Benzyl Quinolone Carboxylic Acid Research Articles

Quantum Dot loaded BQCA‐Octadecylamine conjugate nanosystems for theranostic applications in Alzheimer’s Disease.

AbstractBackgroundAlzheimer’s Disease (AD) is one of the most common types of dementias in the world and is characterized by progressive, irreversible neurodegeneration. Benzyl quinolone carboxylic acid was shown to have beneficial in management of AD. The current challenge with BQCA is its low brain half‐life, permeability and rapid elimination. The above challenge can be answered by formulating lipid drug conjugates of BQCA, with octadecyl amine (BQCA‐ODA). Quantum dots (QDs) with their unique optical properties, with high sensitivity and stability emerging as theranostic agents in several neurodegenerative diseases. Hypothesizing the co‐delivery of QDs and BQCA for the effective treatment of AD, in the present study we have formulated lipid nano carriers loaded with quantum dots (QDs) surface modified with Polysorbate 80, called PS80‐QD‐BQCA‐ODA‐NPs, for integrating imaging and therapy for effective management of AD.MethodBQCA was conjugated to Octadecyl amine (ODA), to form BQCA‐ODA LDC conjugate. The amphiphilic nature of the BQCA‐ODA conjugate allows the formation of self‐assembled nanoparticles. Here, we have employed solvent injection method followed by sonication, while addition of QDs in the organic phase and P80 in the aqueous phase to form PS80‐QD‐BQCA‐ODA‐NPs . The optimized formulation was evaluated for physicochemical properties (particle size, zeta potential and polydispersity index) surface morphology and in vitro drug release. The in vitro biocompatibility of the PS80‐QD‐BQCA‐ODA‐NPs was assessed on RBC and SHSY‐5Y cell lines. Further the application of PS80‐QD‐BQCA‐ODA‐NPs was assessed by in vivo imaging and behavioural studies on STZ induced mice models of AD.ResultThe formation of BQCA‐ODA conjugate formation was confirmed 13C‐NMR, 1H‐NMR, LC‐MS. The optimized PS80‐QD‐BQCA‐ODA‐NPs were found to have a near spherical shape with a particle size (PS) of 152.62 ± 1.82 nm, zeta potential (ZP) of 18.59 ± 0.56 mV and polydispersity index (PDI) of 0.214±0.08. PS80‐QD‐BQCA‐ODA‐NPs. The NPs shown improved brain permeability, with greater fluorescence intensity in mouse brains when compared to free QDs. In the in vivo efficiency studies PS80‐QD‐BQCA‐ODA‐NPs shown a significant improvement in memory in mice models.ConclusionThe PS80‐QD‐BQCA‐ODA‐NPs nanomedicine was successfully prepared, thoroughly characterized and has shown theronostic benefits in AD mice models.

Activation of basal forebrain cholinergic neurons improves colonic hyperpermeability through the vagus nerve and adenosine A2B receptors in rats

Intestinal barrier dysfunction, a leaky gut, contributes to the pathophysiology of various diseases such as dementia and irritable bowel syndrome (IBS). We recently clarified that orexin, ghrelin, or adenosine A2B signaling in the brain improved leaky gut through the vagus nerve. The present study was performed to clarify whether basal forebrain cholinergic neurons (BFCNs) are implicated in the central regulation of intestinal barrier function. We activated BFCNs using benzyl quinolone carboxylic acid (BQCA), a positive muscarinic M1 allosteric modulator, and evaluated colonic permeability by quantifying the absorbed Evans blue in rat colonic tissue. Intracisternal (not intraperitoneal) injection of BQCA blocked the increased colonic permeability in response to lipopolysaccharide. Vagotomy blocked BQCA-induced improvement of colonic hyperpermeability. Intracisternally administered pirenzepine, a muscarinic M1 selective antagonist, prevented intestinal barrier function improvement by intravenously administered 2-deoxy-d-glucose, central vagal stimulant. Adenosine A2B receptor antagonist but not dopamine or opioid receptor antagonist prevented BQCA-induced blockade of colonic hyperpermeability. Additionally, intracisternal injection of pirenzepine blocked orexin- or butyrate-induced intestinal barrier function improvement. These results suggest that BFCNs improve leaky gut through adenosine A2B signaling and the vagal pathway. Furthermore, BFCNs mediate orexin- or butyrate-induced intestinal barrier function improvement. Since BFCNs play a role in cognitive function and a leaky gut is associated with dementia, the present finding may lead us to speculate that BFCNs are involved in the development of dementia by regulating intestinal barrier function.

