Biology subjects

Wright, M.

Publications and source records attributed to Wright, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Phosphorylation of spleen tyrosine kinase Y130 positively regulates intracellular signaling and functional responses in platelets

Syk is a non-receptor type protein-tyrosine kinase (PTK), which is associated with platelets surface receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor II-type (CLEC-2). Syk is also expressed in most hematopoietic lineage cells and other cells, such as fibroblasts and neuronal cells. Syk has two tandem SH2 motifs and a C-terminal kinase domain, which are interrupted by interdomains A and B containing multiple tyrosine residues playing a regulatory role upon phosphorylation. This study aims to evaluate the role of Y130 in Syk signaling in platelets. Syk(Y130F) knock-in (KI) mice we generated using the CRISPR-Cas9 technique represent the first in-vivo model harboring this mutation. Using this system, we compared the platelet signaling and responses in wild-type (WT) and Syk(Y130F) littermates. Platelets from homozygous Syk(Y130F) mice showed a decrease in functional responses after activation with CRP, a GPVI agonist, and CLEC-2 crosslinking compared to WT littermates with no significant differences in responses to PAR-4 or purinergic receptor agonists. Key signaling events triggered via both GPVI and CLEC-2, including phosphorylation LAT and PLC-2, were also reduced in Syk(Y130F) platelets at low agonist concentrations. Consistent with these findings, the time to occlusion in the FeCl3 injury model and bleeding time in the tail bleeding assay were significantly enhanced in Syk(Y130F) mice compared to WT littermates. Thus, phosphorylation of Syk Y130 enhances GPVI- and CLEC-2-mediated signaling and functional responses in platelets affecting thrombosis and hemostasis.

molecular biology↗

bioRxiv2026

Orthogonal Transposons for Iterative Genome Engineering of Mammalian Cells.

The contemporary shift toward multispecific antibodies, antibody-drug conjugates (ADCs), and bespoke glycoengineered therapeutics have exposed the limitations of standard genomic engineering tools. This paper presents a novel iterative engineering paradigm utilizing the Leap-In Transposase(R) platform. By leveraging a suite of three mutually orthogonal transposase-transposon systems, we demonstrate the sequential modification of the Chinese Hamster Ovary (CHO) genome to achieve three distinct functional outcomes: (i) First, the creation of a glutamine synthetase (GS)-deficient host (CHO-K1-GS) via targeted knockdown, (ii) Second, the integration of multiple copies of a model therapeutic IgG1 for expression, and (iii) Third, the subsequent knockdown of the fucosylation pathway to modulate the glycan profile of the expressed IgG1. Genetic stability (copy number & sequence) of each integration event was confirmed using Targeted Locus Amplification (TLA) and Next-Generation Sequencing (NGS). Functional stability (expression levels, metabolic phenotype, and glycan phenotypes) was confirmed using standard cell culture and analytical techniques. Crucially, the truly orthogonal nature of the transposase-transposon pairs prevents cross-mobilization and ensures the structural and functional integrity of previously integrated cargo. This study establishes a "What You See Is What You Get" (WYSIWYG) methodology that provides a robust, scalable, and predictable framework for developing next-generation complex biopharmaceutical manufacturing cell lines.

bioengineering↗

bioRxiv2025

Seminal Fluid Adipokinetic Hormone Increases Insemination Refractoriness in Female Aedes aegypti

Mating often changes behavior and physiology of female insects. In many species, these changes have been attributed to receipt of seminal fluid molecules (SFMs). SFMs influence phenotypes including feeding, egg production, and response to male courtship and insemination attempts. These same phenotypes are potential targets for management of insect pests. Aedes aegypti mosquitoes are the primary vector of several pathogens including dengue, Zika, and chikungunya. Within an hour after an initial insemination, female Ae. aegypti are generally refractory to subsequent inseminations, a response attributed to SFMs. However, the specific molecules involved in inducing long-term insemination refractoriness have not been identified. In a previous study, we identified adipokinetic hormone (AKH) precursor protein as an SFM in Ae. albopictus. AKH is a well-studied insect neuropeptide that impacts phenotypes including those related to metabolism, locomotion, and reproduction. In this study, we investigated whether AKH is an SFM in Ae. aegypti and whether it impacts female re-insemination patterns. We first established that AKH is produced in the male reproductive tract and transferred to females during mating, and is, therefore, an SFM. We then created an AKH-null line which allowed us to demonstrate that seminal fluid AKH contributes to long-term insemination refractoriness of females. Together, these results have established a novel expression pattern for AKH and identified AKH as a contributor to Ae. aegypti insemination refractoriness, laying the groundwork for understanding the evolution and mode of action of novel seminal fluid proteins as well as for investigating novel pathways or approaches for mosquito control.

