Biology subjects

Majumdar, A.

Publications and source records attributed to Majumdar, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Multi-point convective delivery overcomes mass transport barriers for myocardial therapeutics

Angiogenesis-promoting macromolecules reduce adverse remodeling and preserve cardiac function in rodents following myocardial infarctions, yet repeatedly fail to translate across length scales in humans. Through mass transport studies in human and swine myocardium, we found that dense, anisotropic myocardial fibers limit therapeutic diffusion and convection to millimeter scales for existing approaches including bolus intramyocardial injections, shear-thinning hydrogels, and epicardial patches. Furthermore, distributions are confined to one dimension along fibers. To increase myocardial drug distribution to centimeter length scales in vivo in swine, we engineered a three-dimensional multi-injection drug delivery array. Our device performs up to 40 simultaneous 120 {micro}L injections of functional macromolecules, hydrogels, or mRNA lipid nanoparticles. Injections are precisely placed in relation to fiber alignment, achieving near-complete coverage of the left ventricular myocardium.

bioengineering↗

bioRxiv2026

A multitask single-cell analysis framework with knowledge graph as a prior

Single-cell RNA sequencing has reshaped cancer biology by revealing the cellular and molecular diversity of the tumor microenvironment, yet most analysis pipelines still treat clustering, annotation, pathway scoring, metabolic inference, and cell-cell communication as separate tasks. Here, we present scKNIFE (Knowledge graph-based NMF for Inference of Functional Entities), a unified graph-regularized non-negative matrix factorization framework that jointly reconstructs single-cell expression profiles and infers activities of diverse biological entities, including pathways, cell-type markers, metabolic reactions and tasks, ligand-receptor interactions, and cell-state programs. By embedding a heterogeneous knowledge graph directly into the factorization objective, scKNIFE propagates information between biologically related entities through Laplacian regularization while sparsity constraints preserve interpretability. The integrated prior graph spans 47,274 nodes and 506,620 edges, assembled from complementary resources including Gene Ontology, Reactome, Hallmark gene sets, Human-GEM, CellChat, PanglaoDB, Cytopus, and Tabula Muris-derived annotations. Across multiple cancer-focused single-cell datasets, scKNIFE yields competitive to leading performance for clustering and cell-type annotation, while also recovering biologically coherent metabolic programs that agree with dedicated metabolism-focused methods and capture therapy-response-associated states. In addition, the framework supports downstream inference of cell-type-specific biological activities from a single latent representation. Together, these results establish scKNIFE as a modular and extensible framework for end-to-end biological interpretation of single-cell cancer transcriptomics

cancer biology↗

bioRxiv2026

Testing for gene-environment (GxE) interaction using p-value aggregation identifies many GxE loci

Genome-wide gene-environment (GxE) interaction studies have seen limited success in detecting reliable GxE signals. A standard genome-wide GxE scan assumes a single genetic mode of inheritance, such as an additive model. It can lead to reduced statistical power when the true genetic model is non-additive, such as a recessive model. We propose a robust GxE testing approach that uses Cauchy p-value aggregation. It combines the p-values from GxE tests based on the additive, dominant, and recessive genetic models. Using extensive simulation studies, we demonstrate that the p-value combination strategy offers a robust and powerful approach to identifying GxE interactions regardless of the underlying genetic model. The method is substantially more powerful than the additive model when the true genetic model is recessive. It is also more powerful than the general two-degree-of-freedom genotypic test for GxE interaction. We apply our approach to analyze GxE interactions in the UK Biobank data across several combinations of phenotypes and environmental factors. For glycated hemoglobin (HbA1c) level, treating cumulative smoking exposure as the lifestyle factor, our approach identified 82 independent GxE loci while controlling FDR at 5%. The GxE test based on the additive genetic model detected 24 loci. For type 2 diabetes with sleep duration as a lifestyle factor, the proposed approach detected 563 independent GxE loci at 5% FDR, substantially exceeding the number of discoveries by the other approaches.

genomics↗

bioRxiv2026

Distinct control of T cell proliferation and effector function by partitioning of intracellular sulfur from cysteine

Delineating how acquired nutrients are partitioned into different intracellular pathways, and how these various fates support distinct functions in T cells is limited. We show that CD8+ T cells acquire cysteine to serve both as a substrate for glutathione (GSH) production, which modulates effector functions, and to cede its sulfur for NFS1-dependent FeS-cluster synthesis, which supports proliferation. NFS1 deletion in activated CD8+ T cells promotes exhaustion and dampens anti-cancer immunity, while blocking cysteine flux into GSH, or enforcing FeS metabolism, enhance tumor control. This role for disrupted FeS metabolism in T cell exhaustion is echoed in data from human HCC. Elucidating how different intracellular pathways use cysteine enables targeted control of cysteine flux to retain beneficial effects of cysteine while abolishing those that restrain function. We illustrate this concept for one metabolite, cysteine, but it is likely to apply to other metabolites relevant for immune cell function.

