Biology subjects

Jung, J.

Publications and source records attributed to Jung, J..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Hero11 Unlocks TDP-43 Condensate Fluidity via Targeting Inter-Helical Interactions

Biomolecular condensates formed by intrinsically disordered proteins (IDPs) rely on a balance of sequence-encoded interactions and secondary-structure elements. TDP-43, a disease-associated protein, undergoes liquid-liquid phase separation (LLPS) through its low-complexity domain, whereas Hero11 has been proposed to modulate its condensate properties. However, the molecular mechanisms by which Hero11 affects the internal organization and dynamics of TDP-43 condensates remain unknown. Here, using multi-microsecond explicit-solvent all-atom simulations spanning single chains to =~100-chain condensates, we show that the TDP-43 -helix, which is only marginally stable in isolation, becomes a major structural hub within the condensate, forming a percolated helix-helix interaction network whose contact lifetimes are substantially longer than those of the surrounding disordered contacts. Hero11 selectively dismantles this network: it binds preferentially near the helical region, reduces the helix-helix coordination number, and shortens helix-helix contact lifetimes. This targeted disruption lowers condensate density, increases both water and ion infiltration, and enhances TDP-43 diffusion within the dense phase. Notably, dimer simulations reveal that the interactions between TDP-43 and Hero11 are too weak to persist under dilute conditions, indicating that the regulatory effect emerges only through multivalent contacts in the condensed phase. These results establish the -helix as a selectively vulnerable structural element within the TDP-43 condensate and provide an atomic-level mechanism for how a highly charged disordered protein can tune condensate material properties by targeting its longest-lived interaction nodes.

biochemistry↗

bioRxiv2026

Time-resolved cryo-EM reveals conformational trajectory of allosteric activation in isocitrate lyase

Isocitrate lyase 2 (ICL2) from Mycobacterium tuberculosis undergoes dramatic conformational rearrangements upon binding to the allosteric effector acetyl-CoA. Time-resolved cryo-EM captured conformational states along the ICL2 activation trajectory, revealing how acetyl-CoA binding at the allosteric sites leads to asymmetric, half-of-site activity at the catalytic centres. These findings support a conformational selection model of allostery, whereby acetyl-CoA binding shifts the pre-existing equilibrium towards an active state of the enzyme.

biochemistry↗

bioRxiv2026

Transcutaneous auricular Vagus nerve stimulation for working memory enhancement: A comparative study of electrical and ultrasound stimulation

ObjectivesTranscutaneous auricular vagus nerve stimulation (taVNS) is a non-invasive neuromodulation technique that has shown potential to enhance cognitive function, including working memory. This study investigated the acute effects of both electrical (E-taVNS) and ultrasound (U-taVNS) modalities on working memory using a 3-back task in healthy young adults. We hypothesized that active taVNS would enhance working memory performance relative to sham, and that both stimulation modalities would engage similar neuromodulatory mechanisms. Materials and MethodsFifty-nine participants underwent a single-blind, sham-controlled, within-subject design study, with working memory performance assessed using a 3-back task before and after stimulation. Primary performance measures included correct rejection rate, error false alarm, and sensitivity (d'). Statistical analyses compared pre- and post-stimulation performance across modalities. ResultsE-taVNS significantly enhanced working memory performance through an increase in correct rejection rate and sensitivity (measured by d), alongside a reduction in error of false alarm. U-taVNS showed a similar directional trend across performance measures, although these effects did not reach statistical significance. Baseline anxiety levels significantly predicted individual responsiveness to taVNS. In terms of tolerability, a higher proportion of participants receiving E-taVNS reported skin irritation compared to those receiving U-taVNS. ConclusionsE-taVNS can acutely enhance working memory performance, while U-taVNS may offer a comparable, better tolerated alternative. Our findings highlight the potential of taVNS to support memory function, while showing the importance of further research to clarify modality-specific effects and optimize stimulation parameters.