Restoring Agonist Function at a Chemogenetically Modified M1 Muscarinic Acetylcholine Receptor.

Designer receptors exclusively activated by designer drugs (DREADDs) have been successfully employed to activate signaling pathways associated with specific muscarinic acetylcholine receptor (mAChR) subtypes. The M1 DREADD mAChR displays minimal responsiveness to the endogenous agonist acetylcholine (ACh) but responds to clozapine-N-oxide (CNO), an otherwise pharmacologically inert ligand. We have previously shown that benzyl quinolone carboxylic acid (BQCA), an M1 mAChR positive allosteric modulator (PAM), can rescue ACh responsiveness at these receptors. However, whether this effect is chemotype specific or applies to next-generation M1 PAMs with distinct scaffolds is unknown. Here, we reveal that new M1 PAMs restore ACh function at the M1 DREADD while modulating ACh binding at the M1 wild-type mAChR. Importantly, we demonstrate that the modulation of ACh function by M1 PAMs is translated in vivo using transgenic M1 DREADD mice. Our data provide important insights into mechanisms that define allosteric ligand modulation of agonist affinity vs efficacy and how these effects play out in the regulation of in vivo responses.

Novel BQCA- and TBPB-Derived M1 Receptor Hybrid Ligands: Orthosteric Carbachol Differentially Regulates Partial Agonism.

Recently, investigations of the complex mechanisms of allostery have led to a deeper understanding of G protein-coupled receptor (GPCR) activation and signaling processes. In this context, muscarinic acetylcholine receptors (mAChRs) are highly relevant due to their exemplary role in the study of allosteric modulation. In this work, we compare and discuss two sets of putatively dualsteric ligands, which were designed to connect carbachol to different types of allosteric ligands. We chose derivatives of TBPB [1-(1'-(2-tolyl)-1,4'-bipiperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one] as M1 -selective putative bitopic ligands, and derivatives of benzyl quinolone carboxylic acid (BQCA) as an M1 positive allosteric modulator, varying the distance between the allosteric and orthosteric building blocks. Luciferase protein complementation assays demonstrated that linker length must be carefully chosen to yield either agonist or antagonist behavior. These findings may help to design biased signaling and/or different extents of efficacy.

Forebrain Cholinergic Signaling Regulates Innate Immune Responses and Inflammation.

The brain regulates physiological functions integral to survival. However, the insight into brain neuronal regulation of peripheral immune function and the neuromediator systems and pathways involved remains limited. Here, utilizing selective genetic and pharmacological approaches, we studied the role of forebrain cholinergic signaling in the regulation of peripheral immune function and inflammation. Forebrain-selective genetic ablation of acetylcholine release and vagotomy abolished the suppression of serum TNF by the centrally-acting cholinergic drug galantamine in murine endotoxemia. Selective stimulation of acetylcholine action on the M1 muscarinic acetylcholine receptor (M1 mAChR) by central administration of the positive allosteric modulator benzyl quinolone carboxylic acid (BQCA) suppressed serum TNF (TNFα) levels in murine endotoxemia. This effect was recapitulated by peripheral administration of the compound. BQCA also improved survival in murine endotoxemia and these effects were abolished in M1 mAChR knockout (KO) mice. Selective optogenetic stimulation of basal forebrain cholinergic neurons innervating brain regions with abundant M1 mAChR localization reduced serum TNF in endotoxemic mice. These findings reveal that forebrain cholinergic neurons regulate innate immune responses and inflammation, suggesting the possibility that in diseases associated with cholinergic dysfunction, including Alzheimer's disease this anti-inflammatory regulation can be impaired. These results also suggest novel anti-inflammatory approaches based on targeting forebrain cholinergic signaling in sepsis and other disorders characterized by immune dysregulation.

Positive Allosteric Modulation of Cholinergic Receptors Improves Spatial Learning after Cortical Contusion Injury in Mice.

