Biology subjects

Edwards, A.

Publications and source records attributed to Edwards, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Semisynthesis of Oxalyl-Coenzyme A for Enzymatic Assays

Oxalyl-coenzyme A (oxalyl-CoA) is a key intermediate in oxalate metabolism in plants, fungi and oxalate-degrading bacteria, but its limited availability has restricted biochemical investigations of oxalyl-CoA-dependent enzymes. Here, we describe a practical semisynthetic procedure for the preparation of oxalyl-CoA based on rapid oxalyl transfer from S-oxalyl p-thiocresol to coenzyme A. The reaction was monitored directly by 1H NMR spectroscopy, allowing optimisation of pD and reaction conditions. Following removal of thiocresol and purification by reversed-phase HPLC, oxalyl-CoA was obtained in 39% yield as determined by quantitative 1H NMR. The product was characterised by high-resolution electrospray mass spectrometry and comprehensive 1H, 13C and 31P NMR spectroscopy, confirming its structure unequivocally. During the study, the limited stability of oxalyl-CoA in aqueous solution was documented, leading to recommendations for its purification and storage. The semisynthetic protocol provides a convenient source of analytically pure oxalyl-CoA suitable for biochemical assays and supplies reference spectroscopic data for its unambiguous identification. The biological utility of the semisynthetic oxalyl-CoA was demonstrated by its application as an acyl donor substrate in assays of PnBAHD15, enabling quantitative kinetic characterisation of the enzyme and illustrating its suitability for biochemical studies of oxalyl-CoA-dependent enzymes.

biochemistry↗

bioRxiv2026

Whole genome sequencing and variant discovery in 344 global grasspea (Lathyrus sativus L.) lines

The rapid expansion of genomic data resources for major crops is opening new options for crop improvement, while resources for most underutilised crops lag behind, risking a widening gap in crop improvement. One of these underutilised crops is grasspea (Lathyrus sativus), an ancient crop with modern cultivation centred on South Asia and Ethiopia. We conducted whole genome shotgun sequencing on a global collection of 344 grasspea lines, producing over 152 billion reads. Following variant discovery and filtering we created a single nucleotide polymorphism (SNP) marker set of over 1.5 million SNPs. This is a resource of major significance for this crop which can help unlock its breeding potential through marker development and the identification of genes controlling agronomically important traits.

genomics↗

bioRxiv2026

The pattern of mammal change in Garig Gunak Barlu (Cobourg) National Park suggests a fire-mediated pathway to decline and one for recovery

ContextFire is a key driver of wildlife pattern in northern Australia, and in recent decades there has been concerted efforts to return burning practices from unmanaged wildfire to approaches more in tune with traditional approaches to fire management undertaken by First Nations people. AimsWe examined fire pattern (early and late dry season) and distance from coast in relation to mammal pattern on a Cobourg peninsula, as there was some evidence in variable patterns in fire history and mammal persistence in this landscape. MethodsIn this study we examined data collected from Garig Gunak Barlu (Cobourg) National Park, Northern Territory, between 2004 and 2017. We investigated the relationship between all native mammal abundance and species richness, and abundance for eight species, with the three environmental variables (distance from coast, preceding early dry and late dry season burning frequency), using an information theoretic model selection and averaging framework. Key resultsThere is a distinctive change in the fire and mammal pattern with respect to distance from the coast; that is, early and late dry season burning increasing moving inland from the coast, and a concomitant decrease or persistence in some key species on this fire gradient. ConclusionsOur results suggest that fire management in northern Australia still needs targeted research linked to active management to more effectively prevent declines of mammal populations. ImplicationsThe location and shape of Garig Gunak Barlu (Cobourg) National Park provide an excellent opportunity to undertake some natural experiments in strategic burning to investigate improved approaches to conservation in fire-prone landscapes.

ecology↗

bioRxiv2026

Monitoring data indicates some annual change in the mammal fauna at Nitmiluk National Park between 2005 and 2018 but a reduction in effort confounds any interpretation

