Characterization of AN317, a novel selective agonist of α6β2-containing nicotinic acetylcholine receptors

Sandager‐Nielsen, Karin; Ahring, Philip K.; Klein, Jessica; Hout, Marloes van; Thaneshwaran, Siganya; Santos, Altair Brito dos; Jacobsen, Thomas A.; Amrutkar, Dipak Vasantrao; Peters, Dan; Jensen, Anders A.; Kohlmeier, Kristi A.; Christophersen, Palle; Dyhring, Tino

doi:10.1016/j.bcp.2019.113786

Cited by 5 publications

(5 citation statements)

References 83 publications

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“…To address this question further, the properties of analogues 4c and 8j were studied in a radioligand competition binding assay using α4β2-HEK293 cell membranes and the orthosteric nAChR radioligand [ 3 H]epibatidine. In agreement with previous findings, 37 nicotine mediated concentration-dependent inhibition of [ 3 H]epibatidine binding to α4β2, exhibiting a K i value of 9.1 nM (pK i ± S.E.M. : 8.04 ± 0.07, n = 3).…”

Section: Sar Analysis Of Compound 1 Analoguessupporting

confidence: 92%

“…The [ 3 H]epibatidine competition binding assay was performed essentially as previously described. 37 α4β2-HEK293 cells were collected at ∼90% confluency by scraping into ice-cold assay buffer (140 mM NaCl, 1.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 25 mM HEPES, pH 7.4) and homogenized for 10 s using an Ultra-Turrax homogenizer (IKA Labortechnik, Staufen, Germany) and centrifuged 30 min at 47,810 × g at 4 °C. The resulting membrane pellets were resuspended in ice-cold assay buffer, homogenized, and centrifuged for another 30 min under the same conditions, after which the cell membranes were stored at −80 °C until use.…”

Section: ■ Discussion and Conclusionmentioning

confidence: 99%

“…The [ 3 H]epibatidine competition binding assay was performed essentially as previously described . α4β2-HEK293 cells were collected at ∼90% confluency by scraping into ice-cold assay buffer (140 mM NaCl, 1.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 25 mM HEPES, pH 7.4) and homogenized for 10 s using an Ultra-Turrax homogenizer (IKA Labortechnik, Staufen, Germany) and centrifuged 30 min at 47,810 × g at 4 °C.…”

Section: Methodsmentioning

confidence: 99%

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Discovery of a Novel Class of Benzimidazole-Based Nicotinic Acetylcholine Receptor Modulators: Positive and Negative Modulation Arising from Overlapping Allosteric Sites

Zhou,

Dau,

Jensen

2023

J. Med. Chem.

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Here, we present the discovery of a novel class of benzimidazole-based allosteric modulators of nicotinic acetylcholine receptors (nAChRs). The modulators were developed based on a compound (1) exhibiting positive modulatory activity at α4β2 nAChR in a compound library screening by functional characterization of 100 analogues of 1 at nAChRs. Two distinct series of positive and negative allosteric modulators (PAMs and NAMs, respectively) comprising benzimidazole as a shared structural moiety emerged from this SAR study. The PAMs mediated weak modulation of α4β2 and α6β2β3, whereas the NAMs exhibited essentially equipotent inhibition of α4β2, α6β2β3, α6β4β3, and α3β4 nAChRs, with analogue 9j [2-(2,4-dichlorophenoxy)-1,3-dimethyl-1-H-benzo[d]imidazole-3-ium] displaying high-nanomolar and low-micromolar IC 50 values at the β2and β4-containing receptor subtypes, respectively. We propose that the PAMs and NAMs act through overlapping sites in the nAChR, and these findings thus underline the heterogenous modes of modulation that can arise from a shared allosteric site in the receptor.

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Section: Sar Analysis Of Compound 1 Analoguessupporting

confidence: 92%

Section: ■ Discussion and Conclusionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Discovery of a Novel Class of Benzimidazole-Based Nicotinic Acetylcholine Receptor Modulators: Positive and Negative Modulation Arising from Overlapping Allosteric Sites

Zhou,

Dau,

Jensen

2023

J. Med. Chem.

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“…The α6/α3β4 nAChR function has not been examined and is, to some extent, affected by species differences in the receptor. The human-rat species differences in α6/α3β4 nAChR provide a pharmacological basis for drug development targeting this receptor, which may lead to successful development [23,34].…”