We examined benzyl quinolone carboxylic acid (BQCA), a novel M1 muscarinic-positive allosteric modulator, for improving memory and motor dysfunction after cerebral cortical contusion injury (CCI). Adult mice received unilateral motorsensory cortical CCI or sham injury. Benzyl quinolone carboxylic acid (BQCA; 5, 10, and 20 mg/kg, intraperitoneally [i.p.] × 2/day × 3-4 weeks) or vehicle (Veh) were administered, and weekly evaluations were undertaken using a battery of motor tests, as well as the Morris water maze. Thereafter, cerebral metabolic activation was investigated in awake animals during walking with [14C]-2-deoxygIucose autoradiography, comparing CCI mice previously treated with BQCA (20 mg/kg) or vehicle. Relative changes in local cerebral glucose uptake (rCGU) were evaluated in three-dimensional-reconstructed brains using statistical parametric mapping. CCI resulted in mild hyperactivity in the open field, and modest significant motor deficits, as well as significantly decreased spatial learning at 3 weeks. BQCA in CCI mice resulted in significantly improved spatial recall during the third week, with minimal effects on motor outcomes. CCI significantly decreased rCGU in the ipsilesional basal ganglia-thalamocortical circuit and in somatosensory regions, with relative increases noted contralaterally, as well as in the cerebellum. Significant decreases in rCGU were noted in subregions of the ipsilesional hippocampal formation, with significant increases noted contralesionally. BQCA compared to vehicle-treated mice showed modest, though significantly increased, rCGU in motor regions, as well as a partial reversal of lesion-related rCGU findings in subregions of the hippocampal formation. rCGU in ipsilesional posterior CA1 demonstrated a significant inverse correlation with latency to find the submerged platform. BQCA at 20 mg/kg had no significant effect on general motor activity, body weight, or acute motor, secretory, or respiratory symptoms. Results suggest that BQCA is a candidate compound to improve learning and memory function after brain trauma and may not suffer the associated central nervous system side effects typically associated with even modest doses of other cholinergic enhancers.

The pharmacologic characterization of allosteric molecules: Gq protein activation

Context: Drugs such as positive allosteric modulators (PAMs) produce complex behaviors when acting on tissues in different physiological contexts in vivo.Objective: This study describes the use of functional assays of varying receptor sensitivity to unveil the various behaviors of PAMs and thus quantify allosteric effect through system independent scales.Materials and methods: Muscarinic receptor activation with acetylcholine (ACh) was used to the demonstrate activity of the PAM agonist 1–(4-methoxybenzyl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid, Benzyl quinolone carboxylic acid (BQCA) in terms of direct agonism, potentiation of ACh affinity, and ACh efficacy. Concentration–response curves were fit to the functional allosteric model to yield indices of agonism (τB), effects on affinity (α cooperativity), and efficacy (β cooperativity).Results: It is shown that a highly sensitive functional assay revealed the direct efficacy of BQCA as an agonist and relatively insensitive cells (produced by chemical alkylation of muscarinic receptor with phenoxybenzamine) revealed a positive allosteric effect of BQCA on ACh efficacy. A wide range of functional assay sensitivities produced a complex pattern of behavior for BQCA all of which was accurately quantified through the system-independent parameters of the functional allosteric model.Conclusions: The study of complex allosteric molecules in a range of functional assays of varying sensitivity allows the measurement of the complete array of activities of these molecules on receptors and also better predicts which will be seen with these in vivo where a range of tissue sensitivities is encountered.

Probing the binding site of novel selective positive allosteric modulators at the M1 muscarinic acetylcholine receptor