neuroscience↗

bioRxiv2026

Structural Basis for C8 methylation of 23S ribosomal RNA by Cfr

Cfr methylates C8 of adenosine 2503 (A2503) in 23S ribosomal RNA (rRNA) and will also methylate C2 of A2503 after methylating C8. C8 methylation confers resistance to more than five classes of clinically used antibiotics, highlighting it as a worrisome mechanism of antibiotic resistance. Here, we report the structure of Cfr, determined by cryogenic electron microscopy (Cryo-EM). Despite its small size ([~]36 kDa), we exploit a transient protein-RNA crosslink that forms during catalysis, which requires Cys105 to resolve. Using a Cfr Cys105Ala variant and an 87-nucleotide strand of rRNA, we isolate the crosslinked species and determine its structure to 3.0 [A] resolution. Notably, the 87-mer rRNA adopts an L-shaped conformation characteristic of tRNAs, rather than the conformation it assumes in the ribosome. One Sentence SummaryCryo-EM structure of Cfr, a radical S-adenosylmethionine methylase that confers antibiotic resistance

biochemistry↗

bioRxiv2026

Comparing Electrical and Ultrasound Transcutaneous Vagus Nerve Stimulation (taVNS) on Associative Memory

Associative memory, the ability to bind and retrieve relationships between unrelated elements, is a cornerstone of human cognition and a primary target for neurorehabilitation. Vagus nerve stimulation (VNS) has emerged as a promising method to modulate the locus coeruleus-norepinephrine (LC-NE) system and hippocampal-prefrontal circuits essential for memory. However, the comparative efficacy of non-invasive modalities such as electrical (E-taVNS) and the emerging field of ultrasound (U-taVNS) remains poorly understood in the context of active recall. In this study, participants performed a crossmodal video-word associative memory task before and after receiving either E-taVNS or U-taVNS in active and sham conditions. We investigated whether these modalities enhance cued recall accuracy and retrieval reaction time. Our results revealed that neither E-taVNS nor U-taVNS significantly improved recall accuracy. However, E-taVNS significantly accelerated response times specifically for correctly recalled items. These findings suggest that while taVNS may not increase the likelihood of recalling associative memories, electrical stimulation may enhances the efficiency in which we do so. These findings suggest that electrical taVNS is a viable tool for facilitating memory search processes, though further research is required to optimize ultrasound parameters and validate mechanistic pathways through physiological monitoring.

neuroscience↗

bioRxiv2026

Ultrasound transcutaneous auricular vagus nerve stimulation enhances semantic processing

Efficient access to conceptual knowledge is central to everyday cognition, yet the effects of non-invasive neuromodulation on semantic retrieval remain insufficiently characterised. Transcutaneous auricular vagus nerve stimulation (taVNS) offers a mechanistically distinct route to modulate distributed cognitive systems via ascending vagal afferent pathways, but its effects on semantic processing has not been explored. Here, we tested whether ultrasound-based taVNS delivered to the right cymba conchae can acutely enhance semantic retrieval efficiency in healthy adults. Twenty-seven participants completed a single blind, sham-controlled study comprising two counterbalanced sessions (active vs. sham) in a within-subject design, separated by at least five days. In each session, participants performed a semantic association task and a number-judgement control task before and immediately after 30 minutes of stimulation. Active taVNS produced selective post-stimulation improvements in semantic retrieval performance relative to sham and baseline, while performance on the control task remained unchanged. Adverse effects were minimal and did not differ between active and sham conditions. These findings provide causal evidence that ultrasound-based taVNS can acutely improve the efficiency of semantic processing, expanding the cognitive profile of taVNS beyond predominantly episodic and emotional memory domains and supporting its feasibility as a scalable approach for memory enhancement.