ContextThe decline, extirpation and extinction of Australias mammal fauna is without peer. In recent decades long term monitoring has revealed tragic patterns in iconic locations such as Kakadu National Park. AimsPast studies have described universal patterns across multiple national parks in the Northern Territory, linking the declines to fire management; however, much of the data in Nitmiluk National Park do not cover the period that encompasses this decline. We examine this data separately to look for patterns with respect to fire history. MethodsWe examined standardised mammal survey data collected in Nitmiluk from 2005 to 2018, to look for correlates with year of survey, time since last burnt, and the frequency of early season and late season burns. Key resultsDespite subtle difference in methods over the years, we found there were some annual changes in the mammals, but little discernible pattern of change correlated to fire regime (which itself indicated an improvement in seasonality, and the extent of burning overall over this period). Instead, there was a distinct reduction in the survey effort. ConclusionsOur study provides further support for the pitfalls of ecological monitoring losing focus, being decoupled from management actions, and allowing ill-conceived changes in design. We recommend that future monitoring at Nitmiluk should focus on key management questions (i.e., fire), best methods for target taxa (i.e. camera traps), more regular and flexible sampling linked to a conceptual model, and integration and co-design with the local land managers, rangers and Traditional Owners. Short SummaryRe-examination of the mammal data collected in monitoring sites at Nitmiluk National Park between 2005 to 2018 indicated little pattern with fire regime over time, and instead indicated that a reduction in effort, had reduced the ability to find pattern With sparse and declining mammal population in northern Australia, future monitoring needs to be focused on key management questions.

ecology↗

bioRxiv2026

Tracks, Maps and Gaps: A Testable Research Definition for Critical Chondrichthyan Areas, European Atlantic Insights

Designing effective spatial management for chondrichthyans (sharks, skates, rays and chimaeras) requires incorporating critical areas, sites essential for population maintenance, such as reproductive and feeding areas. Yet most area-based measures have been developed without consideration of chondrichthyan habitat use. The Important Shark and Ray Area (ISRA) initiative has been pivotal in designating priority areas through a rigorous, consultative process. To complement this, our study offers researchers a testable definition for generating robust evidence to strengthen future critical area delineations and related management decisions. We define critical areas using three criteria: 1) relative frequency of use, (2) extended within-year occupancy and (3) repeated use across years. This framework enables objective comparison among candidate sites and is generalisable across different critical area types. The definition builds upon established early-life-stage habitat concepts and applies these to broader life-history functions. The utility of this framework is then demonstrated through a systematic review of contemporary peer-reviewed literature of critical chondrichthyan areas in the European Atlantic. The review highlighted 62 critical areas with Strong evidence and 41 areas of Moderate strength evidence, which informed the European Atlantic ISRA selection process. Research effort was concentrated in inshore areas, particularly around the British Isles and Portugal, with biases towards large, threatened and commercially valuable species, whilst chimaeras were notably underrepresented. Early-life stage areas were most frequently identified, whereas resting areas were rarely documented. Evidence patterns and biases are examined in the context of evolving critical area concepts to advance their development and improve the quality and breadth of future research. By outlining a testable definition, identifying key knowledge gaps, and proposing research and reporting guidelines, this work enhances the consistency, comparability, and spatial coverage of future chondrichthyan habitat research to support its application to conservation planning.

ecology↗

bioRxiv2026

Pharmacological METTL3 inhibition attenuates HIV-1 latency reversal in CD4+ T cells