Subtype-selective allosteric modulation of the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) is an attractive approach for the treatment of numerous disorders, including cognitive deficits. The discovery of benzyl quinolone carboxylic acid, BQCA, a selective M1 mAChR positive allosteric modulator (PAM), spurred the subsequent development of newer generation M1 PAMs representing diverse chemical scaffolds, different pharmacodynamic properties and, in some instances, improved pharmacokinetics. Key exemplar molecules from such efforts include PF-06767832 (N-((3R,4S)-3-hydroxytetrahydro-2H-pyran-4-yl)-5-methyl-4-(4-(thiazol-4-yl)benzyl)pyridine-2-carboxamide), VU6004256 (4,6-difluoro-N-(1S,2S)-2-hydroxycyclohexyl-1-((6-(1-methyl-1H-pyrazol-4-yl)pyridine-3-yl)methyl)-1H-indole-3-carboxamide) and MIPS1780 (3-(2-hydroxycyclohexyl)-6-(2-((4-(1-methyl-1H-pyrazol-4-yl)-benzyl)oxy)phenyl)pyrimidin-4(3H)-one). Given these diverse scaffolds and pharmacodynamics, the current study combined pharmacological analysis and site-directed mutagenesis to explore the potential binding site and function of newer M1 mAChR PAMs relative to BQCA. Interestingly, the mechanism of action of the novel PAMs was consistent with a common model of allostery, as previously described for BQCA. Key residues involved in the activity of BQCA, including Y179 in the second extracellular loop (ECL) and W4007.35 in transmembrane domain (TM) 7, were critical for the activity of all PAMs tested. Overall, our data indicate that structurally distinct PAMs share a similar binding site with BQCA, specifically, an extracellular allosteric site defined by residues in TM2, TM7 and ECL2. These findings provide valuable insights into the structural basis underlying modulator binding, cooperativity and signaling at the M1 mAChR, which is essential for the rational design of PAMs with tailored pharmacological properties.

Assessment of the Molecular Mechanisms of Action of Novel 4-Phenylpyridine-2-One and 6-Phenylpyrimidin-4-One Allosteric Modulators at the M1 Muscarinic Acetylcholine Receptors.

Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism but whose molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, benzyl quinolone carboxylic acid (BQCA), in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist ACh on three different signaling pathways [extracellular signal-regulated kinases 1/2 phosphorylation, inositol monophosphate (IP1) accumulation, and β-arrestin-2 recruitment], revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling, and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated.

The concept of hybrid molecules of tacrine and benzyl quinolone carboxylic acid (BQCA) as multifunctional agents for Alzheimer's disease

Novel tacrine-benzyl quinolone carboxylic acid (tacrine-BQCA) hybrids were designed based on multi-target directed ligands (MTLDs) paradigm, synthesized and evaluated in vitro as inhibitors of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). Tacrine moiety is represented herein as 7-methoxytacrine, 6-chlorotacrine or unsubstituted tacrine forming three different families of seven members, i.e. 21 compounds in overall. Introducing BQCA, a positive modulator of M1 muscarinic acetylcholine receptors (mAChRs), the action of novel compounds on M1 mAChRs was evaluated via Fluo-4 NW assay on the Chinese hamster ovarian (CHO-M1WT2) cell line. All the novel tacrine-BQCA hybrids were able to block the action of hAChE and hBChE in micromolar to nanomolar range. The hAChE kinetic profile of 5p was found to be mixed-type which is consistent with our docking experiments. Moreover, selected ligands were assessed for their potential hepatotoxicity on HepG2 cell line and presumable permeation through the blood-brain barrier by PAMPA assay. Expected agonistic profile towards M1 mAChRs delivered by BQCA moiety was not confirmed. From all the hybrids, 5o can be highlighted as non-selective cholinesterase inhibitor (hAChE IC50 = 74.5 nM; hBChE IC50 = 83.3 nM) with micromolar antagonistic activity towards M1 mAChR (IC50 = 4.23 μM). A non-selective pattern of cholinesterase inhibition is likely to be valuable during the onset as well as later stages of AD.

The allosteric site regulates the voltage sensitivity of muscarinic receptors

Muscarinic receptors (M-Rs) for acetylcholine (ACh) belong to the class A of G protein–coupled receptors. M-Rs are activated by orthosteric agonists that bind to a specific site buried in the M-R transmembrane helix bundle. In the active conformation, receptor function can be modulated either by allosteric modulators, which bind to the extracellular receptor surface or by the membrane potential via an unknown mechanism. Here, we compared the modulation of M1-Rs and M3-Rs induced by changes in voltage to their allosteric modulation by chemical compounds. We quantified changes in receptor signaling in single HEK 293 cells with a FRET biosensor for the Gq protein cycle. In the presence of ACh, M1-R signaling was potentiated by voltage, similarly to positive allosteric modulation by benzyl quinolone carboxylic acid. Conversely, signaling of M3-R was attenuated by voltage or the negative allosteric modulator gallamine. Because the orthosteric site is highly conserved among M-Rs, but allosteric sites vary, we constructed “allosteric site” M3/M1-R chimeras and analyzed their voltage dependencies. Exchanging the entire allosteric sites eliminated the voltage sensitivity of ACh responses for both receptors, but did not affect their modulation by allosteric compounds. Furthermore, a point mutation in M3-Rs caused functional uncoupling of the allosteric and orthosteric sites and abolished voltage dependence. Molecular dynamics simulations of the receptor variants indicated a subtype-specific crosstalk between both sites, involving the conserved tyrosine lid structure of the orthosteric site. This molecular crosstalk leads to receptor subtype-specific voltage effects.