biochemistry↗

bioRxiv2025

APOL1 G1 and G2 risk alleles modulate severity of diet-induced obesity in a transgenic mouse model

APOL1 G1 and G2 risk alleles are associated with an increased risk of chronic kidney disease. However, a causal relationship between these alleles and cardiometabolic traits has not been experimentally validated. To address this gap, we placed transgenic APOL1 G0, G1, and G2 FVB/NJ mice on a high-fat diet and analyzed them for weight gain as well as obesity-related cardiometabolic phenotypes. To test whether APOL1 risk alleles modulate the inflammatory basis of obesity, we also exposed bone marrow derived macrophages from these mice to pro-inflammatory, pro-hypertensive, and dyslipidemic conditions. APOL1 high-risk allele female mice gained fat mass more readily than their low-risk female counterparts, while APOL1 high-risk male mice gained fat mass less readily than low-risk males. A parallel sex difference was seen in expression of higher levels of Abca1, Hmox1, and Srebf1 in lipid-loaded female bone-marrow derived macrophages expressing G1 and G2 APOL1, along with minor differences in cardiac function. However, this finding occurred independently of hypertension and insulin resistance, and with only minor albuminuria. Thus, our results highlight the importance of sex as a biological variable in future APOL1 experiments.

genomics↗

bioRxiv2025

Fine-scale ecological biomonitoring in a large, complex agriculturally impacted watershed via eDNA metabarcoding

DNA-based approaches utilizing high-throughput sequencing (HTS) (e.g. DNA metabarcoding) have revolutionized ecological biomonitoring by providing higher sample throughput, greater reproducibility, and better cost-benefits compared to traditional morphology-based bioassessment studies. Here, we utilized DNA metabarcoding in a watershed in Ontario (Canada) dominated by agricultural land uses. Our aim is to understand patterns of biodiversity in benthic taxa from data generated and inferred at various taxonomic scales and to compare these findings with over a decade of traditional morphological data. We sampled 18 watercourses during summer and fall 2023, spanning a forested-to-agricultural land-use gradient. We found significant differences between metabarcoding and historical morphology data where DNA provided more richness values at both the species (p = 2x10-5) and order (p = 0.008) levels. Whereas the morphology dataset contained many unresolved taxa, DNA metabarcoding captured a broader taxonomic breadth with diverse genetic profiles among taxa. Non-metric multidimensional scaling (NMDS) analyses on DNA metabarcoding data produced tighter clusters, more precise separation by land use, and greater consistency across taxonomic scales. Both urban context and land use had significant associations with metabarcoding patterns observed, with differences being strongest between agriculturally-dominated and primarily forested sites (median R{superscript 2} {approx} 0.08-0.11). We also found strong, consistent environmental signals linked to agricultural settings, such as water conductivity and turbidity, and pH. Altogether, our DNA-based results demonstrate the differences in community composition among different land uses in this watershed. Importantly, our work highlights the need for more taxonomic resolution (obtained through DNA analysis) to decipher community changes linked to anthropogenic and environmental drivers, as morphological data alone may lack the precision needed to capture these patterns.

ecology↗

bioRxiv2025

Modular Noncovalent Functionalization of Electrospun Piezoelectric Scaffolds with Bioactive Nanocarriers

Electrospun scaffolds offer a promising platform for immune-instructive materials, but stable and modular functionalization with bioactive signals remains a technical challenge. Here, we develop a surface coating strategy for electrospun scaffolds that consist of poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE), a piezoelectric polymer, using electrostatic adsorption of charged nanoparticles. We show that under certain conditions, these piezoelectric scaffolds are suitable substrates for electrostatic self-assembly, and that the density of nanoparticle coatings can be tuned by adjusting buffer pH, ionic strength, and nanoparticle concentration. This approach enables robust and uniform coating of both polymeric nanoparticles and soft nanocarriers such as liposomes, without requiring covalent surface modification. Liposome-coated scaffolds are cytocompatible with adherent epithelial and suspension immune cells and support lipid exchange at the cell-material interface. Using a supramolecular tethering strategy, we use liposome coatings to present interleukin-15 (IL-15) from the scaffold surface and demonstrate localized, sustained cytokine signaling. Together, these findings establish a modular approach for post-fabrication, noncovalent scaffold functionalization with bioactive nanocarriers, offering new opportunities for tissue and immune engineering.

bioengineering↗

bioRxiv2025

Training and match running distances and perceived exertion at sea-level and altitude in elite North American soccer: A natural longitudinal cross-over case series.

