Design, Synthesis, and Pharmacological Evaluation of Analogues Derived from the PLEV Tetrapeptide as Protein–Protein Interaction Modulators of Voltage-Gated Sodium Channel 1.6

Wang, Pingyuan; Wadsworth, Paul A.; Dvorak, Nolan M.; Singh, A.K.; Chen, Haiying; Liu, Zhiqing; Zhou, Richard; Holthauzen, Luis Marcelo F.; Zhou, Jia; Laezza, Fernanda

doi:10.1021/acs.jmedchem.0c00531

Cited by 8 publications

(17 citation statements)

References 57 publications

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“…To evaluate the hypothesis that PLEV and/or EYYV could modulate FGF14:Nav1.6 complex assembly, an in-cell assay was used with a HEK293 cell line (hereafter coded as "Clone V" cells) stably expressing both CLuc-FGF14 and CD4-Nav1.6-NLuc (Shavkunov et al, 2012;Shavkunov et al, 2013;Shavkunov et al, 2015;Ali et al, 2016Ali et al, , 2018Wadsworth et al, 2019Wadsworth et al, , 2020Dvorak et al, 2020Dvorak et al, , 2021Singh et al, 2020;Wang et al, 2020). In this assay, upon binding of FGF14 to the Nav1.6 C-terminal tail, there is reconstitution of the NLuc and CLuc halves of the luciferase enzyme, which produces luminescence in the presence of substrate D-luciferin (Figures 2A,D).…”

Section: In-cell Testing Of Plev and Eyyv Using The Split Luciferase Complementation Assaymentioning

confidence: 99%

Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

Singh

Dvorak

Tapia

et al. 2021

Front. Mol. Biosci.

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The voltage-gated Na+ (Nav) channel is a primary molecular determinant of the initiation and propagation of the action potential. Despite the central role of the pore-forming α subunit in conferring this functionality, protein:protein interactions (PPI) between the α subunit and auxiliary proteins are necessary for the full physiological activity of Nav channels. In the central nervous system (CNS), one such PPI occurs between the C-terminal domain of the Nav1.6 channel and fibroblast growth factor 14 (FGF14). Given the primacy of this PPI in regulating the excitability of neurons in clinically relevant brain regions, peptides targeting the FGF14:Nav1.6 PPI interface could be of pre-clinical value. In this work, we pharmacologically evaluated peptides derived from FGF14 that correspond to residues that are at FGF14’s PPI interface with the CTD of Nav1.6. These peptides, Pro-Leu-Glu-Val (PLEV) and Glu-Tyr-Tyr-Val (EYYV), which correspond to residues of the β12 sheet and β8-β9 loop of FGF14, respectively, were shown to inhibit FGF14:Nav1.6 complex assembly. In functional studies using whole-cell patch-clamp electrophysiology, PLEV and EYYV were shown to confer differential modulation of Nav1.6-mediated currents through mechanisms dependent upon the presence of FGF14. Crucially, these FGF14-dependent effects of PLEV and EYYV on Nav1.6-mediated currents were further shown to be dependent on the N-terminal domain of FGF14. Overall, these data suggest that the PLEV and EYYV peptides represent scaffolds to interrogate the Nav1.6 channel macromolecular complex in an effort to develop targeted pharmacological modulators.

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Section: In-cell Testing Of Plev and Eyyv Using The Split Luciferase Complementation Assaymentioning

confidence: 99%

Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

Singh

Dvorak

Tapia

et al. 2021

Front. Mol. Biosci.

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“…HEK293 cells were cultured as previously described [26,[29][30][31], with different concentrations of G418 (Invitrogen, Carlsbad, CA, USA) added to the media to ensure stable Na v 1.2 and Na v 1.6 expression.…”

Section: Cell Culturementioning

confidence: 99%

“…Clampex 9.2 software (Molecular Devices) was used to set experimental parameters, and electrophysiological equipment was interfaced to this software using a Digidata 1200 analog-digital interface (Molecular Devices). Acquired data was then analyzed as previously described [27,28,30,31,33].…”

