Publications by authors named "Paul A Wadsworth"

11 Publications

  • Page 1 of 1

Development of Allosteric Modulators of Voltage-Gated Na Channels: A Novel Approach for an Old Target.

Curr Top Med Chem 2021 ;21(10):841-848

Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, 77555, United States.

Given their primacy in governing the action potential (AP) of excitable cells, voltage-gated Na+ (Nav) channels are important pharmacological targets of therapeutics for a diverse array of clinical indications. Despite historically being a traditional drug target, therapeutics targeting Nav channels lack isoform selectivity, giving rise to off-target side effects. To develop isoform-selective modulators of Nav channels with improved target-specificity, the identification and pharmacological targeting of allosteric sites that display structural divergence among Nav channel isoforms represents an attractive approach. Despite the high homology among Nav channel α subunit isoforms (Nav1.1-Nav1.9), there is considerable amino acid sequence divergence among their constituent C-terminal domains (CTD), which enables structurally and functionally specific protein: protein interactions (PPI) with auxiliary proteins. Although pharmacological targeting of such PPI interfaces between the CTDs of Nav channels and auxiliary proteins represents an innovate approach for developing isoform-selective modulators of Nav channels, appreciable modulation of PPIs using small molecules has conventionally been difficult to achieve. After briefly discussing the challenges of modulating PPIs using small molecules, this current frontier review that follows subsequently expounds on approaches for circumventing such difficulties in the context of developing small molecule modulators of PPIs between transmembrane ion channels and their auxiliary proteins. In addition to broadly discussing such approaches, the implementation of such approaches is specifically discussed in the context of developing small molecule modulators between the CTD of Nav channels and auxiliary proteins. Developing allosteric modulators of ion channels by targeting their PPI interfaces with auxiliary proteins represents an innovative and promising strategy in ion channel drug discovery that could expand the "druggable genome" and usher in first-in-class PPI-targeting therapeutics for a multitude of channelopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1568026621666210525105359DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8272397PMC
January 2021

Bioluminescence Methodology for Ion Channel Studies.

Methods Mol Biol 2021 ;2188:191-228

Department of Pharmacology and Toxicology, The University of Texas Medical Branch, Galveston, TX, USA.

As key players in cell function, ion channels are important targets for drug discovery and therapeutic development against a wide range of health conditions. Thus, developing assays to reconstitute ion channel macromolecular complexes in physiological conditions and screen for chemical modifiers of protein-protein interactions within these complexes is timely in drug discovery campaigns. For most ion channels, expressing their pore-forming subunit in heterologous mammalian cells has now become a routine procedure. However, reconstituting protein-channel complexes in physiological environments is still challenging, limiting our ability to identify tools and probes based on allosteric mechanisms, which could lead to more targeted and precise modulation of the channel function. Here, we describe the assay development steps to stably reconstitute the interaction between voltage-gated Na (Nav) channel Nav1.6 and its accessory protein, fibroblast growth factor 14 (FGF14) using the split-luciferase complementation assay (LCA), followed by assay miniaturization and optimization in 384-well plates for in-cell high-throughput screening (HTS) against protein-channel interactions. This optimized LCA can subsequently be used for rapid estimation of hit potency and efficacy via dose-dependency studies, enabling ranking of hits prior to more labor-intensive validation studies. Lastly, we introduce the methodology for rapid functional hit validation studies using semi-automated planar patch-clamp electrophysiology. Our robust, in-cell HTS platform can be adapted to any suitable ion channel complex to explore regulatory pathways of cellular signaling using kinase inhibitors, as well as to screen small molecules for probe development and drug repurposing toward new targets/areas of medicine. Overall, the flexibility of this assay allows users to broadly explore therapeutic options for channelopathy-associated diseases at a fast pace, enabling rapid hypothesis generation in early phase drug discovery campaigns and narrowing down targets prior to more labor-intensive in vivo studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-0818-0_10DOI Listing
March 2021

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

J Med Chem 2020 10 15;63(20):11522-11547. Epub 2020 Oct 15.

The voltage-gated Na (Na) channel is the molecular determinant of excitability. Disruption of protein-protein interactions (PPIs) between Na1.6 and fibroblast growth factor 14 (FGF14) leads to impaired excitability of neurons in clinically relevant brain areas associated with channelopathies. Here, we designed, synthesized, and pharmacologically characterized new peptidomimetics based on a PLEV tetrapeptide scaffold derived from the FGF14:Na1.6 PPI interface. Addition of an N-terminal 1-adamantanecarbonyl pharmacophore significantly improved peptidomimetic inhibitory potency. Surface plasmon resonance studies revealed that while this moiety was sufficient to confer binding to FGF14, altering the C-terminal moiety from methoxy () to π bond-containing ( and ) or cycloalkane substituents () abrogated the binding to Na1.6. Whole-cell patch-clamp electrophysiology subsequently revealed that had functionally relevant interactions with both the C-terminal tail of Na1.6 and FGF14. Collectively, these findings support that () may serve as a promising lead to develop target-selective neurotherapeutics by modulating protein-channel interactions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acs.jmedchem.0c00531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8317200PMC
October 2020

Mobility but Not Balance Limitations Are Associated With Cognitive Decline among Older Hispanics.