An Approach to Discovering Novel Muscarinic M1 Receptor Positive Allosteric Modulators with Potent Cognitive Improvement and Minimized Gastrointestinal Dysfunction

Activation of muscarinic M1 receptor (M1R) is a promising approach for improving cognitive impairment in Alzheimer's disease. However, an M1R-selective positive allosteric modulator (PAM), benzyl quinolone carboxylic acid (BQCA), at 30 mg/kg, induced diarrhea in wild-type mice, but not in M1R knockout mice. Moreover, BQCA (0.1-1000 nM) augmented electric field stimulation (EFS)-induced ileum contraction in an in vitro Magnus assay. Thus, we decided to establish a drug-screening strategy to discover novel M1 PAMs producing potent cognitive improvement with minimized gastrointestinal (GI) dysfunction. We assessed PAM parameters of various M1 PAMs with ≥100-fold selectivity over other muscarinic receptor subtypes by using in vitro binding and functional analysis. Evaluation of these M1 PAMs in the Magnus assay revealed a significant correlation between percentage of ileum contractions at 1 μM and their α-value, a PAM parameter associated with the binding cooperativity between acetylcholine and M1 PAM. M1 PAMs with lower α-value showed lower impact on EFS-induced ileum contraction. Next, we characterized in vivo profiles of two M1 PAMs: compound A (log α = 1.18) and compound B (log α = 3.30). Compound A, at 30 mg/kg, significantly improved scopolamine-induced cognitive deficits without prominent signs of diarrhea at up to 1000 mg/kg in mice. In contrast, compound B, at 10 mg/kg, showed both significant improvement of scopolamine-induced cognitive deficits and severe diarrhea. Thus, fine adjustment of the α-values could be a key to discovering M1 PAMs yielding potent cognitive improvement with a lower risk of GI effects.

FRET Studies of Quinolone-Based Bitopic Ligands and Their Structural Analogues at the Muscarinic M1 Receptor.

Aiming to design partial agonists as well as allosteric modulators for the M1 muscarinic acetylcholine (M1AChR) receptor, two different series of bipharmacophoric ligands and their structural analogues were designed and synthesized. The hybrids were composed of the benzyl quinolone carboxylic acid (BQCA)-derived subtype selective allosteric modulator 3 and the orthosteric building block 4-((4,5-dihydroisoxazol-3-yl)oxy)-N,N-dimethylbut-2-yn-1-amine (base of iperoxo) 1 or the endogenous ligand 2-(dimethylamino)ethyl acetate (base of acetylcholine) 2, respectively. The two pharmacophores were linked via alkylene chains of different lengths (C4, C6, C8, and C10). Furthermore, the corresponding structural analogues of 1 and 2 and of modified BQCA 3 with varying alkyl chain length between C2 and C10 were investigated. Fluorescence resonance energy transfer (FRET) measurements in a living single cell system were investigated in order to understand how these compounds interact with a G protein-coupled receptor (GPCR) on a molecular level and how the single moieties contribute to ligand receptor interaction. The characterization of the modified orthosteric ligands indicated that a linker attached to an orthoster rapidly attenuates the receptor response. Linker length elongation increases the receptor response of bitopic ligands, until reaching a maximum, followed by a gradual decrease. The optimal linker length was found to be six methylene groups at the M1AChR. A new conformational change is described that is not of inverse agonistic origin for long linker bitopic ligands and was further investigated by exceptional fragment-based screening approaches.

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.

M1 muscarinic allosteric modulators slow prion neurodegeneration and restore memory loss.

