Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT3 receptors

Olander, Emma Rie; Madjroh, Nawid; Bunch, Lennart; Söderhielm, Pella Cecilia; Jensen, Anders A.

doi:10.1016/j.bcp.2016.04.004

Cited by 15 publications

(9 citation statements)

References 67 publications

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“…TMPPAA has been proposed to bind to an allosteric site in the TMD 17 , but no clearly interpretable density is found in our data to model the compound. We tested the TMPPAA agonist activity on a set of ~45 single-point mutants of the human receptor, which collectively reveal that the drug binds between M4, M1 and M3, into an intra-subunit cavity skirted by lipids of the outer leaflet in the upper part of the TMD (Extended Data Fig.…”

contrasting

confidence: 63%

“… c. Functional properties exhibited by serotonin and TMPPAA as agonists at 5-HT 3 receptors expressed in tsA201 cell in a fluorescence-based membrane potential (FMP) assay. The human 5-HT 3 A receptor was used for these experiments, since TMPPAA evokes a more robust agonist response through this receptor than through mouse 5-HT 3 A in this assay 17 . The color code is similar to that of panel b. n.d., not determinable, w.a., weak activity.…”

Section: Extended Datamentioning

confidence: 99%

“…We finally collected a dataset in the presence of 30 μM serotonin and 100 μM trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA, a compound showing agonist and positive allosteric modulator properties on the human receptor 17 ), a combination that yields weakly desensitizing currents (Extended Data Fig. 1f).…”

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confidence: 99%

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Conformational transitions of the serotonin 5-HT3 receptor

Polovinkin

Hassaı̈ne

Perot

et al. 2018

Nature

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142

185

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The serotonin 5-HT3 receptor is a pentameric ligand-gated ion channel (pLGIC). It belongs to a large family of receptors that function as allosteric signal transducers across the plasma membrane1,2: upon binding of neurotransmitter molecules to extracellular sites, the receptors undergo complex conformational transitions, which result in transient opening of a pore permeable to ions. 5-HT3 receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression3. Despite the recent accumulation in pLGIC structures4–8, no clear unifying view has emerged on conformational transitions involved in channel gating. Here we report four cryo-EM structures of the full-length mouse 5-HT3 receptor, ranging from 3.2 Å to 4.5 Å resolution, obtained in complex with the anti-emetic drug tropisetron, with serotonin, with serotonin and a positive allosteric modulator. The tropisetron-bound structure resembles those obtained with an inhibitory nanobody5 or without ligand9. The other structures represent new conformations, including that of an open state and of two novel ligand-bound states. We present computational insights into the dynamics of the structures, their pore hydration and free-energy profiles; we characterize motions at the gate level and cation accessibility in the pore. Put together, the data deepen our understanding of the gating mechanism of pLGICs, while capturing ligand binding in unprecedented detail.

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contrasting

confidence: 63%

Section: Extended Datamentioning

confidence: 99%

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Conformational transitions of the serotonin 5-HT3 receptor

Polovinkin

Hassaı̈ne

Perot

et al. 2018

Nature

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142

185

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“…Thymol and carvacrol are structurally closely related positive allosteric modulators and channel activators of the h5-HT 3A receptor. Their binding site is proposed to be located in the transmembrane domain at the subunit interface (Lansdell et al, 2015), which also has been suggested as the binding site for the positive allosteric modulator trans-3-(4methoxyphenyl)-N-(pentan-3-yl)acrylamide (Gasiorek et al, 2016;Polovinkin et al, 2018), which has not been explored in the present study. The M223C mutant showed slight, but significant increases in fluorescence in response to both compounds (Fig.…”

Section: Discussionmentioning

confidence: 91%

Conformational Changes in the 5-HT_3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry

Munro

Ladefoged²,

Padmanathan

et al. 2019

Mol Pharmacol

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The 5-hydroxytryptamine (5-HT) type 3 receptor is a member of the cysteine (Cys)-loop receptor super family of ligand-gated ion channels in the nervous system and is a clinical target in a range of diseases. The 5-HT 3 receptor mediates fast serotonergic neurotransmission by undergoing a series of conformational changes initiated by ligand binding that lead to the rapid opening of an intrinsic cation-selective channel. However, despite the availability of high-resolution structures of a mouse 5-HT 3 receptor, many important aspects of the mechanistic basis of 5-HT 3 receptor function and modulation by drugs remain poorly understood. In particular, there is little direct evidence for the specific conformational changes predicted to occur during ligand-gated channel activation and desensitization. In the present study, we used voltage-clamp fluorometry (VCF) to measure conformational changes in regions surrounding the orthosteric binding site of the human 5-HT 3A (h5-HT 3A) receptor during binding of 5-HT and different classes of 5-HT 3 receptor ligands. VCF utilizes parallel measurements of receptor currents with photon emission from fluorescent reporter groups covalently attached to specific positions in the receptor structure. Reporter groups that are highly sensitive to the local molecular environment can, in real time, report conformational changes as changes in fluorescence that can be correlated with changes in receptor currents reporting the functional states of the channel. Within the loop C, D, and E regions that surround the orthosteric binding site in the h5-HT 3A receptor, we identify positions that are amenable to tagging with an environmentally sensitive reporter group that reports robust fluorescence changes upon 5-HT binding and receptor activation. We use these reporter positions to characterize the effect of ligand binding on the local structure of the orthosteric binding site by agonists, competitive antagonists, and allosterically acting channel activators. We observed that loop C appears to show distinct fluorescence changes for ligands of the same class, while loop D reports similar fluorescence changes for all ligands binding at the orthosteric site. In contrast, the loop E reporter position shows distinct changes for agonists, antagonists, and allosteric compounds, suggesting the conformational changes in this region are specific to ligand function. Interpretation of these results within the framework of current models of 5-HT 3 and Cys-loop mechanisms are used to expand the understanding of how ligand binding in Cys-loop receptors relates to channel gating. SIGNIFICANCE STATEMENT The 5-HT 3 receptor is an important ligand-gated ion channel and drug target in the central and peripheral nervous system. Determining how ligand binding induced conformational changes in the receptor is central for understanding the structural mechanisms underlying 5-HT 3 receptor function. Here, we employ voltage-gated fluorometry to characterize conformational changes in the extracellular domain of the human 5-...

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“…The negative allosteric modulations of bupropion and hydroxybupropion on 5-HT 3A Rs provide a novel pharmacological basis for their antidepressant activity. On the other hand, TMPPAA is a 5-HT 3A R PAM that enhanced serotonin-evoked currents and also directly activated the receptors in the absence of serotonin . Chimera and mutational analyses suggested that the ago-PAM property of TMPPAA was ascribed to an allosteric binding site in the TMD of 5-HT 3A Rs.…”

Section: Allosteric Modulations Of Ligand-gated Ion Channels (Lgics)mentioning

confidence: 99%

Drug Development in Channelopathies: Allosteric Modulation of Ligand-Gated and Voltage-Gated Ion Channels

Zhang

Wang

2020

J. Med. Chem.

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Ion channels have been characterized as promising drug targets for treatment of numerous human diseases. Functions of ion channels can be fine-tuned by allosteric modulators, which interact with channels and modulate their activities by binding to sites spatially discrete from those of orthosteric ligands. Positive and negative allosteric modulators have presented a plethora of potential therapeutic advantages over traditionally orthosteric agonists and antagonists in terms of selectivity and safety. This thematic review highlights the discovery of representative allosteric modulators for ligand-gated and voltage-gated ion channels, discussing in particular their identifications, locations, and therapeutic uses in the treatment of a range of channelopathies. Additionally, structures and functions of selected ion channels are briefly described to aid in the rational design of channel modulators. Overall, allosteric modulation represents an innovative targeting approach, and the corresponding modulators provide an abundant but challenging landscape for novel therapeutics targeting ligand-gated and voltage-gated ion channels.

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Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT3 receptors

Cited by 15 publications

References 67 publications

Conformational transitions of the serotonin 5-HT3 receptor

Conformational transitions of the serotonin 5-HT3 receptor

Conformational Changes in the 5-HT_3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry

Drug Development in Channelopathies: Allosteric Modulation of Ligand-Gated and Voltage-Gated Ion Channels

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Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT3 receptors

Cited by 15 publications

References 67 publications

Conformational transitions of the serotonin 5-HT3 receptor

Conformational transitions of the serotonin 5-HT3 receptor

Conformational Changes in the 5-HT3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry

Drug Development in Channelopathies: Allosteric Modulation of Ligand-Gated and Voltage-Gated Ion Channels

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Conformational Changes in the 5-HT_3A Receptor Extracellular Domain Measured by Voltage-Clamp Fluorometry