PurposeThere is limited real-world data on differential perceptions of exertion in professional soccer at altitude. In a natural cross-over design, we quantified responses to training and matches at sea level and altitude. MethodsTotal, and high-speed, running distance and differential session ratings of perceived exertion for breathlessness (s-RPE-B) and leg exertion (s-RPE-L) were collected from 19 professional soccer players (age: 27.3 {+/-} 3.4 years, stature: 181.2 {+/-} 5.9cm, mass: 78.7 {+/-} 8.1kg) at sea-level and altitude. Data were collected for matches raining sessions one and two days pre match. ResultsDifferences between altitude conditions were estimated through mixed-linear modeling, with 95% confidence intervals representing values highly compatible with our observed data. In matches, total distance may have been reduced (95% confidence intervals 43 to -833 m, surprisal [s] = 3.74) and s-RPE-B may have increased (-0.6 to 10.7, s = 3.66) at altitude. A clear perceptual shift was observed with s-RPE-B increasing relative to s-RPE-L (-13.4 to -7.17, s >13) at altitude. Training at altitude included higher total (318 to 661 m, s > 13), and high-speed (33.1 to 93 m, s > 13), running distances but s-RPE-L reduced (-14.2 to -1.01, s = 5.39) and s-RPE-B increased relative to s-RPE-L (-15.1 to -3.93, s = 10.3). ConclusionsOur key finding was breathlessness increased relative to leg exertion at altitude.

physiology↗

bioRxiv2025

G1 and G2 ApolipoproteinL1 modulate macrophage inflammation and lipid accumulation through the polyamine pathway

The G1 and G2 variants of the gene encoding Apolipoprotein L1 (APOL1) increase risk for kidney disease and cardiometabolic traits. While previous studies have elucidated key mechanisms by which G1 and G2 APOL1 cause cellular inflammation and cytotoxicity, it remains unclear whether these mechanisms drive inflammation in G1 and G2 macrophages. In this study, we used mouse bone-marrow-derived macrophages and human induced pluripotent stem cell-derived macrophages to identify altered immune signaling and inflammatory activation caused by G1 and G2 APOL1. We demonstrated that G1 and G2 APOL1 increased lipid accumulation, pro-inflammatory cytokine expression, and inflammasome signaling; this inflammatory response was sustained when treated with anti-inflammatory cytokines IL-4 and IL-10. Additionally, in G1 and G2 macrophages we observed increased mitochondrial size and elongation, oxidative phosphorylation, and glycolysis. Finally, we used unbiased metabolite analysis to identify an accumulation of polyamine spermidine and the enrichment of the spermidine synthesis pathway in G1 and G2 macrophages. When treated with polyamine inhibitor -difluoromethylornithine (DFMO), lipid accumulation and inflammasome gene expression decreased in G1 and G2 macrophages. Together, these findings establish the pro-inflammatory effects of G1 and G2 APOL1 in macrophages and identify a novel pathway which ameliorates G1 and G2 effects on cellular inflammation.

cell biology↗

bioRxiv2025

Cholinergic neuron circadian clock mediates RNA-binding protein function and contributes to ALS disease phenotypes