neuroscience↗

bioRxiv2026

Systemic and Local Delivery of siRNA to the CNS and Periphery via Anti-IGF1R Antibody Conjugation

siRNA delivery platforms capable of accessing both central and peripheral tissues are critically needed to expand the therapeutic potential of oligonucleotides. To address this, we developed a novel siRNA-antibody conjugate by attaching an Hprt-targeting siRNA to an engineered antibody shuttle. This shuttle targets the insulin-like growth factor 1 receptor (IGF1R) using a fused antibody fragment (Clone F) and utilizes an antibody backbone with no tissue-relevant binding in this study. The resulting conjugate, designated Clone F-Hprt, demonstrated robust in vivo knockdown across multiple tissues. Clone F-Hprt demonstrated enhanced penetration into central nervous system (CNS) tissues compared to unconjugated siRNA following intracerebroventricular (ICV) and intravenous (IV) administration. In peripheral tissues, Clone F-Hprt achieved widespread knockdown in muscle, heart, and lung, consistent with IGF1R expression. The conjugate was well tolerated across all routes, including with repeated dosing. Although several receptor-mediated approaches for CNS delivery are progressing to the clinic (e.g., targeting the transferrin receptor), clinical validation remains to be demonstrated. Our findings highlight IGF1R as an alternative receptor capable of supporting delivery across both central and peripheral tissues, offering a complementary strategy for expanding the therapeutic landscape of oligonucleotide delivery.

neuroscience↗

bioRxiv2026

OSTM1 is a ubiquitin E3 ligase that suppresses B-cell malignancy by activating the cAMP/PKA/CREB pathway

Osteoclastogenesis-associated transmembrane protein 1 (OSTM1) is a glycosylated, membrane-integral protein that regulates lysosomal homeostasis, with loss-of-function mutations causing autosomal recessive osteopetrosis. Through a whole-genome CRISPR/Cas9 screen, we identified OSTM1 as a previously unrecognized tumor suppressor in B-cell malignancies. Consistent with this role, OSTM1 is frequently deleted or downregulated across a broad spectrum of human B-cell cancers. In mice, B-cell-specific monoallelic or biallelic ablation of Ostm1 cooperates with Cdkn2a loss to drive lymphomagenesis with near-complete penetrance. Mechanistically, we uncover a cytosolic, non-glycosylated fraction of OSTM1 that functions as a ubiquitin E3 ligase to promote proteasomal degradation of phosphodiesterase 3B (PDE3B). As PDE3B hydrolyzes cAMP and suppresses the tumor-protective PKA/CREB/CREBBP signaling axis, loss of OSTM1 leads to PDE3B stabilization, attenuation of cAMP signaling, and enhanced oncogenic transformation. Together, our findings establish OSTM1 as a critical suppressor of B-cell lymphomagenesis through regulation of the cAMP/PKA/CREB pathway.

cancer biology↗

bioRxiv2026

PersonaAI: An Interactive Agentic-AI Framework for Autonomous Hypothesis Generation and Validation in Aging

Elucidating the mechanisms of aging is impeded by its stochastic, multi-scale nature and cellular heterogeneity, challenges that are compounded by the overwhelming volume of biomedical literature and the complexity of genome-wide datasets. To overcome these barriers, we present PersonaAI, an interactive agentic-AI framework that acts as a digital co-scientist. By integrating literature-based reasoning with autonomous in silico validation, PersonaAI synthesizes over 560,000 aging-related publications via retrieval-augmented generation (RAG) to propose mechanistic hypotheses. These hypotheses are subsequently validated by autonomous agents utilizing single-cell RNA-seq data. Using a temporal cutoff strategy restricted to pre-2020 literature, we demonstrate that PersonaAI can generate hypotheses effectively validated by post-2021 discoveries, proving its capacity for inference beyond simple information retrieval. In application, the system identified senescent Cirbp+ hepatocytes as a liver-intrinsic aging program and uncovered a middle-aged, male-specific decline in adipose stem and progenitor cells, driven by vascular niche deterioration and disrupted VEGF-VEGFR signaling. These results establish PersonaAI as a scalable platform that augments human intuition with autonomous data-driven validation, providing a generalizable platform for accelerating discovery in aging biology.

