying the extinction and reinstatement of conditioned place preference (CPP) induced by morphine. Male Wistar rats underwent surgical implantation of bilateral cannulas in the NAc and were assessed in a CPP protocol. In study 1 at the same time as the extinction phase, the rats were given varying doses of S-3,4-DCPG (0.03, 0.3, and 3 μg/0.5 μl). In study 2, rats that had undergone CPP extinction were given S-3,4-DCPG (0.03, 0.3, and 3 μg/0.5 μl) five minutes prior to receiving a subthreshold dose of morphine (1 mg/kg) in order to reactivate the previously extinguished morphine response. The findings demonstrated that administering S-3,4-DCPG directly into the accumbens nucleus resulted in a decrease in the duration of the CPP extinction phase. Moreover, dose-dependent administration of S-3,4-DCPG into the NAc inhibited CPP reinstatement. The observations imply that microinjection of S-3,4-DCPG as a potent orthosteric agonist with high selectivity for the mGlu8 receptor into the NAc promotes the process of extinction while concurrently exerting inhibitory effects on the reinstatement of morphine-induced CPP. This effect may be associated with the modulation of glutamate engagement within the NAc and the plasticity of reward pathways at the synaptic level.

Development of a Fluorescent Ligand for the Intracellular Allosteric Binding Site of the Neurotensin Receptor 1.

The membrane protein family of G protein-coupled receptors (GPCRs) represents a major class of drug targets. Over the last years, the presence of additional intracellular binding sites besides the canonical orthosteric binding pocket has been demonstrated for an increasing number of GPCRs. Allosteric modulators harnessing these pockets may represent valuable alternatives when targeting the orthosteric pocket is not successful for drug development. Starting from SBI-553, a recently discovered intracellular allosteric modulator for neurotensin receptor subtype 1 (NTSR1), we developed the fluorescent molecular probe 14. Compound 14 binds to NTSR1 with an affinity of 0.68 μM in the presence of the agonist NT(8-13). NanoBRET-based ligand binding assays with 14 were established to derive the affinity and structure-activity relationships for allosteric NTSR1 modulators in a direct and nonisotopic manner, thereby facilitating the search for and optimization of novel allosteric NTSR1 ligands. As a consequence of cooperativity between the ligands binding to the allosteric and orthosteric pocket, compound 14 can also be used to investigate orthosteric NTSR1 agonists and antagonists. Moreover, employing 14 as a probe in a drug library screening, we identified novel chemotypes as binders for the intracellular allosteric SBI-553 binding pocket of NTSR1 with single-digit micromolar affinity. These hits may serve as interesting starting points for the development of novel intracellular allosteric ligands for NTSR1 as a highly interesting yet unexploited drug target in the fields of pain and addiction disorder therapy.

Bitter taste receptor activation by cholesterol and an intracellular tastant.

Bitter taste sensing is mediated by type 2 taste receptors (TAS2Rs (also known as T2Rs)), which represent a distinct class of G-protein-coupled receptors1. Among the 26 members of the TAS2Rs, TAS2R14 is highly expressed in extraoral tissues and mediates the responses to more than 100 structurally diverse tastants2-6, although the molecular mechanisms for recognizing diverse chemicals and initiating cellular signalling are still poorly understood. Here we report two cryo-electron microscopy structures for TAS2R14 complexed with Ggust (also known as gustducin) and Gi1. Both structures have an orthosteric binding pocket occupied by endogenous cholesterol as well as an intracellular allosteric site bound by the bitter tastant cmpd28.1, including a direct interaction with the α5 helix of Ggust and Gi1. Computational and biochemical studies validate both ligand interactions. Our functional analysis identified cholesterol as an orthosteric agonist and the bitter tastant cmpd28.1 as a positive allosteric modulator with direct agonist activity at TAS2R14. Moreover, the orthosteric pocket is connected to the allosteric site via an elongated cavity, which has a hydrophobic core rich in aromatic residues. Our findings provide insights into the ligand recognition of bitter taste receptors and suggest activities of TAS2R14 beyond bitter taste perception via intracellular allosteric tastants.

Activation of mGlu 2/3 receptors with the orthosteric agonist LY-404,039 alleviates dyskinesia in experimental parkinsonism.

LY-404,039 is an orthosteric agonist at metabotropic glutamate 2 and 3 (mGlu 2/3 ) receptors, with a possible additional agonist effect at dopamine D 2 receptors. LY-404,039 and its pro-drug, LY-2140023, have previously been tested in clinical trials for psychiatric indications and could therefore be repurposed if they were shown to be efficacious in other conditions. We have recently demonstrated that the mGlu 2/3 orthosteric agonist LY-354,740 alleviated L-3,4-dihydroxyphenylalanine (L-DOPA)-induced abnormal involuntary movements (AIMs) in the 6-hydroxydopamine (6-OHDA)-lesioned rat without hampering the anti-parkinsonian action of L-DOPA. Here, we seek to take advantage of a possible additional D 2 -agonist effect of LY-404,039 and see if an anti-parkinsonian benefit might be achieved in addition to the antidyskinetic effect of mGlu 2/3 activation. To this end, we have administered LY-404,039 (vehicle, 0.1, 1 and 10 mg/kg) to 6-OHDA-lesioned rats, after which the severity of axial, limbs and oro-lingual (ALO) AIMs was assessed. The addition of LY-404,039 10 mg/kg to L-DOPA resulted in a significant reduction of ALO AIMs over 60-100 min (54%, P < 0.05). In addition, LY-404,039 significantly enhanced the antiparkinsonian effect of L-DOPA, assessed through the cylinder test (76%, P < 0.01). These results provide further evidence that mGlu 2/3 orthosteric stimulation may alleviate dyskinesia in PD and, in the specific case of LY-404,039, a possible D 2 -agonist effect might also make it attractive to address motor fluctuations. Because LY-404,039 and its pro-drug have been administered to humans, they could possibly be advanced to Phase IIa trials rapidly for the treatment of motor complications in PD.