Circadian clocks are encoded by a transcription-translation feedback loop that aligns physiological processes with the solar cycle. Previous work linking the circadian clock to the regulation of RNA-binding proteins (RBPs) and alternative splicing provides a foundation for the vital examination of their mechanistic connections in the context of amyotrophic lateral sclerosis (ALS)--a fatal neurodegenerative disease commonly marked by disrupted RBP function. Here, we reveal that the spinal cord cholinergic neuron rhythmic transcriptome is enriched for genes associated with ALS and other neurodegenerative diseases. We show that there is time-of-day-dependent expression of ALS-linked RBP transcripts and rhythmic alternative splicing of genes involved in fundamental neuronal processes, such as microtubule cytoskeleton organization, intracellular trafficking, and synaptic function. We demonstrate clock-dependent expression of ALS-linked RBP Ataxin 2 in this neuronal subtype. Further, through in silico analysis of RNA sequencing data from sporadic ALS patients, we find that gene expression profiles altered in disease correspond with rhythmic gene networks. Finally, we report that clock disruption through cholinergic neuron-specific deletion of clock activator BMAL1 (i) increases lumbar spinal cord motor neuron loss and sciatic nerve axon degeneration and (ii) drives time-of-day-dependent alternative splicing of genes associated with RNA metabolism, including genes encoding ALS-linked RBPs (e.g., Matr3, Srsf7, and Ythdf2). Our results establish a role for the cholinergic neuron circadian clock in RNA metabolism and mediating neurodegeneration.

neuroscience↗

bioRxiv2025

Design and Characterisation of Photoactivatable and Lysine Reactive o-Nitrobenzyl Alcohol-Based Crosslinkers

Photoreactive groups are invaluable tools in structural proteomics, offering reagent-free activation and temporal control of protein labelling. However, traditional UV-activatable functional groups often produce unstable intermediates and diverse products, making these chemistries difficult to deploy at scale. In this study, we performed a systematic analysis of ortho-nitrobenzyl alcohol (oNBA) reactivity for integration into novel reagents for chemical crosslinking-mass spectrometry. oNBA photochemistry represents a promising alternative to traditional photoactivatable crosslinkers due to its unique specificity towards lysine residues. Here, we synthesised two molecules comprising oNBA functional groups with different substituents and assessed their labelling efficiency against a model protein. To ensure high labelling yields while maintaining a short irradiation time, we constructed a high power 365 nm irradiation device which improves the efficiency of oNBA photolysis. Our studies identified an amide-substituted probe that labels proteins with high efficiency. We next incorporated this optimised oNBA moiety into a homo-bifunctional crosslinker and a hetero-bifunctional crosslinker in combination with an NHS ester, which both resulted in high yields of crosslinked products. Our findings highlight that optimised oNBA-based reactive groups are viable UV-activated warheads that can deliver high labelling yields and efficient protein crosslinking, unlocking a wealth of potential structural proteomics applications.

biophysics↗

bioRxiv2025

Context-dependent Interactors Regulate TDP-43 Dysfunction in ALS/FTLD

TDP-43 mislocalization, aggregation, and loss of splicing function are neuropathological hallmarks in over 97% of Amyotrophic Lateral Sclerosis (ALS), 45% of Frontotemporal Lobar Degeneration (FTLD), and 60% of Alzheimers Disease, which has been reclassified as LATE-NC. However, the mechanisms underlying TDP-43 dysfunction remain elusive. Here, we utilize APEX2-driven proximity labeling and mass spectrometry to characterize the context-dependent TDP-43 interactome in conditions of cytoplasmic mislocalization, impaired RNA-binding contributing to aggregation, and oxidative stress. We describe context-dependent interactors, including disrupted interactions with splicing-related proteins and altered biomolecular condensate (BMC) associations. By integrating ALS and FTLD snRNA-seq data, we uncover disease-relevant molecular alterations and validate our dataset through a functional screen that identifies key TDP- 43 regulators. We demonstrate that disrupting nuclear speckle integrity, particularly through the downregulation of the splicing factor SRRM2, promotes TDP-43 mislocalization and loss of function. Additionally, we identify NUFIP2 as an interactor associated with mislocalization that sequesters TDP-43 into cytoplasmic aggregates and co-localizes with TDP-43 pathology in patient tissue. We also highlight HNRNPC as a potent TDP-43 splicing regulator, where precise modulation of TDP-43 or HNRNPC can rescue cryptic exon splicing. These findings provide mechanistic insights and potential therapeutic targets for TDP-43 dysfunction.

molecular biology↗

bioRxiv2024

The ALS-associated co-chaperone DNAJC7 mediates neuroprotection against proteotoxic stress by modulating HSF1 activity