The current frontline symptomatic treatment for Alzheimer’s disease (AD) is whole-body upregulation of cholinergic transmission via inhibition of acetylcholinesterase. This approach leads to profound dose-related adverse effects. An alternative strategy is to selectively target muscarinic acetylcholine receptors, particularly the M1 muscarinic acetylcholine receptor (M1 mAChR), which was previously shown to have procognitive activity. However, developing M1 mAChR–selective orthosteric ligands has proven challenging. Here, we have shown that mouse prion disease shows many of the hallmarks of human AD, including progressive terminal neurodegeneration and memory deficits due to a disruption of hippocampal cholinergic innervation. The fact that we also show that muscarinic signaling is maintained in both AD and mouse prion disease points to the latter as an excellent model for testing the efficacy of muscarinic pharmacological entities. The memory deficits we observed in mouse prion disease were completely restored by treatment with benzyl quinolone carboxylic acid (BQCA) and benzoquinazoline-12 (BQZ-12), two highly selective positive allosteric modulators (PAMs) of M1 mAChRs. Furthermore, prolonged exposure to BQCA markedly extended the lifespan of diseased mice. Thus, enhancing hippocampal muscarinic signaling using M1 mAChR PAMs restored memory loss and slowed the progression of mouse prion disease, indicating that this ligand type may have clinical benefit in diseases showing defective cholinergic transmission, such as AD.

Optogenetic stimulation of brain cholinergic networks suppresses inflammation

Abstract Brain cholinergic muscarinic acetylcholine receptor (mAChR) signaling regulates peripheral inflammation via the inflammatory reflex (Nat Rev Endocrinol, 2012, 8:743). Here we provide further insight into this regulation by utilizing optogenetic stimulation, a method allowing precise spatiotemporal neuronal activation. Laser light (473 nm) and transgenic mice expressing light activated channelrhodopsin-2 coupled to a fluorescent protein (ChR2-YFP) under the control of the choline acetyl transferase (ChAT) promoter were used. Optogenetic stimulation of the medial septum, a major source of brain cholinergic output to areas with abundant M1 mAChR localization significantly suppressed serum TNF levels, as compared to sham stimulation (P<0.0398) in ChAT-ChR2-YFP mice during endotoxemia. In contrast to ChAT-ChR2-YFP mice, using the same approach in control (non-carrier) endotoxemic mice did not alter serum TNF. In addition, intracerebroventricular administration of the selective M1 mAChR agonist benzyl quinolone carboxylic acid (5 μg/kg) significantly decreased serum TNF, as compared to vehicle administration (P<0.0180) in C57Bl/6 mice during endotoxemia. These results from ongoing studies reveal a role for forebrain cholinergic M1 mAChR-mediated signaling in controlling peripheral inflammation. These findings may be of interest for development of new approaches for selective brain modulation for the treatment of inflammatory conditions.

Mechanistic Insights into Allosteric Structure-Function Relationships at the M1 Muscarinic Acetylcholine Receptor

Benzylquinolone carboxylic acid (BQCA) is the first highly selective positive allosteric modulator (PAM) for the M1 muscarinic acetylcholine receptor (mAChR), but it possesses low affinity for the allosteric site on the receptor. More recent drug discovery efforts identified 3-((1S,2S)-2-hydroxycyclohexyl)-6-((6-(1-methyl-1H-pyrazol-4-yl)pyridin-3-yl)methyl)benzo[h]quinazolin-4(3H)-one (referred to herein as benzoquinazolinone 12) as a more potent M1 mAChR PAM with a structural ancestry originating from BQCA and related compounds. In the current study, we optimized the synthesis of and fully characterized the pharmacology of benzoquinazolinone 12, finding that its improved potency derived from a 50-fold increase in allosteric site affinity as compared with BQCA, while retaining a similar level of positive cooperativity with acetylcholine. We then utilized site-directed mutagenesis and molecular modeling to validate the allosteric binding pocket we previously described for BQCA as a shared site for benzoquinazolinone 12 and provide a molecular basis for its improved activity at the M1 mAChR. This includes a key role for hydrophobic and polar interactions with residues Tyr-179, in the second extracellular loop (ECL2) and Trp-400(7.35) in transmembrane domain (TM) 7. Collectively, this study highlights how the properties of affinity and cooperativity can be differentially modified on a common structural scaffold and identifies molecular features that can be exploited to tailor the development of M1 mAChR-targeting PAMs.