bioinformatics↗

bioRxiv2026

A Direct DNA Extraction Workflow for Metabarcoding Fungal Bioaerosols from Adhesive Samplers

Monitoring fungal bioaerosols is essential for understanding their impacts on human health and crop productivity. However, current monitoring approaches rely on culture-dependent techniques, which underestimate the diversity of fungal exposures. Adhesive-coated films can effectively capture airborne particles but have been regarded as unsuitable for DNA downstream analysis, owing to the lack of standardised DNA extraction methods for the adhesive capture surface. Here, we present an optimised DNA extraction protocol for adhesive film, which we developed and validated to enable direct and culture-independent ITS2-based metabarcoding of fungal bioaerosols collected on a 96 well plate sealing film ( Sticky sampler). This validated approach was applied to field samples collected from four agricultural farms and from a domestic garden to evaluate seasonal and spatial changes in fungal bioaerosols. Our analysis uncovered pronounced site-specific contrasts linked to geography and land management, as well as clear seasonal shifts in garden fungal communities. This study demonstrates that adhesive samplers, combined with optimised DNA extraction, provide a practical and inexpensive tool for fungal bioaerosol research by resolving ecologically meaningful spatiotemporal dynamics of airborne fungi. This protocol opens new opportunities for highly scalable surveillance of environmental bioaerosols.

microbiology↗

bioRxiv2026

Siglec-7 and -15 recognize repeated clustered sulfo-sialo O-glycan motifs on select O-glycoproteins

Siglecs are key immunoreceptors in immune homeostasis and cancer immunosuppression. Diverse endogenous and exogenous sialoglycan ligands induce Siglec-mediated immunoregulation, but the nature of endogenous Siglec ligands and how seemingly ubiquitous sialoglycans achieve selectivity is unclear. Here, we employed a HEK293 cell-based array and glycoprotein reporters designed from human mucins and mucin-like proteins with O-glycans in natural clusters and repeat motifs to dissect specificities of Siglec-7 and -15. We first used precomplexed Siglec-Fc chimera to demonstrate that CHST1-mediated 6-O-sulfation markedly enhanced binding to select O-glycoproteins, namely MAdCAM1, CD43, and PSGL-1. Probing binding properties of Siglec-7 expressed exogenously on CHO cells or endogenously on human monocytes by reverse assays employing fluorophore-tagged O-glycoprotein reporters revealed specific binding to the sulfo-sialyl core1 O-glycoform of the same O-glycoproteins. Our study indicates that Siglec-7 on immune cells employs multivalent interactions with repeated motifs of clustered sulfo-sialyl O-glycans to drive selectivity in ligand interactions and Siglec-induced immunosuppression.

molecular biology↗

bioRxiv2026

Hyperactive BMP and Mechanosignaling Remodel Chromatin to Drive Aberrant Osteogenesis in FOP