N6-methyladenosine (m6A) is a major epitranscriptomic modification that regulates RNA metabolism and affects the replication and latency reversal of human immunodeficiency virus type 1 (HIV-1) in cells. Methyltransferase-like 3 (METTL3) is the principal catalytic enzyme responsible for m6A deposition, and its pharmacological inhibition has emerged as a potential therapeutic strategy for cancer and viral infections. However, the relative potency of METTL3 inhibitors in reducing m6A levels and their effects on HIV-1 latency reversal remain undefined. Here, we compared three commercially available METTL3 inhibitors (STM2457, STM3006, and STC-15) to evaluate their ability to reduce RNA m6A levels, suppress HIV-1 latency reversal, and affect cell viability in latently infected J-Lat cells and primary CD4+ T cells. In J-Lat cells, STM3006 and STC-15 were more potent than STM2457 in reducing RNA m6A levels at 24 and 48 hours post-treatment, as reflected by lower half-maximal inhibitory concentrations (IC50). However, STM3006 and STC-15 exhibited significant cytotoxicity at concentrations above 2 {micro}M at 48 hours post-treatment, whereas STM2457 displayed minimal toxicity across all tested doses. In primary CD4+ T cells from three healthy donors, all three inhibitors reduced RNA m6A levels but induced greater cytotoxicity compared with J-Lat cells, with comparable effects at optimized concentrations. Notably, reduced RNA m6A levels correlated with diminished HIV-1 latency reversal in both J-Lat cells and a primary central memory CD4+ T cell model. Together, these findings demonstrate differential potency and cytotoxicity among METTL3 inhibitors and support a critical role for m6A RNA modification in regulating HIV-1 latency reversal.

microbiology↗

bioRxiv2026

Myc inhibition triggers GM-CSF-driven regression of pancreatic tumours

The complex aetiology of pancreatic ductal adenocarcinoma (PDAC), together with its desmoplastic and hypoxic microenvironment, hinders effective treatment and contributes to its rapid lethality1. Using a reversibly switchable genetic mouse model that closely recapitulates human PDAC phenotype progression, we previously showed that selective deactivation of oncogenic Myc in PDAC epithelial cells triggers rapid disassembly of advanced PDACs2, both tumor cells and their associated immune microenvironment. Here, leveraging spatiotemporal genomics and multiplex immune profiling we determine the mechanism underpinning this regression programme and identify granulocyte-macrophage colony-stimulating factor (GM-CSF) as its key instructive cytokine, transiently released by pancreatic ductal epithelial cells rapidly following Myc inactivation, that initiates tumour regression. We further demonstrate that type 1 conventional dendritic cells (cDC1s) act as critical effectors in Myc-OFF GM-CSF-driven tumour regression. Both antibody-mediated blockade of GM-CSF and genetic ablation of cDC1s via Batf3 knockout bone marrow transplantation prevent PDAC regression. Conversely, transient systemic administration of recombinant GM-CSF to PDAC-bearing mice promotes rapid cDC1 infiltration and induces marked tumour regression in the sustained presence of Myc activity. Together, these findings reveal that PDAC regression induced by Myc de-activation is mediated by a latent morphogenic programme that is initiated by transient release of GM-CSF from tumour cells. This regression programme is rapid, tissue-specific, involves both neoplastic cells and attendant desmoplastic stroma, is reliant on innate immunity and provides a novel framework for therapeutic intervention in PDAC.

cancer biology↗

bioRxiv2025

Nucleic acid strand length governs mitochondrial reprogramming and mtROS-associated antiviral responses following TLR3 engagement

Mitochondria are important rheostats that regulate innate sensing processes by producing energy, biosynthetic precursors, and bioactive molecules that affect cellular signaling. When viral nucleic acids engage endosomal and cytosolic pattern recognition receptors (PRR), antiviral immune responses are supported by mitochondrial remodeling but the role of mitochondria in fine tuning ligand-specific responses remains incompletely understood. For example, endosomal TLR3 can detect various lengths of dsRNA (0.4-8 kb) ranging from viral segmented genomes or endogenous nucleic acids have been shown to induce distinct cytokine profiles. However, it is unclear if these differences are associated with differential mitochondrial remodeling. Here, we report that TLR3 engagement with both high (HMW; 1.5-8 kb) or low molecular weight (LMW; 0.2-1 kb) Polyinosinic:polycytidylic acid (Poly(I:C)) is associated with reduced but sustained oxidative phosphorylation (OXPHOS) activity and increased mitochondrial reactive oxygen species (mtROS) production/accumulation to support antiviral responses in bone marrow-derived macrophages (BMDM). They differed in the amount of mtROS production, their spare respiratory capacity (SRC) and their mitochondrial membrane potential (MMP). Interestingly, while uncoupling protein 2 (UCP2) was found required for antiviral cytokine production, it did not contribute to ligand specific responses. Dynamic modulation of complex I of the electron transport chain (ETC), however resulted in the differential accumulation of mtROS (HMW>LMW). Further, selectively targeting the mtROS derived from Complex I leads to augmented type I IFN production. Overall, these findings highlight that targeting specific sources of mtROS without affecting electron flow may be a potential avenue for specific augmentation of antiviral responses during viral infections.