immunology↗

bioRxiv2026

Temperature-dependent biofilm and sublancin production immobilise arsenic and antibiotic resistance gene mobility in soil system

Climate change-induced warming and arsenic soil contamination synergistically threaten agricultural sustainability by restructuring microbial communities and accelerating antibiotic resistance evolution. Here, we demonstrate that Bacillus subtilis 168-derived sublancin, a temperature-optimised glycosylated antimicrobial peptide, simultaneously maximises microbial arsenic sequestration and suppresses antibiotic resistance gene (ARG) horizontal transfer. Temperature-dependent biofilm formation increased progressively from 25-35{degrees}C (absorbance: 0.37-1.2), correlating with enhanced arsenic accumulation within the extracellular polymeric substance matrix (74% increase in weight percentage). Time-of-flight secondary ion mass spectrometry confirmed maximum arsenic sequestration at 35{degrees}C (total counts: 4.725x10). Sublancin production peaked at 30.8{degrees}C (129.72 mg/L) and significantly reduced soil microbial biomass carbon by 33% (p < 0.001), with selective suppression of Gram-positive bacteria (42%-28% survival reduction). Conjugation assays revealed sublancin bio-amendment dramatically suppressed horizontal ARG transfer, reducing HGT frequency by 74.7% (6.64 x 10-3 to 1.68 x 10-3, p < 0.001) through dual mechanisms: decreased donor viability and reduced conjugation efficiency. Gene ontology enrichment identified coordinated upregulation of exopolysaccharide synthesis (FDR ~1.0x10-{superscript 2}), phosphotransferase systems, and arsenic resistance pathways, alongside downregulation of conventional antibiotic resistance genes. These findings establish sublancin as a dual-function soil amendment simultaneously addressing two critical agricultural threats: heavy metal bioaccumulation and antibiotic resistance dissemination under climate warming.

microbiology↗

bioRxiv2026

Birth by Cesarean Section Remodels Neonatal Brain Barriers and Associated Immune and Metabolic Pathways in Mice

Delivery mode shapes early-life physiology through multiple pathways, including stress and hormonal signalling, neonatal oxygenation, and exposure to maternal microbes. Cesarean section (CS) bypasses several of these processes and has been associated with altered immune maturation and neurodevelopmental outcomes in humans and animal models. Given that CS is known to disrupt gut barrier integrity and that gut and brain barriers operate as a coordinated network, we investigated whether CS affects early postnatal brain barriers. Using a mouse model, we compared CS-delivered pups to vaginally born (VB) controls at postnatal days 8-9. Brain barrier permeability and structure were assessed using tracer extravasation and vascular and tight junction analyses, choroid plexus immune populations were profiled, and metabolomics was performed in cortex and plasma. The blood-brain barrier in the medial prefrontal cortex was mostly preserved. In contrast, the blood-cerebrospinal fluid barrier (BCSFB) exhibited structural alterations in vascular and epithelial tight junction compartments. These changes were accompanied by immune remodelling, including expansion of antigen-presenting border-associated macrophages and reduced parenchymal microglia density. CS delivery increased brain tracer accumulation, consistent with altered barrier function during this developmental window. Metabolomic analyses revealed lower levels of key brain metabolites, including N-acetylaspartate, a marker of neuronal metabolic state. Together, these findings demonstrate that CS delivery induces early alterations in brain barrier function, BCSFB structure, the immune landscape, and metabolic profiles in mice. This identifies delivery mode as a key perinatal variable shaping neonatal brain physiology and establishes a framework for dissecting how birth context may influence long-term neurodevelopmental trajectories. HIGHLIGHTSO_LICesarean section (CS) reveals the neonatal BCSFB as a vulnerable interface shaped by birth mode C_LIO_LICS increases brain tracer accumulation despite largely preserved BBB structural markers at the mPFC C_LIO_LICS induces region-specific vascular, epithelial, and immune remodeling in the choroid plexus C_LIO_LICesarean birth increases the proportion of antigen-presenting choroid plexus macrophages C_LIO_LICS shifts mPFC metabolic profiles, including reduced N-acetylaspartate and malate C_LI

neuroscience↗

bioRxiv2026

Dorsal striatal dopamine integrates sensory and reward prediction errors to guide perceptual decisions