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder in which a recurrent ACVR1 (R206H) mutation drives progressive heterotopic ossification (HO). While aberrant BMP hypersensitivity has been studied, how this mutation enforces a persistent pro-osteogenic state remains unclear. Here, we combined super-resolution stochastic optical reconstruction microscopy (STORM), transposase-accessible chromatin with sequencing (ATAC-Seq), and RNA sequencing (RNA-Seq) to investigate how Acvr1R206H remodels chromatin to promote osteogenic transcriptional programs. Mutant mouse embryonic fibroblasts (MEFs) exhibited globally decondensed chromatin and increased accessibility at developmental and osteogenic loci enriched for HOX, TEAD, and RUNX motifs. Integration of ATAC-Seq and RNA-Seq data identified transcriptional networks primed for osteochondrogenic gene expression, including ossification, extracellular matrix organization, and cell adhesion pathways, consistent with enhanced BMP-SMAD and mechanotransduction activity. Time-course experiments revealed heightened responses to BMP ligands in Acvr1R206H/+ MEFs compared to wild-type, highlighting ligand hypersensitivity. Importantly, pharmacological modulation showed that chromatin alterations were dynamic and reversible: activation of Rho/ROCK in wild-type cells reproduced the mutant chromatin state, while inhibition of Rho/ROCK or BMP-SMAD signaling restored condensation to wild-type levels in mutant cells. Together, these findings establish that Acvr1R206H enforces a pro-osteogenic chromatin landscape through convergent BMP-SMAD and Rho/ROCK signaling, predisposing progenitors to aberrant differentiation trajectories. Our study reframes FOP as a disorder of persistent, but reversible, chromatin states and identifies novel therapeutic opportunities to restore mesenchymal cell homeostasis and prevent pathological bone formation. Significance StatementFibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder in which a mutation in ACVR1/ALK2 drives progressive heterotopic ossification. However, how this mutation enforces a persistent pro-osteogenic state is unclear. Here, we show that the Acvr1R206H mutation remodels chromatin architecture and accessibility through hyperactive BMP-SMAD and Rho/ROCK signaling, activating transcription factor networks that drive osteochondrogenic gene expression. These chromatin changes are dynamic and reversible with targeted pathway inhibition, revealing therapeutic potential to restore mesenchymal cell plasticity and prevent pathological bone formation.

bioengineering↗

bioRxiv2025

Predicting and Designing Red Fluorescent Protein Variants Using Sequence-to-Function Machine Learning Models

Fluorescent proteins (FPs) are widely used reporters for visualizing cellular structures and processes. Traditional wet-lab strategies for FP engineering (rational design and directed evolution) have enabled substantial improvements in photophysical performance but are limited by their requirement for deep expert knowledge or labor-intensive screening. AI-driven approaches have recently gained traction for engineering variants of green FPs, yet applications to red fluorescent proteins (RFPs) remain scarce. Here, we applied machine learning models to an RFP sequence-function dataset and trained these models to predict functional single-mutation variants of the state-of-the-art RFP mScarlet-I3. Guided by model predictions, we identified variants exhibiting red-shifted emission peaks, large Stokes shifts, or brightness comparable to the parental protein. Our findings show that even lightweight, data-efficient models can extract actionable design principles for improving RFPs. This work demonstrates the feasibility of AI-guided design for RFPs and provides a reliable benchmark for future development of more powerful AI-driven strategies for FP engineering.

molecular biology↗

bioRxiv2025

Interpretable Thermodynamic Score-based Classification of Relaxation Excursions

Classifying cellular states from high-dimensional molecular and genomic measurements requires methods that provide not only accurate predictions but also calibrated uncertainty and interpretability. Current nonlinear classifiers offer accuracy but often lack uncertainty quantification and mechanistic insights into the features that matter most. We introduce Keeping SCORE, a framework that transforms conditional diffusion models into probabilistic engines for classification and regression by computing exact likelihoods along stochastic noising trajectories. We first benchmark Keeping SCORE on image recognition tasks (handwritten digits, natural photos). We then apply Keeping SCORE to single-cell transcriptomics across a 22-million-cell atlas, classifying 164 cell types with accuracy matching or exceeding state-of-the-art methods, while uniquely providing posterior probability estimates and prediction confidence. For genetic perturbation mapping across 100 CRISPRi conditions in a multi-study Perturb-seq dataset, our approach again matches or surpasses discriminative baselines, with feature-level attributions identifying which genomic features drive each decision. Applied to large-scale protein sequence data, our framework accurately regresses mutational stability effects, attributing them quantitatively to positions along the input sequence. Keeping SCORE requires no retraining or architectural changes to existing diffusion models, providing portable, interpretable, and uncertainty-aware predictions for biological discovery.

