Carbachol dimers as homobivalent modulators of muscarinic receptors

Matucci, Rosanna; Nesi, Mascia; Martino, Maria Vittoria; Bellucci, Cristina; Manetti, Dina; Ciuti, Elisa; Mazzolari, Angelica; Dei, Silvia; Guandalini, Luca; Teodori, Elisabetta; Vistoli, Giulio; Romanelli, Maria Novella

doi:10.1016/j.bcp.2016.03.012

Cited by 8 publications

(23 citation statements)

References 32 publications

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“…Cell membrane preparations (0.5–0.7 mg/ml) were used for saturation binding assay with [ 3 H]NMS (0.05–1.6 nM; Perkin-Elmer Life and Analytical Science), or competition binding assay (0.2 nM) with the muscarinic antagonist metoctramine (0.1 nM–0.1 mM; Sigma-Aldrich) as previously described (Matucci et al, 2016 ). Nonspecific binding was defined using 10 μM Atropine (Sigma-Aldrich).…”

Section: Methodsmentioning

confidence: 99%

Young Human Cholinergic Neurons Respond to Physiological Regulators and Improve Cognitive Symptoms in an Animal Model of Alzheimer’s Disease

Morelli

Sarchielli

Guarnieri

et al. 2017

Front. Cell. Neurosci.

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The degeneration of cholinergic neurons of the nucleus basalis of Meynert (NBM) in the basal forebrain (BF) is associated to the cognitive decline of Alzheimer’s disease (AD) patients. To date no resolutive therapies exist. Cell-based replacement therapy is a strategy currently under consideration, although the mechanisms underlying the generation of stem cell-derived NBM cholinergic neurons able of functional integration remain to be clarified. Since fetal brain is an optimal source of neuronal cells committed towards a specific phenotype, this study is aimed at isolating cholinergic neurons from the human fetal NBM (hfNBMs) in order to study their phenotypic, maturational and functional properties. Extensive characterization confirmed the cholinergic identity of hfNBMs, including positivity for specific markers (such as choline acetyltransferase) and acetylcholine (Ach) release. Electrophysiological measurements provided the functional validation of hfNBM cells, which exhibited the activation of peculiar sodium (INa) and potassium (IK) currents, as well as the presence of functional cholinergic receptors. Accordingly, hfNBMs express both nicotinic and muscarinic receptors, which were activated by Ach. The hfNBMs cholinergic phenotype was regulated by the nerve growth factor (NGF), through the activation of the high-affinity NGF receptor TrkA, as well as by 17-β-estradiol through a peculiar recruitment of its own receptors. When intravenously administered in NBM-lesioned rats, hfNBMs determined a significant improvement in memory functions. Histological examination of brain sections showed that hfNBMs (labeled with PKH26 fluorescent dye prior to administration) reached the damaged brain areas. The study provides a useful model to study the ontogenetic mechanisms regulating the development and maintenance of the human brain cholinergic system and to assess new lines of research, including disease modeling, drug discovery and cell-based therapy for AD.

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Section: Methodsmentioning

confidence: 99%

Young Human Cholinergic Neurons Respond to Physiological Regulators and Improve Cognitive Symptoms in an Animal Model of Alzheimer’s Disease

Morelli

Sarchielli

Guarnieri

et al. 2017

Front. Cell. Neurosci.

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“…Data from one-point kinetic experiments were analyzed in order to obtain estimates of the affinity of an allosteric ligand for the [ 3 H]-NMS occupied receptor (log K occ ) in a single step, using an equation introduced in GraphPad Prism 5.02 [33,34].…”

Section: Discussionmentioning

confidence: 99%

“…Dissociation binding assays were conducted in assay buffer at room temperature as above described [33]. Membranes were preincubated with 2 nM [ 3 H]-NMS for 60 min at room temperature (mix membrane).…”

Section: Full Time Coursementioning

confidence: 99%

“…Off-rate assays [34] were performed as described elsewhere [33] to estimate the affinity of the ligands for the [ 3 H]-NMS-occupied receptor (log K occ ). Briefly, a high concentration of membranes (about 0.1 mg/mL) was incubated with a high concentration of [ 3 H]-NMS (2 nM) for 60 min at room temperature (mix membrane).…”

Section: One Point Kinetic Assaysmentioning

confidence: 99%

“…Later (80 min for hM 1 , 20 min for hM 2 and 70 min for hM 5 ), the samples were filtered and counted as previously reported. Non specific binding was measured as previously reported [33].…”

Section: One Point Kinetic Assaysmentioning

confidence: 99%

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Repositioning Dequalinium as Potent Muscarinic Allosteric Ligand by Combining Virtual Screening Campaigns and Experimental Binding Assays

Mazzolari

Gervasoni

Pedretti

et al. 2020

IJMS

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Structure-based virtual screening is a truly productive repurposing approach provided that reliable target structures are available. Recent progresses in the structural resolution of the G-Protein Coupled Receptors (GPCRs) render these targets amenable for structure-based repurposing studies. Hence, the present study describes structure-based virtual screening campaigns with a view to repurposing known drugs as potential allosteric (and/or orthosteric) ligands for the hM2 muscarinic subtype which was indeed resolved in complex with an allosteric modulator thus allowing a precise identification of this binding cavity. First, a docking protocol was developed and optimized based on binding space concept and enrichment factor optimization algorithm (EFO) consensus approach by using a purposely collected database including known allosteric modulators. The so-developed consensus models were then utilized to virtually screen the DrugBank database. Based on the computational results, six promising molecules were selected and experimentally tested and four of them revealed interesting affinity data; in particular, dequalinium showed a very impressive allosteric modulation for hM2. Based on these results, a second campaign was focused on bis-cationic derivatives and allowed the identification of other two relevant hM2 ligands. Overall, the study enhances the understanding of the factors governing the hM2 allosteric modulation emphasizing the key role of ligand flexibility as well as of arrangement and delocalization of the positively charged moieties.

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Dibenzodiazepinone-type muscarinic receptor antagonists conjugated to basic peptides: Impact of the linker moiety and unnatural amino acids on M2R selectivity

Weinhart

Wifling

Schmidt

et al. 2021

European Journal of Medicinal Chemistry

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Carbachol dimers as homobivalent modulators of muscarinic receptors

Cited by 8 publications

References 32 publications

Young Human Cholinergic Neurons Respond to Physiological Regulators and Improve Cognitive Symptoms in an Animal Model of Alzheimer’s Disease

Young Human Cholinergic Neurons Respond to Physiological Regulators and Improve Cognitive Symptoms in an Animal Model of Alzheimer’s Disease

Repositioning Dequalinium as Potent Muscarinic Allosteric Ligand by Combining Virtual Screening Campaigns and Experimental Binding Assays

Dibenzodiazepinone-type muscarinic receptor antagonists conjugated to basic peptides: Impact of the linker moiety and unnatural amino acids on M2R selectivity

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