IndraLab
Statements
sparser
"Therefore, in summary, our study presents three key findings to support this novel mechanism: i ) inhibition of JAK2 limits FGF14:Nav1.6 complex assembly, potentiates FGF14 homodimerization, and abolishes FGF14-dependent modulation of Nav1.6 currents, contingent upon the presence of Y158; ii ) JAK2 phosphorylates FGF14 at Y158; and iii ) in native brain slices, inhibition of JAK2 reduces neuronal excitability, contingent upon the presence of FGF14."
sparser
"Primary hits that fulfilled the following two criteria were promoted for further studies: (1) inhibition or stimulation of the FGF14:Nav1.6 complex by at least 40% (equivalent to 60% or 140% luminescence when normalized to DMSO controls, respectively) and Z-score ≥ 3 for enhancers or ≤ −5 for inhibitors, and (2) the primary kinase target was targeted by two or more compounds meeting the prior hit selection criteria (i.e., at least two inhibitors of JAK2 observed to modulate FGF14:Nav1.6 complex assembly by ≥ 40%)."
sparser
"Following inhibition of JAK2, but not Src, FGF14 homodimerization increased in a manner directly inverse to FGF14:Nav1.6 complex formation with a comparable degree of both efficacy (minimum vs. maximum percent luminescence for FGF14:Nav1.6 compared to FGF14:FGF14 dimerization, respectively), as well as potency (inhibitor IC 50 against the FGF14:Nav1.6 complex vs. EC 50 against the FGF14:FGF14 dimer) ( xref and xref )."
sparser
"From the Selleck library, two STAT3 inhibitors (S3I-201 and Ursolic Acid) had minimal impact (115.3% and 103.5% luminescence, respectively), while the inhibitor Stattic resulted in almost complete inhibition of the FGF14:Nav1.6 complex (6.0% luminescence), but was excluded due to cell toxicity (57.2% fluorescence from the CTB assay)."
sparser
"Briefly, altering the N -terminal substituent to Cbz and adding a Boc protective group to the constituent lysine residue of the protected FLPK scaffold produced ZL181 ( 4 , xref A), which inhibited FGF14:Nav1.6 complex assembly (half maximal inhibitory concentration (IC 50 ) = 63 µM) and decreased maximal and instantaneous firing frequencies in MSNs of the NAc [ xref ]."
sparser
"Select kinases targeted by ≥4 hits from the HTS against the FGF14:Nav1.6 complex were counter-screened against the FGF14:FGF14 homodimer, using ≥2 selected compounds per target based on hypothesis-driven target analysis, as well as inhibitor selectivity, potency, and availability."
sparser
"For the FGF14:Nav1.6 assay, it would exist as a dimer of CLuc-FGF14:CLuc-FGF14, yielding no luminescence in the presence of luciferin; increases in this dimer would reduce the FGF14 available for binding to CD4-Nav1.6-NLuc, thereby reducing reconstituted luciferase enzyme capable of generating luminescence."
sparser
"The docking results demonstrated that FLPK can be well docked into monomeric FGF14 (Figure xref ) within the binding pocket formed by the β9 and β12 strands, specifically by interactions with hotspot residues that are similarly found at the interface of the FGF14:Nav1.6 complex (Ali et al., xref , xref )."
sparser
"Overall, these SAR studies using the LCA identified three potential positive allosteric modulators (PAMs; 17 , 19 , and 22 ) and one negative allosteric modulator (NAM; 12 ) of FGF14:Nav1.6 complex assembly, the four of which were selected for protein:ligand binding studies using SPR."
sparser
"While JAK2 inhibitors resulted in increased stability of the FGF14:FGF14 dimer and inhibition of the FGF14:Nav1.6 complex, Src kinase inhibitors were largely only effective on the FGF14:Nav1.6 complex, resulting in only moderate inhibition of the FGF14 dimer, and only at high concentrations."
sparser
"Next, we compared the double stable cell line, hereafter referred to as Clone V, to transiently transfected HEK293 cells after treatment with the peptidomimetic ZL181 (negative control 1), a rationally designed inhibitor of the FGF14:Nav1.6 interaction xref , and the Akt inhibitor triciribine (positive control 1), which enhances the interaction of this complex presumably by increasing GSK3-dependent phosphorylation of the complex xref , xref ."
sparser
"Parallel studies lead us to explore the effect of TNF-α (positive control 2) on the FGF14:Nav1.6 complex, and we found that it is a more efficacious enhancer of the complex (mean and SD: TNF-α, 210.8% ± 18.6%; triciribine, 142.5% ± 10.7%; triciribine, 142.5% ± 10.7%; Fig. xref ); despite moderately higher variance, the mean effect of TNF-α is over 2-fold greater than that of triciribine."