The degeneration of neurons in patients with amyotrophic lateral sclerosis (ALS) is commonly associated with accumulation of misfolded, insoluble proteins. Heat shock proteins (HSPs) are central regulators of protein homeostasis as they fold newly synthesized proteins and refold damaged proteins. Heterozygous loss-of- function mutations in the DNAJC7 gene that encodes an HSP co-chaperone were recently identified as a cause for rare forms of ALS, yet the mechanisms underlying pathogenesis remain unclear. Using mass spectrometry, we found that the DNAJC7 interactome in human motor neurons (MNs) is enriched for RNA binding proteins (RBPs) and stress response chaperones. MNs generated from iPSCs with the ALS-associated mutation R156X in DNAJC7 exhibit increased insolubility of its client RBP HNRNPU and associated RNA metabolism alterations. Additionally, DNAJC7 haploinsufficiency renders MNs increasingly susceptible to proteotoxic stress and cell death as a result of an ablated HSF1 stress response pathway. Critically, expression of HSF1 in mutant DNAJC7 MNs is sufficient to rescue their sensitivity to proteotoxic stress, while postmortem ALS patient cortical neurons exhibit a reduction in the expression of HSF1 pathway genes. Taken together, our work identifies DNAJC7 as a crucial mediator of HNRNPU function and stress response pathways in human MNs and highlights HSF1 as a therapeutic target in ALS.

neuroscience↗

bioRxiv2024

Variations in human trigeminal and facial nerve branches and foramina identified by dissection and microcomputed tomography

The aim of this study was to identify branches of the trigeminal and facial nerves relevant to surgical incisions and injections and the scalp block techniques in the frontotemporal region, and to determine their relationships with superficial vascular structures and bony landmarks. Half-heads from consenting embalmed donors (6 male, 2 female, mean age at death 78.4 years) were used in this study. Detailed dissection was carried out to identify the position of the auriculotemporal nerve (ATN) relative to the superior temporal artery (STA) and the facial nerve (FN) in six subjects (5 male, 1 female). The results provide a minimum safe distance of 5 mm between the STA and the frontotemporal branches of the FN at the level of the low edge of zygoma and 8mm between the low edge of zygoma and the FN trunk, providing a pre-auricular triangle of safety for incisions and injections. Variability between subjects was up to 60%. Microcomputed tomography (microCT) scans were taken from all eight subjects and the three-dimensional reconstructions were used to identify the supraorbital notch (SON), the zygomaticotemporal foramen (ZTF) and the zygomaticofacial foramen (ZFF). The volume and relative locations of these foramina were calculated for 5-8 subjects. The closest distance between ZTF and the FZS ranged from 9 to 21mm (26% variation); 3 subjects had a single ZTF while 5 subjects had two ZTF. The angle at the centre of the orbit between ZFF and the FZS ranged from 156 to 166 degrees (2.5% variation). These findings demonstrate that both traditional cadaveric dissection methods as well as contemporary microCT methods can be used to investigate the relative locations of nerves or their foramina in the human head. The findings provide anatomical considerations for fronto-temporal incisions and local anaesthesia.

neuroscience↗

bioRxiv2024

Myocardial infarction injury is exacerbated by nicotine in vape aerosol exposure

RationaleVaping is touted as a safer alternative to traditional cigarette smoking but the full spectrum of harm reduction versus comparable risk remains unresolved. Elevated bioavailability of nicotine in vape aerosol together with known risks of nicotine exposure may result in previously uncharacterized cardiovascular consequences of vaping. ObjectiveAssess the impact of nicotine exposure via vape aerosol inhalation upon myocardial response to infarction injury. Methods and ResultsFlavored vape juice containing nicotine (5 mg / ml) or vehicle alone (0 mg) was delivered using identical 4-week treatment protocols. Mice were subjected to acute myocardial infarction injury and evaluated for outcomes of cardiac structure and function. Findings reveal that nicotine exposure leads to worse outcomes with respect to contractile performance regardless of sex. Non-myocyte interstitial cell accumulation following infarction significantly increased with exposure to vape aerosol alone, but a comparable increase was not present when nicotine was included. ConclusionsMyocardial function after infarction is significantly decreased after exposure to nicotine vape aerosol irrespective of sex. Comparable loss of contractile function was not observed in mice exposed to vape aerosol alone, highlighting the essential role of nicotine in loss of contractile function. Increased vimentin immunoreactivity was observed in the vape alone group compared to control and vape nicotine. The correlation between vaping, interstitial cell responses, and cardiac remodeling leading to impaired contractility warrants further investigation. Public health experts seeking to reduce vaping-related health risks should consider messaging that highlights the increased cardiovascular risk especially with nicotine-containing aerosols.