Gerontol Geriatr Med 2020 Jan-Dec;6:2333721420947952. Epub 2020 Aug 13.

The University of Texas Medical Branch, Galveston, TX, USA.

Aging is associated with changes in lower-body functioning. The extent to which lower-body function is associated with cognitive changes over time is unclear, especially among older Hispanics, a high-risk population for declines in physical and cognitive functioning. We sought to determine if the association between lower-body functioning and cognitive decline over 9-years differentially varied with respect to balance, gait speed, lower-body strength (chair stands), or a summary score of the three measures. This retrospective cohort study used clinical performance data from the Hispanic Established Populations for the Epidemiologic Study of the Elderly (H-EPESE). Cognitive function was measured using the Mini-Mental Status Exam. Linear mixed modeling was used to investigate the association between lower-body function and cognitive decline, controlling for patients' demographic and health characteristics. We found that gait speed and timed chair stands but not balance were associated with accelerated cognitive decline in Mexican-Americans age 75 years and older. These parameters of lower-body function can be feasibly measured in any clinic. As limitations in lower-body functioning may be an early marker of cognitive decline, this suggests an opportunity for the development of interventions to slow cognitive and physical disablement and promote successful aging among persons older than 75 years.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1177/2333721420947952DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7427021PMC
August 2020

Bidirectional Modulation of the Voltage-Gated Sodium (Nav1.6) Channel by Rationally Designed Peptidomimetics.

Molecules 2020 Jul 24;25(15). Epub 2020 Jul 24.

Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 75901, USA.

Disruption of protein:protein interactions (PPIs) that regulate the function of voltage-gated Na (Nav) channels leads to neural circuitry aberrations that have been implicated in numerous channelopathies. One example of this pathophysiology is mediated by dysfunction of the PPI between Nav1.6 and its regulatory protein fibroblast growth factor 14 (FGF14). Thus, peptides derived from FGF14 might exert modulatory actions on the FGF14:Nav1.6 complex that are functionally relevant. The tetrapeptide Glu-Tyr-Tyr-Val (EYYV) mimics surface residues of FGF14 at the β8-β9 loop, a structural region previously implicated in its binding to Nav1.6. Here, peptidomimetics derived from EYYV () were designed, synthesized, and pharmacologically evaluated to develop probes with improved potency. Addition of hydrophobic protective groups to and truncation to a tripeptide () produced a potent inhibitor of FGF14:Nav1.6 complex assembly. Conversely, addition of hydrophobic protective groups to followed by addition of an -terminal benzoyl substituent () produced a potentiator of FGF14:Nav1.6 complex assembly. Subsequent functional evaluation using whole-cell patch-clamp electrophysiology confirmed their inverse activities, with and reducing and increasing Nav1.6-mediated transient current densities, respectively. Overall, we have identified a negative and positive allosteric modulator of Nav1.6, both of which could serve as scaffolds for the development of target-selective neurotherapeutics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/molecules25153365DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7435778PMC
July 2020

Mapping of the FGF14:Nav1.6 complex interface reveals FLPK as a functionally active peptide modulating excitability.

Physiol Rep 2020 07;8(14):e14505

Department of Pharmacology & Toxicology, University of Texas Medical Branch, Galveston, TX, USA.

The voltage-gated sodium (Nav) channel complex is comprised of pore-forming α subunits (Nav1.1-1.9) and accessory regulatory proteins such as the intracellular fibroblast growth factor 14 (FGF14). The cytosolic Nav1.6 C-terminal tail binds directly to FGF14 and this interaction modifies Nav1.6-mediated currents with effects on intrinsic excitability in the brain. Previous studies have identified the FGF14 residue within the FGF14 core domain as a hotspot for the FGF14:Nav1.6 complex formation. Here, we used three short amino acid peptides around FGF14 to probe for the FGF14 interaction with the Nav1.6 C-terminal tail and to evaluate the activity of the peptide on Nav1.6-mediated currents. In silico docking predicts FLPK to bind to FGF14 with the expectation of interfering with the FGF14:Nav1.6 complex formation, a phenotype that was confirmed by the split-luciferase assay (LCA) and surface plasmon resonance (SPR), respectively. Whole-cell patch-clamp electrophysiology studies demonstrate that FLPK is able to prevent previously reported FGF14-dependent phenotypes of Nav1.6 currents, but that its activity requires the FGF14 N-terminal tail, a domain that has been shown to contribute to Nav1.6 inactivation independently from the FGF14 core domain. In medium spiny neurons in the nucleus accumbens, where both FGF14 and Nav1.6 are abundantly expressed, FLPK significantly increased firing frequency by a mechanism consistent with the ability of the tetrapeptide to interfere with Nav1.6 inactivation and potentiate persistent Na currents. Taken together, these results indicate that FLPK might serve as a probe for characterizing molecular determinants of neuronal excitability and a peptide scaffold to develop allosteric modulators of Nav channels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.14814/phy2.14505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363588PMC
July 2020