bioinformatics↗

bioRxiv2025

Transcutaneous auricular vagus nerve stimulation enhances emotional bias towards happiness in healthy young adults: A comparative study of electrical and ultrasound stimulation

Transcutaneous auricular vagus nerve stimulation (taVNS) is an emerging neuromodulation technique demonstrating promise in emotional regulation. This study investigated the acute effects of both electrical (E-taVNS) and ultrasound (U-taVNS) modalities on emotional bias using a facial emotion categorization task in healthy young adults. Fifty-nine participants underwent a single-blind, sham-controlled, within-subject design, with emotional bias assessed at pre-, during-, and post-stimulation phases. Our findings revealed that both E-taVNS and U-taVNS significantly enhanced emotional bias scores, shifting the perception of neutral and ambiguous faces towards positive interpretations and reducing negative emotional bias. No significant differences in efficacy were observed between the two stimulation modalities. Furthermore, individual differences in interoceptive awareness were found to be associated with the observed taVNS effects. These results suggest that both electrical and ultrasound taVNS can acutely modulate emotional regulation, highlighting the potential of taVNS as a non-invasive, well-tolerated alternative for interventions targeting emotional bias and mood disorders. HighlightsO_LItaVNS reduces negative emotional bias toward ambiguous stimuli. C_LIO_LIBoth electrical and ultrasound taVNS shifted emotional bias toward more positive interpretations of ambiguous facial expressions. C_LIO_LIUltrasound taVNS (U-taVNS) demonstrated comparable efficacy to electrical taVNS (E-taVNS). C_LIO_LIThe subscale of the Multidimensional Assessment of Interoceptive Awareness predicted individual variability in taVNS-induced emotional bias change. C_LI

neuroscience↗

bioRxiv2025

Deoxyguanosine Kinase Deficiency Couples Purine Metabolism to Innate Immune Activation and Lipid Accumulation in Hepatocytes

Mitochondrial DNA depletion syndromes (MDS) encompass a heterogeneous set of metabolic disorders caused by defects in enzymes responsible for maintaining mitochondrial nucleotide pools and genome integrity. Among these, deoxyguanosine kinase (DGUOK) acts within the mitochondrial purine-salvage pathway and loss-of-function mutations give rise to DGUOK deficiency, a severe hepatocerebral form of MDS marked by liver failure, neurodevelopmental impairment, and systemic metabolic inflammation. Although the clinical manifestations of DGUOK deficiency have been primarily ascribed to defective mitochondrial DNA (mtDNA) replication, some patients exhibit hepatic steatosis and inflammation despite preserved mtDNA content, suggesting that DGUOK deficiency may deregulate additional metabolic and immune pathways. Here we show that DGUOK depletion reprograms hepatocellular metabolism and innate immune signaling through a purine-dependent mechanism operating independently of mtDNA depletion. In human hepatocellular carcinoma hepatocytes (HEPG2) subjected to siRNA-mediated DGUOK silencing, mitochondrial architecture and respiration remained intact but cells exhibited pronounced lipid droplet accumulation and a robust cell-intrinsic innate immune type I interferon response. Bulk RNA sequencing revealed widespread transcriptional reprogramming, including upregulation of human endogenous retroviruses (HERVs) and interferon-stimulated genes (ISGs), suppression of lipid metabolic pathways, and changes in purine, methionine, and methylation-associated gene networks. Perturbing purine homeostasis through deoxyadenosine (dAdo) supplementation in wild-type cells phenocopied DGUOK disruption, causing global DNA hypomethylation and activation of viral mimicry pathways. Together, these findings identify DGUOK as a central regulator of the purine-regulated lipid-immune axis in hepatocytes, demonstrating that mitochondrial nucleotide salvage preserves hepatic immune and metabolic homeostasis beyond its canonical role in mtDNA synthesis. By linking purine imbalances to steatosis and type I interferon activation, this study establishes a mechanistic framework for immunometabolic pathology in DGUOK deficiency.