Section: Discussionmentioning

confidence: 99%

Molecular Determinants of Species Specificity of α-Conotoxin TxIB towards Rat and Human α6/α3β4 Nicotinic Acetylcholine Receptors

Xie

Yuan

Zhao

et al. 2023

IJMS

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Conotoxins are widely distributed and important for studying ligand-gated ion channels. TxIB, a conotoxin consisting of 16 amino acids derived from Conus textile, is a unique selective ligand that blocks rat α6/α3β2β3 nAChR (IC50 = 28 nM) without affecting other rat subtypes. However, when the activity of TxIB against human nAChRs was examined, it was unexpectedly found that TxIB had a significant blocking effect on not only human α6/α3β2β3 nAChR but also human α6/α3β4 nAChR, with an IC50 of 537 nM. To investigate the molecular mechanism of this species specificity and to establish a theoretical basis for drug development studies of TxIB and its analogs, different amino acid residues between human and rat α6/α3 and β4 nAChR subunits were identified. Each residue of the human species was then substituted with the corresponding residue of the rat species via PCR-directed mutagenesis. The potencies of TxIB towards the native α6/α3β4 nAChRs and their mutants were evaluated through electrophysiological experiments. The results showed that the IC50 of TxIB against h[α6V32L, K61R/α3]β4L107V, V115I was 22.5 μM, a 42-fold decrease in potency compared to the native hα6/α3β4 nAChR. Val-32 and Lys-61 in the human α6/α3 subunit and Leu-107 and Val-115 in the human β4 subunit, together, were found to determine the species differences in the α6/α3β4 nAChR. These results also demonstrate that the effects of species differences between humans and rats should be fully considered when evaluating the efficacy of drug candidates targeting nAChRs in rodent models.

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“…The compounds were tested for binding at the human α4β2 and the rat α3β4 nAChRs in a competition [ 3 H]epibatidine binding assay performed essentially as previously described. 44 Membranes were prepared from α4β2or α3β4-expressing HEK293 cell lines 45,46 by harvesting at 90% confluency and homogenization for 10 s using an Ultra-Turrax homogenizer (IKA-Labortechnik, Staufen, Germany) in 30 mL of icecold assay buffer (140 mM NaCl, 1.5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 25 mM HEPES, pH 7.4). After centrifugation at 50,000g at 4 °C for 30 min, the supernatant was discarded.…”

Section: 2]octane-22′-chroman]-4′-one (23b)mentioning

confidence: 99%

Identification of 5-HT2 Serotonin Receptor Modulators through the Synthesis of a Diverse, Tropane- and Quinuclidine-alkaloid-Inspired Compound Library

Yao,

Jensen,

Bryce-Rogers

et al. 2023

J. Med. Chem.

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The recombination of natural product (NP) fragments in unprecedented ways has emerged as an important strategy for bioactive compound discovery. In this context, we propose that privileged primary fragments predicted to be enriched in activity against a specific target class can be coupled to diverse secondary fragments to engineer selectivity among closely related targets. Here, we report the synthesis of an alkaloid-inspired compound library enriched in spirocyclic ring fusions, comprising 58 compounds from 12 tropane-or quinuclidine-containing scaffolds, all of which can be considered pseudo-NPs. The library displays excellent predicted drug-like properties including high Fsp 3 content and Lipinski's rule-of-five compliance. Targeted screening against selected members of the serotonin and dopamine G protein-coupled receptor family led to the identification of several hits that displayed significant agonist or antagonist activity against 5-HT 2A and/or 5-HT 2C , and subsequent optimization of one of these delivered a lead dual 5-HT 2B/C antagonist with a highly promising selectivity profile.

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Characterization of AN317, a novel selective agonist of α6β2-containing nicotinic acetylcholine receptors

Cited by 5 publications

References 83 publications

Discovery of a Novel Class of Benzimidazole-Based Nicotinic Acetylcholine Receptor Modulators: Positive and Negative Modulation Arising from Overlapping Allosteric Sites

Discovery of a Novel Class of Benzimidazole-Based Nicotinic Acetylcholine Receptor Modulators: Positive and Negative Modulation Arising from Overlapping Allosteric Sites

Molecular Determinants of Species Specificity of α-Conotoxin TxIB towards Rat and Human α6/α3β4 Nicotinic Acetylcholine Receptors

Identification of 5-HT2 Serotonin Receptor Modulators through the Synthesis of a Diverse, Tropane- and Quinuclidine-alkaloid-Inspired Compound Library

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