JAK2 regulates Nav1.6 channel function via FGF14 phosphorylation.

Biochim Biophys Acta Mol Cell Res 2020 10 26;1867(10):118786. Epub 2020 Jun 26.

Department of Pharmacology & Toxicology, The University of Texas Medical Branch, Galveston, TX, USA; Center for Addiction Research, The University of Texas Medical Branch, Galveston, TX, USA. Electronic address:

Background: Protein interactions between voltage-gated sodium (Nav) channels and accessory proteins play an essential role in neuronal firing and plasticity. However, a surprisingly limited number of kinases have been identified as regulators of these molecular complexes. We hypothesized that numerous as-of-yet unidentified kinases indirectly regulate the Nav channel via modulation of the intracellular fibroblast growth factor 14 (FGF14), an accessory protein with numerous unexplored phosphomotifs and required for channel function in neurons.

Methods: Here we present results from an in-cell high-throughput screening (HTS) against the FGF14: Nav1.6 complex using >3000 diverse compounds targeting an extensive range of signaling pathways. Regulation by top kinase targets was then explored using in vitro phosphorylation, biophysics, mass-spectrometry and patch-clamp electrophysiology.

Results: Compounds targeting Janus kinase 2 (JAK2) were over-represented among HTS hits. Phosphomotif scans supported by mass spectrometry revealed FGF14, a site previously shown to mediate both FGF14 homodimerization and interactions with Nav1.6, as a JAK2 phosphorylation site. Following inhibition of JAK2, FGF14 homodimerization increased in a manner directly inverse to FGF14:Nav1.6 complex formation, but not in the presence of the FGF14 mutant. Patch-clamp electrophysiology revealed that through Y158, JAK2 controls FGF14-dependent modulation of Nav1.6 channels. In hippocampal CA1 pyramidal neurons, the JAK2 inhibitor Fedratinib reduced firing by a mechanism that is dependent upon expression of FGF14.

Conclusions: These studies point toward a novel mechanism by which levels of JAK2 in neurons could directly influence firing and plasticity by controlling the FGF14 dimerization equilibrium, and thereby the availability of monomeric species for interaction with Nav1.6.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamcr.2020.118786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984254PMC
October 2020

High-throughput screening against protein:protein interaction interfaces reveals anti-cancer therapeutics as potent modulators of the voltage-gated Na channel complex.

Sci Rep 2019 11 15;9(1):16890. Epub 2019 Nov 15.

Department of Pharmacology & Toxicology, The University of Texas Medical Branch, Galveston, Texas, 77555, USA.

Multiple voltage-gated Na (Nav) channelopathies can be ascribed to subtle changes in the Nav macromolecular complex. Fibroblast growth factor 14 (FGF14) is a functionally relevant component of the Nav1.6 channel complex, a causative link to spinocerebellar ataxia 27 (SCA27) and an emerging risk factor for neuropsychiatric disorders. Yet, how this protein:channel complex is regulated in the cell is still poorly understood. To search for key cellular pathways upstream of the FGF14:Nav1.6 complex, we have developed, miniaturized and optimized an in-cell assay in 384-well plates by stably reconstituting the FGF14:Nav1.6 complex using the split-luciferase complementation assay. We then conducted a high-throughput screening (HTS) of 267 FDA-approved compounds targeting known mediators of cellular signaling. Of the 65 hits initially detected, 24 were excluded based on counter-screening and cellular toxicity. Based on target analysis, potency and dose-response relationships, 5 compounds were subsequently repurchased for validation and confirmed as hits. Among those, the tyrosine kinase inhibitor lestaurtinib was highest ranked, exhibiting submicromolar inhibition of FGF14:Nav1.6 assembly. While providing evidence for a robust in-cell HTS platform that can be adapted to search for any channelopathy-associated regulatory proteins, these results lay the potential groundwork for repurposing cancer drugs for neuropsychopharmacology.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-53110-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6858373PMC
November 2019

Sex-Specific Proteomic Changes Induced by Genetic Deletion of Fibroblast Growth Factor 14 (FGF14), a Regulator of Neuronal Ion Channels.