immunology↗

bioRxiv2025

Engineering a Biomimetic Multiphasic Suture Anchor System for Enhanced Rotator Cuff Enthesis Regeneration

Conventional suture anchor methods in rotator cuff repair often fail to replicate the native tendon-to-bone interface, leading to re-tears due to stress concentrations and poor biological integration at anchor sites. To address these challenges, we engineered a biomimetic multiphasic scaffold system (BMS) that integrates with standard suture anchors and deliver spatially organized structural and biological cues to enhance enthesis regeneration. The BMS comprises three distinct phases: aligned nanofibrous decellularized bovine Achilles tendon extracellular matrix (dECM) with stiff methacrylated hyaluronic acid (MeHA) for tendon regeneration; nonaligned nanofibrous dECM with soft MeHA for fibrocartilage formation; and a porous, citrate-based composite scaffold with bioactive glass for bone integration. In vitro, the BMS facilitated zone-specific tenogenic, fibrochondrogenic, and chondrogenic differentiation. Further, in vivo, it promoted successful integrative healing, forming distinct tendon, fibrocartilage, and bone regions at the repair site. This advanced multiphasic scaffold replicates native tissue properties, offering a promising strategy to improve rotator cuff repair. Its integration with conventional suture anchors provides an innovative design that enhances mechanical fixation and guides enthesis healing to reduce re-tear rates. Broadly, this platform offers a versatile solution for biointegrative repair strategies across complex soft-to-hard tissue interfaces.

bioengineering↗

bioRxiv2025

Non-Carbohydrate Inhibitors of Sialic Acid-binding Immunomodulatory-type Lectin-7 (Siglec-7) Discovered from Genetically Encoded Bicyclic Peptide Libraries

Glycan-binding proteins (GBP) are among the most difficult to drug targets. This deficiency delays clinical progress for therapeutically important GBPs. We employed bicyclic genetically encoded libraries (BiGELs), produced by chemical modification of phage-displayed libraries of peptides with two-fold symmetric linchpins, to discover inhibitors of therapeutically relevant Siglec-7:GD3 interactions. Next-generation sequencing (NGS) analysis of panning of BiGEL against Siglec-7 yielded 815 candidates from which 23 hits yielded KD = 1-100 {micro}M as determined by surface plasmon resonance (SPR). Competitive enzyme-linked immunosorbent assays (ELISA) identified a subset of leads that disrupted the Siglec-7:GD3 interaction with IC50= 3-300 {micro}M. Machine learning models trained on NGS datasets identified additional inhibitors with equivalent potency. Alanine scans of 8c (SWCRPATVNC, IC50 = 3.8 {micro}M) and 12c (SFCHYPTHVC, IC50= 11 {micro}M), identified key residues as crucial for activity. Ring reshaping studies of compound 8c highlighted the critical role of bicyclic topology produced by analogue 46e (SAAAAAWCRPATVNC, IC50= 9.5 {micro}M). Multivalent display of the lead bicycles alongside [~]100 glycans in Liquid glycan Array (LiGA), made it possible to compare the binding of bicycles and glycans to Siglec-7 expressed on CHO, Jurkat, and Raji cells. LiGA assays confirmed binding of the bicycles to Siglec-7 but revealed considerable non-specific interactions with receptor-negative cells. Saturation transfer difference nuclear magnetic resonance (STD-NMR) revealed 46e binds to Siglec-7 at a site distinct from the V-Ig domain, suggesting it might inhibit binding of glycans to the glycan-binding site of Siglec-7 via an allosteric site. Together these results demonstrate that BiGEL enables the discovery of bicyclic peptides for undruggable Siglec targets but highlights future challenges in molecular discoveries that aim to identify small, non-carbohydrate inhibitors of GBPs.

biochemistry↗