immunology↗

bioRxiv2025

Identification and Characterization of a Small Molecule Ligand for the Huntingtin-HAP40 Complex

Huntingtons disease (HD) is caused by a CAG repeat expansion mutation, giving rise to a polyglutamine expansion in the huntingtin (HTT). However, the explicit molecular functions of HTT and opportunities for direct pharmacological modulation remain incompletely understood. Here, we report the discovery of a small molecule ligand for the full-length HTT protein in complex with its partner, HAP40. Using affinity selection mass spectrometry (AS-MS), we identified a stereoselective binder, whose binding was characterized by surface plasmon resonance, hydrogen-deuterium exchange mass spectrometry, and cryo-electron microscopy at 2.3 [A] resolution. The ligand binds HTT-HAP40 in vitro with single-digit micromolar affinity and one-to-one stoichiometry at a druggable interface previously predicted computationally. In silico studies predicted and experimental analyses confirmed the (R)-enantiomer as the eutomer and initial structure activity relationship was established experimentally. This work details a structurally-validated chemical scaffold and highlights a ligandable pocket which could enable development of chemical probes for probing HTT biology, as well as therapeutics such as degraders and imaging agents for HD.

pharmacology and toxicology↗

bioRxiv2025

Contribution of Mesenchymal-like and Epithelial Cellular Subsets to Chemotherapy Resistance in Triple-Negative Breast Cancer

Background/ObjectivesTriple-negative breast cancer (TNBC) tumors are typically heterogeneous, predominantly epithelial tissues with discrete patches of mesenchymal-like TNBC cells, that differ in their invasiveness, proliferation potential and response to treatment. However, the contribution of mesenchymal-like and epithelial TNBC cells in the persistence of chemotherapy resistant disease remains poorly understood. MethodsMesenchymal-like and epithelial TNBC cell types were detected by multiplex fluorescent immunohistochemistry using antibodies against vimentin, Ki67, and Annexin A6 (AnxA6). Chemotherapy drug resistant mesenchymal-like and epithelial TNBC cell populations were established by pulse exposure and stepwise dose escalation and validated by 3D cultures and unbiased antibody arrays. ResultsAnalysis of the response of stage IV TNBC tumors treated with six common chemotherapy regimens resulted in 36% complete response and 64% partial response with residual tumor sizes ranging from 0.5 to 37.0 mm. Treatment of TNBC cells with chemotherapy agents led to distinct resistance signatures including downregulation of survivin and upregulation of M-CSF and CXCL8/IL-8 in model mesenchymal-like, and upregulation of CCL2/MCP-1, CTSS and DKK-1 in model epithelial TNBC cells. The inhibitory phosphorylation of GSK-3{beta} (p-S9) increased in paclitaxel resistant epithelial cells but decreased in resistant mesenchymal-like TNBC cells. Finally, chemotherapy resistance also activated p90 ribosomal S6 kinases (RSK1/2) in both cell types, while activation of mitogen- and stress-activated kinases (MSK1/2) was only observed in chemotherapy resistant epithelial TNBC cells. ConclusionsThese data reveal that chemotherapy resistance of epithelial and mesenchymal-like TNBC cellular subsets upregulated distinct profiles of proinflammatory and immune cell chemotactic cytokines and modulated the activities of GSK-3{beta}, p90 RSK1/2 and the related MSK1/2. Targeting these factors and/or the associated signaling pathways may help overcome chemotherapy resistance in TNBC. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/685128v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@1c5481dorg.highwire.dtl.DTLVardef@101a766org.highwire.dtl.DTLVardef@1954523org.highwire.dtl.DTLVardef@ee2ddb_HPS_FORMAT_FIGEXP M_FIG C_FIG TNBC tumors are typically heterogeneous, predominantly epithelial tissues with discrete patches of mesenchymal-like TNBC cells, that differ in their invasiveness, proliferation potential and response to treatment. This study reveals that Chemotherapy resistance in epithelial and mesenchymal-like TNBC cells is maintained by distinct signatures of proinflammatory and immune cell chemotactic cytokines, and activities of GSK-3{beta} and p90 ribosomal S6 kinases.