Section: Whole-cell Voltage-clamp Recordings In Heterologous Cellsmentioning

confidence: 99%

“…Having shown that pharmacological inhibition of Wee1 kinase confers modulatory effects on FGF14:Na v 1.2 complex assembly (Figure 1A), we next sought to investigate if these alterations in complex assembly resulted in changes in the function of the Na v 1.2 channel. To do so, HEK293 cells stably expressing the Na v 1.2 channel (HEK-Na v 1.2) [26,29,30] were transiently transfected with either GFP (HEK-Na v 1.2-GFP) or FGF14-GFP (HEK-Na v 1.2-FGF14-GFP). Cells were then incubated for 30 min with either vehicle (0.1% DMSO) or 15 µM of Wee1 inhibitor II, a concentration selected on the basis of it being close to the IC 50 value of the compound as determined in the LCA (Figure 1A).…”

Section: Pharmacological Inhibition Of Wee1 Kinase Modulates Fgf14-mediated Regulation Of Na V 12 Channelsmentioning

confidence: 99%

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Pharmacological Inhibition of Wee1 Kinase Selectively Modulates the Voltage-Gated Na+ Channel 1.2 Macromolecular Complex

Dvorak

Tapia

Baumgartner

et al. 2021

Cells

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Voltage-gated Na+ (Nav) channels are a primary molecular determinant of the action potential (AP). Despite the canonical role of the pore-forming α subunit in conferring this function, protein–protein interactions (PPI) between the Nav channel α subunit and its auxiliary proteins are necessary to reconstitute the full physiological activity of the channel and to fine-tune neuronal excitability. In the brain, the Nav channel isoforms 1.2 (Nav1.2) and 1.6 (Nav1.6) are enriched, and their activities are differentially regulated by the Nav channel auxiliary protein fibroblast growth factor 14 (FGF14). Despite the known regulation of neuronal Nav channel activity by FGF14, less is known about cellular signaling molecules that might modulate these regulatory effects of FGF14. To that end, and building upon our previous investigations suggesting that neuronal Nav channel activity is regulated by a kinase network involving GSK3, AKT, and Wee1, we interrogate in our current investigation how pharmacological inhibition of Wee1 kinase, a serine/threonine and tyrosine kinase that is a crucial component of the G2-M cell cycle checkpoint, affects the Nav1.2 and Nav1.6 channel macromolecular complexes. Our results show that the highly selective inhibitor of Wee1 kinase, called Wee1 inhibitor II, modulates FGF14:Nav1.2 complex assembly, but does not significantly affect FGF14:Nav1.6 complex assembly. These results are functionally recapitulated, as Wee1 inhibitor II entirely alters FGF14-mediated regulation of the Nav1.2 channel, but displays no effects on the Nav1.6 channel. At the molecular level, these effects of Wee1 inhibitor II on FGF14:Nav1.2 complex assembly and FGF14-mediated regulation of Nav1.2-mediated Na+ currents are shown to be dependent upon the presence of Y158 of FGF14, a residue known to be a prominent site for phosphorylation-mediated regulation of the protein. Overall, our data suggest that pharmacological inhibition of Wee1 confers selective modulatory effects on Nav1.2 channel activity, which has important implications for unraveling cellular signaling pathways that fine-tune neuronal excitability.

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“…Similar assays have facilitated the discovery of biased ligands for the nociception opioid peptide (NOP) [62] and serotonin HT1A [63] receptors. Although ion channel/transporter interactions with function-modulating proteins are not as well characterized compared with GPCRs, there is great potential to apply similar assays to those systems, as in a recent example where a peptide disrupting the Na v 1.6/fibroblast growth factor interaction was discovered [64]. In addition, intramolecular fluorescence/bioluminescence resonance energy transfer (FRET/BRET) sensors have been developed to monitor MP conformational changes in response to drug binding/activation [65,66].…”

Section: Hts Approachesmentioning

confidence: 99%

Membrane protein production and formulation for drug discovery

Gulezian

Crivello

Bednenko

et al. 2021

Trends in Pharmacological Sciences

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Design, Synthesis, and Pharmacological Evaluation of Analogues Derived from the PLEV Tetrapeptide as Protein–Protein Interaction Modulators of Voltage-Gated Sodium Channel 1.6

Cited by 8 publications

References 57 publications

Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

Differential Modulation of the Voltage-Gated Na+ Channel 1.6 by Peptides Derived From Fibroblast Growth Factor 14

Pharmacological Inhibition of Wee1 Kinase Selectively Modulates the Voltage-Gated Na+ Channel 1.2 Macromolecular Complex

Membrane protein production and formulation for drug discovery

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