Proteomes 2019 Jan 23;7(1). Epub 2019 Jan 23.

Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX 77555, USA.

Fibroblast growth factor 14 (FGF14) is a member of the intracellular FGFs, which is a group of proteins involved in neuronal ion channel regulation and synaptic transmission. We previously demonstrated that male mice recapitulate the salient endophenotypes of synaptic dysfunction and behaviors that are associated with schizophrenia (SZ). As the underlying etiology of SZ and its sex-specific onset remain elusive, the model may provide a valuable tool to interrogate pathways related to disease mechanisms. Here, we performed label-free quantitative proteomics to identify enriched pathways in both male and female hippocampi from and mice. We discovered that all of the differentially expressed proteins measured in animals, relative to their same-sex wildtype counterparts, are associated with SZ based on genome-wide association data. In addition, measured changes in the proteome were predominantly sex-specific, with the male mice distinctly enriched for pathways associated with neuropsychiatric disorders. In the male Fgf14 mouse, we found molecular characteristics that, in part, may explain a previously described neurotransmission and behavioral phenotype. This includes decreased levels of ALDH1A1 and protein kinase A (PRKAR2B). ALDH1A1 has been shown to mediate an alternative pathway for gamma-aminobutyric acid (GABA) synthesis, while PRKAR2B is essential for dopamine 2 receptor signaling, which is the basis of current antipsychotics. Collectively, our results provide new insights in the role of FGF14 and support the use of the mouse as a useful preclinical model of SZ for generating hypotheses on disease mechanisms, sex-specific manifestation, and therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/proteomes7010005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6473632PMC
January 2019

Intracellular Fibroblast Growth Factor 14: Emerging Risk Factor for Brain Disorders.

Front Cell Neurosci 2017 19;11:103. Epub 2017 Apr 19.

Department of Pharmacology and Toxicology, University of Texas Medical BranchGalveston, TX, USA.

The finely tuned regulation of neuronal firing relies on the integrity of ion channel macromolecular complexes. Minimal disturbances of these tightly regulated networks can lead to persistent maladaptive plasticity of brain circuitry. The intracellular fibroblast growth factor 14 (FGF14) belongs to the nexus of proteins interacting with voltage-gated Na+ (Na) channels at the axonal initial segment. Through isoform-specific interactions with the intracellular C-terminal tail of neuronal Na channels (Na1.1, Na1.2, Na1.6), FGF14 controls channel gating, axonal targeting and phosphorylation in neurons effecting excitability. FGF14 has been also involved in synaptic transmission, plasticity and neurogenesis in the cortico-mesolimbic circuit with cognitive and affective behavioral outcomes. In translational studies, interest in FGF14 continues to rise with a growing list of associative links to diseases of the cognitive and affective domains such as neurodegeneration, depression, anxiety, addictive behaviors and recently schizophrenia, suggesting its role as a converging node in the etiology of complex brain disorders. Yet, a full understanding of FGF14 function in neurons is far from being complete and likely to involve other functions unrelated to the direct regulation of Na channels. The goal of this Mini Review article is to provide a summary of studies on the emerging role of FGF14 in complex brain disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fncel.2017.00103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5396478PMC
April 2017

Systematic identification of single amino acid variants in glioma stem-cell-derived chromosome 19 proteins.

J Proteome Res 2015 Feb 25;14(2):778-86. Epub 2014 Nov 25.

Department of Pharmacology and Toxicology and ‡Biochemistry and Molecular Biology, UTMB Cancer Center, University of Texas Medical Branch , Galveston, Texas 77555, United States.

Novel proteoforms with single amino acid variations represent proteins that often have altered biological functions but are less explored in the human proteome. We have developed an approach, searching high quality shotgun proteomic data against an extended protein database, to identify expressed mutant proteoforms in glioma stem cell (GSC) lines. The systematic search of MS/MS spectra using PEAKS 7.0 as the search engine has recognized 17 chromosome 19 proteins in GSCs with altered amino acid sequences. The results were further verified by manual spectral examination, validating 19 proteoforms. One of the novel findings, a mutant form of branched-chain aminotransferase 2 (p.Thr186Arg), was verified at the transcript level and by targeted proteomics in several glioma stem cell lines. The structure of this proteoform was examined by molecular modeling in order to estimate conformational changes due to mutation that might lead to functional modifications potentially linked to glioma. Based on our initial findings, we believe that our approach presented could contribute to construct a more complete map of the human functional proteome.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/pr500810gDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4324435PMC
February 2015
-->