cancer biology↗

bioRxiv2025

Life in the fast lane: Functional consequences of male-female dynamic differences in the renal autoregulation of flow

Tubuloglomerular feedback (TGF) is essential for the renal auto-regulation of flow. TGF is known to induce spontaneous oscillations in single-nephron tubular fluid flow in male kidneys. However, male-female differences in this dynamic behavior have not been studied. Leveraging intravital two-photon microscopy, resting-state magnetic resonance imaging, ultrasound-based and transdermal recordings, we found TGF-mediated oscillations across spatial scales in the rodent kidney, from single-nephron to whole-organ levels, and that male kidneys exhibited higher operating frequencies than females. To understand the mechanisms involved, we developed a dynamical systems model of TGF that agrees with physiological observations. Analysis of the mathematical model indicated that higher reabsorption rate and fluid flow efficiency in male proximal tubules not only result in higher frequencies, but also render male nephrons more susceptible to lose TGF-mediated oscillations. Furosemide abolished TGF-mediated oscillations in male kidneys and upregulated tubular injury marker KIM-1, suggesting that the propensity to lose TGF-mediated oscillations underlies the heightened risk for injury in males. Our analysis also indicated that SGLT-2 inhibition confers renoprotection by preserving TGF-mediated oscillations in hyperglycemia. Combining quantitative imaging and mathematical modeling, this study provides mechanistic insights into the transition from normal physiology to pathophysiology in the kidney.

physiology↗

bioRxiv2025

Novel monoclonal antibodies against the C-terminal HEAT domain of Huntingtin

BACKGROUNDReliable detection of huntingtin (HTT) is essential for understanding Huntingtons disease (HD) biology and evaluating therapeutic strategies. However, high-quality monoclonal antibodies (mAbs) against the HTT C-terminal domain remain limited. OBJECTIVEWe sought to generate and validate novel monoclonal antibodies targeting the HTT C-terminal HEAT-containing domain to better detect HTT independently of potential effects of polyglutamine length that can impact some N-terminally targeted antibodies. METHODSWe immunized mice with a highly purified, well-characterized recombinant protein corresponding to the HTT C-terminal domain. We generated monoclonal antibody-producing hybridoma cell lines and characterized the antibodies using parental and HTT-knockout cell lines in common immuno-applications. RESULTSThree novel, independent hybridoma lines producing anti-HTT monoclonal antibodies were derived. Using CRISPR-edited HTT knockout cell lines we identified one clone, anti-HTT [2F8], that was specific and effective across Western blot, immunofluorescence, and ELISA assays. All antibodies bound full-length HTT irrespective of HAP40 interaction or polyQ length and showed no cross-reactivity to the N-terminal HEAT domain. CONCLUSIONSThese C-terminal HTT mAbs are thus valuable additional tools for studying endogenous HTT function in both normal and disease contexts.

