Biology subjects

Linley, J.

Publications and source records attributed to Linley, J..

4 recordsLinked to original sources

bioRxiv2025

The Rise and Fall of SARM1 Base-Exchange Inhibitors

The sterile alpha and TIR motif containing 1 (SARM1) enzyme is a key driver of axonal degeneration in response to injury, making it an attractive target for treating chemotherapy-induced peripheral neuropathy (CIPN) and other nervous system diseases. In this study, we identified and optimised a new class of base-exchange inhibitors (BEXi) targeting SARM1 and explored their molecular interactions and conformational effects using cryo-EM, HDX-MS and SAXS. Although BEXi produced robust inhibition across all biochemical and cellular assay formats, application at sub-inhibitory concentrations consistently led to paradoxical SARM1 activation, and in neuronal assays, accelerated neurite degeneration. Further analysis showed that BEXi only delayed, rather than prevented, neurite degeneration when applied to primary neuronal cells, even at exceedingly high inhibitor concentrations. These results prompted us to discontinue BEXi development in favour of alternative strategies, underscoring the complexity of SARM1 as a therapeutic target and the need for comprehensive, mechanistically informed screening cascades.

neuroscience↗

bioRxiv2025

A molecular stabiliser of an inhibitory eIF2B-eIF2(αP) complex activates the Integrated Stress Response

Eukaryotic initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF), promotes protein synthesis by charging translation initiation factor 2 (eIF2) with GTP. Stress-induced phosphorylation of eIF2 on its -subunit [eIF2(P)] inhibits this reaction triggering a protective Integrated Stress Response (ISR). A DNA-encoded chemical library (DEL) screen for modulators of eIF2B, led to the identification of a chemical series that inactivates eIF2B, stimulating the ISR. Cryo-EM of compound-bound eIF2B revealed a conformational switch to the inactive state engaged by eIF2(P). In cells, compound activity was sensitive to eIF2s phosphorylation state and to a competing eIF2B ligand (ISRIB) that activates the GEF allosterically. These findings mark the discovery of a first-in-class drug-like allosteric inhibitor of eIF2B, an ISR activator (ISRAC), paving the way to explore the therapeutic potential of eIF2B-directed ISR activation.

molecular biology↗

bioRxiv2025

Painless Nav1.7 mutations reveal function-critical residuesin the outer vestibule and N-terminus

Chronic pain is a common condition, placing a large cost on society and a heavy burden on the individual. Nav1.7 has emerged as a non-redundant and axiomatically important part of human pain pathways. Biallelic loss-of-function (LOF) mutations in SCN9A, encoding voltage-gated sodium channel Nav1.7, cause the Mendelian disorder Congenital Insensitivity to Pain (CIP). Studying novel missense mutations in this channel has the potential to uncover unrealised functions of critical residues that could enable new strategies for analgesia. Here we describe detailed functional studies of six SCN9A missense variants found in individuals with typical SCN9A-CIP: c.224T>C/p.Leu75Pro, c.1025C>T/p.Thr342Met, c.2686C>T/p.Arg896Trp, c.2732G>A/p.Arg911His, c.5059G>C/p.Ala1687Pro, and c.5173G>C/p.Gly1725Arg. While all variants were extremely rare and predicted to alter function, current tools cannot specify the extent or direction of change in function, nor provide information about the mechanism(s) of pathogenicity. Stable cell lines were generated for each variant, using the isogenic Jump-In T-REx HEK293 system. We investigated how each variant caused molecular dysfunction using multiple approaches: sequence conservation analysis and 3D modelling to examine the structural importance of the affected residues; automated whole-cell patch clamp electrophysiology to assess channel biophysics; and finally, immunocytochemistry along with cell-surface biotinylation to detect any alterations in subcellular localisation. Our studies revealed that all mutations reached the cell membrane but caused a complete LOF through small localised structural changes in the extracellular vestibule, ion selectivity filter and N-terminus that abolished sodium conductance. Functional analysis remains the most reliable method of classifying SCN9A missense variants and can help to pinpoint important subdomains. Online SummaryVoltage-gated sodium channel Nav1.7 is a key modulator of nociceptor excitability and associated with congenital painlessness. Functional analysis and in silico modelling reveal critical roles for residues in the extracellular vestibule, ion selectivity filter and unexpectedly, the N-terminus.

molecular biology↗

bioRxiv2017

Mapping protein interactions of sodium channel NaV1.7 using epitope-tagged gene targeted mice

The voltage-gated sodium channel NaV1.7 plays a critical role in pain pathways. Besides action potential propagation, NaV1.7 regulates neurotransmitter release, integrates depolarizing inputs over long periods and regulates transcription. In order to better understand these functions, we generated an epitope-tagged NaV1.7 mouse that showed normal pain behavior. Analysis of NaV1.7 complexes affinity-purified under native conditions by mass spectrometry revealed 267 NaV1.7 associated proteins including known interactors, such as the sodium channel {beta}3 subunit (Scn3b) and collapsin response mediator protein (Crmp2), and novel interactors. Selected novel NaV1.7 protein interactors membrane-trafficking protein synapototagmin-2 (Syt2), G protein-regulated inducer of neurite outgrowth 1 (Gprin1), L-type amino acid transporter 1 (Lat1) and transmembrane P24 trafficking protein 10 (Tmed10) together with Scn3b and Crmp2 were validated using co-immunoprecipitation and functional assays. The information provided with this physiologically normal epitope-tagged mouse should provide useful insights into the pain mechanisms associated with NaV1.7 channel function.

neuroscience↗