sparser
"Additionally, the three predicted phosphorylation sites in FGF14 (Y158, Y162, and Y211; xref ), as well as the moderate inhibitory effect of Src inhibitors on FGF14:FGF14 dimerization (mean from all four Src inhibitors: FGF14:Nav1.6, 32.6%; FGF14:FGF14, 66.7% luminescence) was indicative of regulation by Src on both complexes."
sparser
"Crucially, this analog displayed markedly improved potency, inhibiting FGF14:Nav1.6 complex assembly with an IC 50 value of 11 µM. Additionally, mechanism of action (MOA) studies using whole-cell patch-clamp electrophysiology revealed that ZL0177 significantly reduced Nav1.6-mediated peak current density and caused a depolarizing shift in voltage-dependence of Nav1.6 channel activation, suggesting that the peptidomimetic’s activity is conferred by functioning as a partial FGF14 mimic [ xref ]."
sparser
"One of these peptides, Phe-Leu-Pro-Lys (FLPK; 1 , xref A), was derived from a four amino acid sequence located on the β12 sheet of FGF14 at the FGF14:Nav1.6 PPI interface, whereas the other, Glu-Tyr-Tyr-Val (EYYV; 5 , xref B), was derived from an amino acid sequence located on the exposed β8–β9 loop of FGF14 [ xref ]."
sparser
"Overall, these results demonstrate that: (1) our assay is capable of reliably detecting both inhibitors and enhancers of the FGF14:Nav1.6 complex from a background signal with low variability; (2) initial effects of identified hits can be reproduced in subsequent studies; and (3) that the screening system is capable of follow-up dose-dependency studies using an identical 384-well plate format with additional replicates for each concentration."
sparser
"Phosphomotif scans revealed Y158 as a potential JAK2 phosphorylation site ( xref ), and homology modeling here as well as in previous studies[ xref , xref ] has demonstrated that this site is at the PPI interface of both the FGF14:FGF14 dimer and the FGF14:Nav1.6 complex ( xref and xref )."
sparser
"e, only 1–2 compounds/target); ii ) kinase target representation was neither complete nor fully distributed (i.e., preference toward Ser/Thr over Tyr kinase inhibitors; not all known kinases were represented, such as multiple tyrosine kinases); and iii ) lack of initial rapid counter-screening studies to explore potential mechanisms, such as comparison of target effects on the FGF14:FGF14 vs FGF14:Nav1.6 complexes."
sparser
"Treatment with 50 µM ZL181 caused a similar inhibitory effect (23.9 vs. 25% luminescence compared to DMSO control), and treatment with 25 µM triciribine resulted in a similar increase in FGF14:Nav1.6 C-tail assembly (144.0% vs. 166.9% luminescence) in transiently transfected cells compared to Clone V cells (Fig. xref )."
sparser
"One possible reason for the less clear patterns observed may be due to fewer available inhibitors specific for STAT3, as well as that changes in STAT3 regulation of the FGF14:Nav1.6 complex may be a less potent form of regulation (i.e. indirect) than that of phosphorylation by JAK2."
sparser
"We found that the FGF14:Nav1.6 C-tail interaction was indirectly inhibited (i.e., the relevant kinase inhibitor acts as antagonist) through targeting S/T kinases including rapidly accelerated fibrosarcoma (c-RAF), protein kinases A, C, and G (PKA, PKC, PKG), rho-associated coiled-coil-containing protein kinase 1 (ROCK1), pyruvate dehydrogenase kinase 1 (PDK1), phosphoinositide 3-kinases (PI3K), polo-like kinase 1 (PLK1), and mitogen-activated protein kinase kinase (MEK1, aka MAP2K1)."
sparser
"While it is potentially somewhat surprising that 12 , 17 , and 19 bind to both FGF14 and the C -terminal tail of Nav1.6, this finding is consistent with a previous investigation that revealed both overlap and structural divergence between the FGF14:FGF14 homodimer and FGF14:Nav1.6 complex PPI interfaces [ xref ]."
sparser
"Building on previous studies in which LCA was conducted using transient transfection, here we created a double stable cell line that expresses the FGF14:Nav1.6 C-tail complex and miniaturized this assay from 96- to 384-well plates to be amenable for HTS of large chemical libraries."