neuroscience↗

bioRxiv2025

Structure and iron-transporting mechanism of brain organic cation transporter 1

Brain organic cation transporter 1 (BOCT1), also known as the solute carrier family 22 member 17 (SLC22A17) or the receptor for lipocalin-2 (LCN2), plays critical roles in health and disease. Its deficiency in mice results in early postnatal mortality and severe neurogenesis impairments. Despite its importance in physiology and pathophysiology, BOCT1s structure and transport mechanism remain elusive. Here, we integrate cryo-electron microscopy (cryo-EM), functional assays, biochemical experiments, and molecular dynamics simulations to elucidate the structure, substrate recognition, and transport mechanism of mouse BOCT1 (mBOCT1). The high-resolution cryo-EM structure reveals a distinctive N-terminal domain with a unique folding pattern dominated by a transmembrane loop atop TM6 (TML6), diverging from both known structures of SLC22 transporters and AlphaFold predictions. Notably, mBOCT1 functions as a high-capacity, low-affinity iron transporter independent of LCN2 binding. Iron transport is facilitated by a substrate gating mechanism involving TML6. These findings establish a structural basis for BOCT1s role as an independent iron transporter, enhancing our understanding of the transport mechanisms within major facilitator superfamily (MFS) transporters and providing new insights into brain iron homeostasis.

biophysics↗

bioRxiv2025

Submicron-Sampling of Living Cells by Macrophages

SUMMARY PARAGRAPHAn effective immune system must sample and appreciate healthy-self identity to prevent autoimmunity and to contrast to pathogenic insults1-3. Self-proteins are presented to T cells in the thymus during immune cell development2,3, and must be presented throughout the body to both maintain regulatory T cell populations4-6 and provide a tonic signal to maintain conventional T cells over time7-9. The ready observations of continuous apoptosis in some organs together with the ingestion of that material by myeloid populations has led to a conventional understanding of ongoing cell-death as a major source of self-antigens10, complemented in some situations by uptake of free-floating cell-derived vesicles. Here, we used a series of companion imaging and vesicular labeling technologies to reveal an alternate process undertaken by macrophages that results in non-destructive and direct sampling of living cells. The process requires cell-cell contact, does not require caspase activation, and takes place via a trogocytosis-like stretching of the target cell into the macrophage, leading to the generation of submicron-sized vesicles containing cytoplasm. Using a high-dimensional flow-based method for labeling vesicles ingested under this versus other conditions, we find that live-sampled material is distinctly processed, is poorly subject to fusion with lysosomes, and produces ensuing differential effects on the presentation of those to CD4 versus CD8 T cells. Disrupting this trafficking by redirecting antigen to the lysosome significantly reduced the associated macrophage-mediated priming of CD8 T cells. This demonstrates an important and substantial sampling of living cells by the immune system, with clear consequences for maintaining the border of immunity.

immunology↗

bioRxiv2025

Co-Activation Patterns in Neonates using High-Density Diffuse Optical Tomography: Insights into Early Dynamic Functional Connectivity

SignificanceDynamic functional connectivity (FC) in neonates is a growing area of interest due to the developmental significance of early functional networks. There are several emerging techniques to measure dynamic FC, adding new perspectives to well-studied static FC networks. Recent dynamic FC studies suggest that adult resting-state networks are driven by key moments of dynamic activity rather than sustained correlations. Co-activation pattern (CAP) analysis leverages this theory, clustering high-activity frames to identify recurring configurations of significant activity. High-density diffuse optical tomography (HD-DOT) is an infant-friendly modality that measures hemodynamic changes in the cortex and has been used to investigate static FC in term-aged infants. CAP analysis has not yet been applied to neonatal HD-DOT and may reveal temporal features of early functional networks that are not apparent from static methods. AimThis study applies CAP analysis to neonatal HD-DOT to characterize transient co-activation states and provide new insight into early functional brain networks beyond what is known from static FC methods alone. ApproachTask-free HD-DOT data were acquired from a cohort of sleeping term newborns at the Rosie Hospital, Cambridge UK (n = 44, postmenstrual age = 40+3 (range: 38+2-42+6) weeks). In each recording, the top 15% of seed-selected frames were clustered using the K-means algorithm for three regions of interest (ROIs: frontal, central, and parietal) to identify significant seed-associated patterns of co-activation or co-deactivation. These co-activation patterns (CAPs) were characterized for each infant using four metrics: consistency, fractional occupancy, dwell time, and transition likelihood. ResultsDistinct CAPs, reflecting the dynamic organization of neonatal cortical networks, were identified for frontal, central, and parietal regions. These CAPs showed high consistency scores, reflecting high intra-cluster spatial correlation and validating the efficacy of CAP analysis for newborn HD-DOT data. The CAP decomposition revealed significant patterns not observed in conventional static functional connectivity analyses. Several neonatal CAPs exhibited frontoparietal co-activation, potentially reflecting early default mode network activity, which is immature and modular for the first year after birth. ConclusionsThis work demonstrates the utility of CAP analysis with newborn HD-DOT and provides new insight into the dynamics of neonatal functional connectivity.