Synthesis and pharmacological evaluation of analogues of benzyl quinolone carboxylic acid (BQCA) designed to bind irreversibly to an allosteric site of the M ₁ muscarinic acetylcholine receptor.

Activation of the M1 muscarinic acetylcholine receptor (mAChR) is a prospective treatment for alleviating cognitive decline experienced in central nervous system (CNS) disorders. Current therapeutics indiscriminately enhance the activity of the endogenous neurotransmitter ACh, leading to side effects. BQCA is a positive allosteric modulator and allosteric agonist at the M1 mAChR that has high subtype selectivity and is a promising template from which to generate higher affinity, more pharmacokinetically viable drug candidates. However, to efficiently guide rational drug design, the binding site of BQCA needs to be conclusively elucidated. We report the synthesis and pharmacological validation of BQCA analogues designed to bind irreversibly to the M1 mAChR. One analogue in particular, 11, can serve as a useful structural probe to confirm the location of the BQCA binding site; ideally, by co-crystallization with the M1 mAChR. Furthermore, this ligand may also be used as a pharmacological tool with a range of applications.

Allosteric Modulation of M1 Muscarinic Acetylcholine Receptor Internalization and Subcellular Trafficking

Allosteric modulators are an attractive approach to achieve receptor subtype-selective targeting of G protein-coupled receptors. Benzyl quinolone carboxylic acid (BQCA) is an unprecedented example of a highly selective positive allosteric modulator of the M1 muscarinic acetylcholine receptor (mAChR). However, despite favorable pharmacological characteristics of BQCA in vitro and in vivo, there is limited evidence of the impact of allosteric modulation on receptor regulatory mechanisms such as β-arrestin recruitment or receptor internalization and endocytic trafficking. In the present study we investigated the impact of BQCA on M1 mAChR regulation. We show that BQCA potentiates agonist-induced β-arrestin recruitment to M1 mAChRs. Using a bioluminescence resonance energy transfer approach to monitor intracellular trafficking of M1 mAChRs, we show that once internalized, M1 mAChRs traffic to early endosomes, recycling endosomes and late endosomes. We also show that BQCA potentiates agonist-induced subcellular trafficking. M1 mAChR internalization is both β-arrestin and G protein-dependent, with the third intracellular loop playing an important role in the dynamics of β-arrestin recruitment. As the global effect of receptor activation ultimately depends on the levels of receptor expression at the cell surface, these results illustrate the need to extend the characterization of novel allosteric modulators of G protein-coupled receptors to encapsulate the consequences of chronic exposure to this family of ligands.

Molecular Determinants of Allosteric Modulation at the M1 Muscarinic Acetylcholine Receptor

Benzylquinolone carboxylic acid (BQCA) is an unprecedented example of a selective positive allosteric modulator of acetylcholine at the M1 muscarinic acetylcholine receptor (mAChR). To probe the structural basis underlying its selectivity, we utilized site-directed mutagenesis, analytical modeling, and molecular dynamics to delineate regions of the M1 mAChR that govern modulator binding and transmission of cooperativity. We identified Tyr-85(2.64) in transmembrane domain 2 (TMII), Tyr-179 and Phe-182 in the second extracellular loop (ECL2), and Glu-397(7.32) and Trp-400(7.35) in TMVII as residues that contribute to the BQCA binding pocket at the M1 mAChR, as well as to the transmission of cooperativity with the orthosteric agonist carbachol. As such, the BQCA binding pocket partially overlaps with the previously described "common" allosteric site in the extracellular vestibule of the M1 mAChR, suggesting that its high subtype selectivity derives from either additional contacts outside this region or through a subtype-specific cooperativity mechanism. Mutation of amino acid residues that form the orthosteric binding pocket caused a loss of carbachol response that could be rescued by BQCA. Two of these residues (Leu-102(3.29) and Asp-105(3.32)) were also identified as indirect contributors to the binding affinity of the modulator. This new insight into the structural basis of binding and function of BQCA can guide the design of new allosteric ligands with tailored pharmacological properties.