sparser
"The presence of FBS completely prevented triciribine from enhancing FGF14:Nav1.6 complementation (10% FBS: 103.1 ± 8.9%, n = 8; 5% FBS: 97.6 ± 8.1%, n = 8; no FBS: 142.5% ± 10.7%, n = 8, p < 0.0001), and a higher concentration of FBS significantly reduced the potency of ZL181 (21.2 ± 2.8%, n = 8, p < 0.0001) compared to media with no FBS (11.2 ± 1.5%, n = 8, p < 0.0001)."
sparser
"All together, these effects demonstrate that PLEV and EYYV display both convergent and divergent effects on Nav1.6 channel activity, which could be attributable to their derivation from different structural motifs of FGF14 and differential interactions with residues at the FGF14:Nav1.6 PPI interface."
sparser
"Additionally, we found that the tyrosine kinase inhibitor MNS (30 µM; negative control 2) significantly reduces FGF14:Nav1.6 interaction, and the effect was greater with lower variance than that of ZL181 (one-way ANOVA; normalized mean and SD: MNS, 10.8% ± 3.1%; ZL181, 25.0% ± 7.3%; p < 0.0001) (Fig. xref )."
sparser
"To test the hypothesis that FLPK, PLEV and/or EYYV in cells could act as FGF14 inhibitors of the FGF14:Nav1.6 complex assembly, the complex was reconstituted using LCA (Ali et al., xref , xref ; Hsu et al., xref , xref ; Shavkunov et al., xref , xref , xref ; Wadsworth et al., xref )."
sparser
"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 ( xref ; xref ; xref ; xref , xref ; xref , xref ; xref , xref ; xref ; xref )."
sparser
"Consistent with the molecular modeling studies shown in xref , these nonoverlapping modulatory effects on Nav1.6 channel activity could be attributable to the tetrapeptides being derived from different structural motifs of FGF14 and resultantly displaying divergent interactions with residues at the FGF14:Nav1.6 PPI interface."
sparser
"The initial hit selection criteria were based on previously identified challenges in detecting potent enhancers of the FGF14:Nav1.6 C-tail interaction xref , xref , and the target profile and chemical attributes of compounds were subsequently analyzed to determine top hits for validation studies (Fig. xref and Table xref )."
sparser
"We sought to discover new regulators and explore potential phosphorylation networks regulating the FGF14:Nav1.6 complex by first conducting a high-throughput screening (HTS) campaign of diverse chemical libraries that was significantly expanded compared to previous studies[ xref , xref ]."
sparser
"Based on this information, we selected five ‘hit’ kinase inhibitors for extensive dose-dependency validation studies using fresh compound samples, including H-90, Critzotinib, BX913, Lestaurtinib, and BI2537, which all acted as antagonists toward the FGF14:Nav1.6 C-tail interaction."
sparser
"These new control compounds demonstrate that our modified LCA is capable of detecting agents that greatly increase or decrease FGF14:Nav1.6 complex formation without modifying the assay output (luminescence) through non-specific effects (i.e., luciferase modulation or changes in protein expression)."
sparser
"As shown in xref , for the FGF14 Y158A :Nav1.6 complex, the effects of Momelotinib and Pacritinib were abolished, while those of TG101209 and Fedratinib were greatly reduced (78.0% and 76.8% luminescence, respectively, with these remnant inhibitory effects being observed only at much higher concentrations compared to FGF14 WT (i.e., Fedratinib IC 50 shift from 9.7 μM to 27 μM)."
sparser
"We have studied the modulatory effects of the tetrapeptides PLEV and EYYV, which correspond to residues of FGF14 that are at its PPI interface with the CTD of the Nav1.6 channel, on FGF14:Nav1.6 complex assembly and the functional activity of the Nav1.6 channel macromolecular complex."
sparser
"A plausible hypothesis to explain its mechanism of action in the native system is that rather than limiting the FGF14:Nav1.6 complex formation, which would result in suppression of Na + currents and decreased firing, FLPK minimizes channel inactivation by stabilizing or altering interactions of FGF14 with other domains of Nav1.6 independently from the core domain like the N‐terminal tail, which is supported by our studies in heterologous cells."
sparser
"Similarly, for the potentiator of FGF14:Nav1.6 complex assembly ( 19 ), its modulatory effects on the PPI could be conferred by both binding to the C -terminal tail of Nav1.6 that makes the FGF14 interaction site increasingly accessible to the regulatory protein, or, conversely, by binding to FGF14 and causing it to undergo a conformational change that affords it with increased accessibility to its interaction site on the C -terminal tail of Nav1.6."