molecular biology↗

bioRxiv2024

First endangered black-footed ferrets, Mustela nigripes, cloned for genetic rescue

An endangered black-footed ferret female that died in 1988 with no living descendants in the current population was successfully cloned from cryopreserved cells using cross-species somatic cell nuclear transfer, producing three healthy kits. Incorporating progeny from these clones would provide an 8th founder to the breeding program and increase genetic variation to the species limited gene-pool. This marks the first time a native U.S. endangered species has been cloned.

zoology↗

bioRxiv2024

ALS-Associated TDP-43 Dysfunction Compromises UPF1-Dependent mRNA Metabolism Pathways Including Alternative Polyadenylation and 3'UTR Length

UPF1-mediated decay entails several mRNA surveillance pathways that play a crucial role in cellular homeostasis. However, the precise role of UPF1 in postmitotic neurons remains unresolved, as does its activity in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease characterized by TDP-43 pathology and disrupted mRNA metabolism. Here, we used human iPSC-derived spinal motor neurons (MNs) to identify mRNAs subject to UPF1 degradation by integrating RNA-seq before and after UPF1 knockdown with RIP-seq to identify RNAs that co-immunoprecipitate with the active form of phosphorylated UPF1. We define a stringent set of bona fide UPF1 targets in MNs that are functionally enriched for autophagy and structurally enriched for GC-rich and long 3 UTRs but not for premature termination codon (PTC)-containing transcripts. TDP-43 depletion in iPSC-derived MNs reduces UPF1 phosphorylation and consequently post-transcriptional upregulation of UPF1 targets, suggesting that TDP-43 dysfunction compromises UPF1-mediated mRNA surveillance. Intriguingly, our datasets reveal that UPF1 and TDP-43 regulate alternative polyadenylation and 3UTR length of mRNAs associated with synaptic and axonal function, a process that we find to be compromised in ALS models in vitro and ALS patient tissue. Our study provides a comprehensive description of UPF1-mediated mRNA decay activity in neurons, reveals overlapping roles between UPF1 and TDP-43 in regulating 3UTR length, and offers novel insight into the intricate interplay between RNA metabolism and neurodegeneration in ALS.

neuroscience↗

bioRxiv2023

BrainLine: An Open Pipeline for Connectivity Analysis of Heterogeneous Whole-Brain Fluorescence Volumes

Whole-brain fluorescence images require several stages of computational processing to fully reveal the neuron morphology and connectivity information they contain. However, these computational tools are rarely part of an integrated pipeline. Here we present BrainLine, an open-source pipeline that interfaces with existing software to provide registration, axon segmentation, soma detection, visualization and analysis of results. By implementing a feedback based training paradigm with BrainLine, we were able to use a single learning algorithm to accurately process a diverse set of whole-brain images generated by light-sheet microscopy. BrainLine is available as part of our Python package brainlit: http://brainlit.neurodata.io/.

bioengineering↗

bioRxiv2022

CryoEM reveals unprecedented binding site for NaV1.7 inhibitors enabling rational design of potent hybrid inhibitors

The voltage-gated sodium (NaV) channel NaV1.7 has been identified as a potential novel pain target due to its striking human genetics. However, clinically available drugs (e.g. lidocaine, carbamazepine, etc.) are not selective among the nine NaV channel subtypes, NaV1.1-NaV1.9, and the two currently known classes of NaV1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. Moreover, understanding of the structure-activity relationships of the acylsulfonamide class of NaV1.7 inhibitors, exemplified by the clinical development candidate GDC-0310, has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor series. To advance inhibitor design targeting the NaV1.7 channel, we established an iterative system to routinely obtain high-resolution ligand-bound NaV1.7 structures using cryogenic electron microscopy (cryo-EM). We report that GDC-0310 engages the NaV1.7 voltage-sensing domain 4 (VSD4) through an unexpected binding mode orthogonal to the arylsulfonamide class binding pose, which identifies a previously unknown ligand binding site in NaV channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, this study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. It also underscores an important role of the membrane bilayer in the discovery of selective NaV channel modulators.

bioengineering↗