neuroscience↗

bioRxiv2025

The Endo-GeneScreen Platform Identifies Drug-Like Probes that Regulate Endogenous Protein Levels within Physiological Contexts

Traditional phenotypic drug discovery platforms have suffered from poor scalability and a lack of mechanistic understanding of newly discovered phenotypic probes. To address this, we created Endo-GeneScreen (EGS), a high-throughput enabled screening platform that identifies bioactive small molecules capable of regulating endogenous protein expression encoded by any preselected target gene within a biologically appropriate context. As a proof-of-concept, EGS successfully identified drug candidates that up-regulate endogenous expression of neuronal Syngap1, a gene that causes a neurodevelopmental disorder when haploinsufficient. For example, SR-1815, a previously unknown and undescribed kinase inhibitor, alleviated major cellular consequences of Syngap1 loss-of-function by restoring normal SynGAP protein levels and dampening neuronal hyperactivity within haploinsufficient neurons. Moreover, we demonstrate that EGS assays accelerate preclinical development of identified drug candidates and facilitate mode-of-action deconvolution studies. Thus, EGS identifies first-in-class bioactive small molecule probes that promote biological discovery and precision therapeutic development.

neuroscience↗

bioRxiv2025

Enantioselective Protein Affinity Selection Mass Spectrometry (EAS-MS)

We report an enantioselective protein affinity selection mass spectrometry screening approach (E-ASMS) that enables the detection of weak binders, informs on selectivity, and generates orthogonal confirmation of binding. After method development with control proteins, we screened 31 human proteins against a designed library of 8,210 chiral compounds. 16 binders to 12 targets, including many proteins predicted to be "challenging to ligand", were discovered and confirmed in orthogonal biophysical assays. 7 binders to 6 targets bound in an enantioselective manner, with KD values ranging from 3 to 20 {micro}M. Binders for four targets (DDB1, WDR91, WDR55, and HAT1) were selected for in-depth characterization using X-ray crystallography. In all four cases, the mechanism for enantioselectivity was readily explained. We conclude E-ASMS can be used to identify and characterize selective and weakly-binding ligands for novel protein targets with unprecedented throughput and sensitivity.

pharmacology and toxicology↗

bioRxiv2024

Structural basis of TMPRSS11D specificity and autocleavage activation

Transmembrane Protease, Serine-2 (TMPRSS2) and TMPRSS11D are human proteases that enable SARS-CoV-2 and Influenza A/B virus infections, but their biochemical mechanisms for facilitating viral cell entry remain unclear. We demonstrate these proteases can spontaneously and efficiently cleave their own zymogen activation motifs, thereby activating their wider protease activity on other cellular substrates. We determined TMPRSS11D co-crystal structures in complexes with a native TMPRSS11D zymogen activation motif and with an engineered activation motif, providing insights into TMPRSS11D autocleavage activation and revealing unique regions of its substrate binding cleft. We further show that a protease inhibitor that underwent clinical trials for TMPRSS2-targeted COVID-19 therapy, nafamostat mesylate, was rapidly cleaved by TMPRSS11D and converted to low activity derivatives. These insights into human protease viral tropism and into liabilities with existing human serine protease inhibition strategies will guide future drug discovery campaigns for these targets.

biochemistry↗