Identification of the Candidate mGlu2 Allosteric Modulator THRX-195518 through In Silico Method and Evaluation of Its Neuroprotective Potential against Glutamate-Induced Neurotoxicity in SH-SY5Y Cell Line.

Glutamate (Glu) toxicity has been an important research topic in toxicology and neuroscience studies. In vitro and in vivo studies have shown that Group II metabotropic Glu2 (mGlu2) activators have cell viability effects. This study aims to determine a candidate ligand with high mGlu2 allosteric region activity among cytotoxicity-safe molecules using the in silico positioning method and to evaluate its cell viability effect in vitro. We investigated the candidate molecule's cell viability effect on the SH-SY5Y human neuroblastoma cell line by MTT analysis. In the study, LY 379268 (agonist) and JNJ-46281222 (positive allosteric modulator; PAM) were used as control reference molecules. Drug bank screening yielded THRX-195518 (docking score being -12.4 kcal/mol) as a potential novel drug candidate that has a high docking score and has not been mentioned in the literature so far. The orthosteric agonist LY 379268 exhibited a robust protective effect in our study. Additionally, our findings demonstrate that JNJ-46281222 and THRX-195518, identified as activating the mGlu2 allosteric region through in silico methods, preserve cell viability against Glu toxicity. Therefore, our study not only emphasizes the positive effects of this compound on cell viability against Glu toxicity but also sheds light on the potential of THRX-195518, acting as a mGlu2 PAM, based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET) data, as a candidate drug molecule. These findings underscore the potential utility of THRX-195518 against both neurotoxicity and Central Nervous System (CNS) disorders, providing valuable insights.

Insight into the mechanism of action of ORG27569 at the cannabinoid type one receptor utilising a unified mathematical model

Allosteric modulation of CB1 is therapeutically advantageous compared to orthosteric activation as it potentially offers reduced on-target adverse effects. ORG27569 is an allosteric modulator that increases orthosteric agonist binding to CB1 but decreases functional signalling. ORG27569 is characterised by a delay in disinhibition of agonist-induced cAMP inhibition (lag); however, the mechanism behind this kinetic lag is yet to be identified. We aimed to utilise a mathematical model to predict data and design in vitro experiments to elucidate mechanisms behind the unique signalling profile of ORG27569. The established kinetic ternary complex model includes the existence of a transitional state of CB1 bound to ORG27569 and CP55940 and was used to simulate kinetic cAMP data using NONMEM 7.4 and Matlab R2020b. These data were compared with empirical cAMP BRET data in HEK293 cells stably expressing hCB1. The pharmacometric model suggested that the kinetic lag in cAMP disinhibition by ORG27569 is caused by signal amplification in the cAMP assay and can be reduced by decreasing receptor number. This was confirmed experimentally, as reducing receptor number through agonist-induced internalisation resulted in a decreased kinetic lag by ORG27569. ORG27569 was found to have a similar interaction with CP55940 and the high efficacy agonist WIN55,212-2, and was suggested to have lower affinity for CB1 bound by the partial agonist THC compared to CP55940. Allosteric modulators have unique signalling profiles that are often difficult to interrogate exclusively in vitro. We have used a combined mathematical and in vitro approach to prove that ORG27569 causes a delay in disinhibition of agonist-induced cAMP inhibition due to large receptor reserve in this pathway. We also used the pharmacometric model to investigate the common phenomenon of probe dependence, to propose that ORG27569 binds with higher affinity to CB1 bound by high efficacy orthosteric agonists.

Structural basis for partial agonism in 5-HT3A receptors.

Hyperactivity of serotonin 3 receptors (5-HT3R) underlies pathologies associated with irritable bowel syndrome and chemotherapy-induced nausea and vomiting. Setrons, a class of high-affinity competitive antagonists, are used in the treatment of these conditions. Although generally effective for chemotherapy-induced nausea and vomiting, the use of setrons for treating irritable bowel syndrome has been impaired by adverse side effects. Partial agonists are now being considered as an alternative strategy, with potentially less severe side effects than full antagonists. However, a structural understanding of how these ligands work is lacking. Here, we present high-resolution cryogenic electron microscopy structures of the mouse 5-HT3AR in complex with partial agonists (SMP-100 and ALB-148471) captured in pre-activated and open-like conformational states. Molecular dynamics simulations were used to assess the stability of drug-binding poses and interactions with the receptor over time. Together, these studies reveal mechanisms for the functional differences between orthosteric partial agonists, full agonists and antagonists of the 5-HT3AR.