sparser
"The p38 MAPK inhibitor SB 203580 was initially found to enhance the FGF14:Nav1.6 interaction in the primary screening (30 µM), but evaluation of dose-dependent behavior (Fig. xref ) revealed mild inhibition at lower concentrations (0.5–2 µM) and stimulation at higher concentrations, indicative of off-target effects."
sparser
"Although these findings support the role of Src family kinases in regulation of the FGF14:Nav1.6 complex, Lck and Syk were not further pursued due to both proportionately low number of hits (relative to total # screened compounds targeting that kinase), as well as lack of observed phosphorylation motifs in FGF14."
sparser
"Additionally, dose‐response studies with FLPK showed that the tetrapetide is able to reduce the FGF14:Nav1.6 complex formation with an apparent IC 50 = 58 μM, but a relatively small efficacy of above 50% compared to control conditions (Figure xref ), indicative of weak inhibition."
sparser
"Although this odd behavior for the FGF14:Nav1.6 complex was less prevalent in follow-up studies with repurchased compound, a similar pattern of linear direction for enhancing the FGF14:FGF14 dimer was observed using Cucurcitabine I, but not S3I-201, which had minimal effect against FGF14:Nav1.6."
sparser
"Targeting these kinases with inhibitors or short-hairpin RNA alters protein complex stability, Nav1.6 currents and excitability xref , xref , xref , xref , xref , while peptidomimetics targeting the FGF14 V160 and FGF14 Y158 residues, which are located at the FGF14:Nav1.6 PPI interface, reduce complex formation, exhibit state-dependent modulation of Nav1.6 currents and suppress excitability of medium spiny neurons in the nucleus accumbens (NAc) xref , xref ."
sparser
"Given the abundant expression of Nav1.6 and its regulatory protein fibroblast growth factor 14 (FGF14) in these neurons, a feasible approach for developing such probes is through the rational design of small molecules targeting the FGF14:Nav1.6 protein:protein interaction (PPI) interface [ xref , xref ]."
sparser
"Comparatively, EYYV and PLEV were predicted to interact with FGF14 at regions of the FGF14:Nav1.6 interaction interface that are more peripheral (Figure xref ), but nonetheless have been shown to play a role in mediating the protein–protein interactions, at least at the FGF14:FGF14 dimer interface."
sparser
"The C- and N-terminal fragments of the P. Pyralis luciferase are fused, respectively, to FGF14 (CLuc-FGF14) and a chimera expressing CD4 fused to the Nav1.6 C-tail (CD4-Nav1.6-NLuc), and FGF14:Nav1.6 C-tail complex formation can be detected in the presence of the luciferase substrate, D-luciferin (Fig. xref )."
sparser
"Corroborated by homology modeling predictions and structural studies of other highly homologous iFGFs (Goetz et al., xref ), these studies led to the conclusion that FGF14 V160 and FGF14 Y158 are part of the PPI interface that confers structure–function specificity to the FGF14:Nav1.6 complex."
"Sodium channel fast inactivation is modulated by alpha subunit interaction with a family of cytoplasmic proteins termed fibroblast growth factor homologous factors (FHFs). In this paper, we report that all A-type FHFs exert rapid onset long-term inactivation on Nav1.6 and other sodium channels."
sparser
"In-cell screening of these analogs revealed that a tripeptide ( 12 ) displayed low micromolar inhibitory activity against FGF14:Nav1.6 complex assembly (IC 50 = 23.7 µM), whereas addition of hydrophobic protective groups to 6 followed by addition of a N-terminal acetyl ( 17 ) or benzoyl ( 19 ) substituent produced potentiators of the complex’s assembly."
sparser
"ROCK1 is a is a regulator of the actomyosin cytoskeleton which promotes contractile force generation xref ; this finding in combination with the numerous microtubule hits observed in our assay serves to reinforce the idea that the cytoskeleton may play a role in controlling FGF14:Nav1.6 interactions."
sparser
"After surface plasmon resonance (SPR) studies revealed that both peptidomimetics exhibited great affinity for Nav1.6, subsequent functional evaluation of the two analogs using whole-cell patch-clamp electrophysiology confirmed their inverse activities, with the inhibitor and potentiator of FGF14:Nav1.6 complex assembly reducing and increasing Nav1.6-mediated transient current densities, respectively."
sparser
"Interestingly, numerous DNA synthesis inhibitors (anti-metabolites), alkylating agents, and microtubule inhibitors enhanced the FGF14:Nav1.6 interaction; while exploration of possible mechanisms for these compounds are subjects for future investigation, the results may not be biologically relevant for neuronal Nav channel function."