Perceptual decisions are shaped by expectations about sensory stimuli and rewards, learned through sensory and reward prediction errors. Dopamine is known to convey reward prediction errors that shape perceptual decisions. However, whether dopamine also signals sensory prediction errors during perceptual decision-making remains unknown. We recorded dopamine release in the dorsal striatum of mice performing a visual decision-making task while manipulating sensory and reward expectations. The two manipulations produced similar behavioral biases but elicited opposite dopamine signals, indicating that dopamine within the same striatal region encoded both sensory and reward prediction errors. Sensory prediction error signaling was specific to dopamine as striatal acetylcholine showed no such error signals. Optogenetically stimulating striatal dopamine at stimulus onset biased subsequent perceptual decisions, consistent with updating sensory expectations. A computational model learning from both sensory and reward prediction errors captured behavioral biases and dopamine data, revealing distinct but spatially overlapping dopaminergic teaching signals.

neuroscience↗

bioRxiv2025

Aromatic residues in mobile regions distal to the active site support the closed conformation of E. coli DXPS

The essential bacterial enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXPS) is absent in humans, making the enzyme an attractive antimicrobial target. Its product DXP sits at a metabolic branchpoint between the biosynthesis of pyridoxal phosphate (PLP), thiamin diphosphate (ThDP), and isoprenoids. DXP is formed via decarboxylation of pyruvate and subsequent carboligation with D-glyceraldehyde-3-phosphate (D-GAP) in a ThDP-dependent manner. In the current mechanistic model, DXPS follows a ligand-gated mechanism. Pyruvate reacts with ThDP to form C2-lactylThDP (LThDP) which coincides with a shift to a closed conformation. The flexible "spoon" and "fork" motifs become ordered, situating the catalytic residue H299 within the active site which supports LThDP persistence and the closed conformation of the E-LThDP complex until binding of D-GAP. Our goal is to understand the molecular basis for stabilization of the E-LThDP complex in its closed conformation in the absence of D-GAP. We propose the conserved aromatic residues Y288, F298, and F304 in the E. coli DXPS spoon and fork motifs form a cluster upon transition from the open to closed form to position H299 within the active site, necessary for LThDP persistence. Here, we conducted mutagenesis studies to elucidate the roles of Y288, F298, and F304 in conformational cycling and catalysis. On each variant, the conformational equilibrium favored an open state, hindered intermediate formation and persistence, and promoted intermediate release from the active site. Our results support a model in which conserved aromatic residues within the mobile, sequence-diverse spoon and fork motifs promote the closed conformation and support catalysis.

biochemistry↗

bioRxiv2025

Biomolecular condensates of Eya drive transcriptional co-activation associated with eye development

Eyes absent (Eya), a transcriptional coactivator essential for eye development in Drosophila, also plays an important role in organ development in mammals and is associated with several diseases. To better understand the mechanism of Eya-mediated transcriptional co-activation, we find that Eya forms biomolecular condensates with liquid-like properties. These condensates function as possible transcription hubs, through the compartmentalization of key eye development regulators, the transcription factors So, Dac, Optix, Ey, and the kinase Nemo, RNA Pol II machinery, P300/CBP, and show the presence of target RNAs. We map the PST-TPM-PST region of Eya, which is critical for transcriptional co-activation and eye development, to be crucial for condensate formation as well. We identify a human deafness-associated mutation mapping to a conserved Drosophila site in the PST-TPM-PST region, which impairs transcriptional co-activation and shifts the material property of the Eya condensates to a less dynamic state. Our findings thus provide evidence that the condensation of Eya and its dynamic state are integral to transcription regulation and its role in development and disease mechanisms.

cell biology↗

bioRxiv2025

Structural insights into mitotic-centrosome assembly

Centrosomal material assembles rapidly in mitosis. In Drosophila, the coiled-coil protein Cnn forms a scaffold that recruits PCM clients; in C.elegans, SPD-5 plays an analogous role. Here we show that full-length Cnn and SPD-5 can both form spherical condensates in vitro, but that the interactions driving their assembly into scaffolds inside cells appear to diverge. We show that the Cnn PReM adopts a helical hairpin fold that autoinhibits CM2 binding but that phosphorylation appears to increase hairpin breathing to permit CM2 engagement and robust scaffold assembly. Phospho-blocking mutations prevent PReM-CM2 interactions and scaffold formation, whereas phospho-mimetic substitutions partially restore function. The human homologue CDK5RAP2 contains a CM2 domain that can partially substitute for fly CM2 in vivo and we identify a candidate CDK5RAP2 PReM region that forms macromolecular networks with human CM2 in vitro. By contrast, the putative PReM and CM2 regions of SPD-5 cannot substitute for their equivalent fly domains and they do not interact detectably, suggesting a distinct assembly mechanism in worms despite conserved PLK1-dependent control of PCM growth.

cell biology↗

bioRxiv2025

Copy number of the blaNDM-1 gene (New Delhi metallo-β-lactamase-1) and meropenem susceptibility in Enterobacterales: Consideration for treatment with meropenem?

Some Enterobacterales carrying blaNDM were still susceptible to meropenem, exhibiting meropenem minimum inhibitory concentration (MIC) values below the clinical breakpoint. As this phenomenon is a rare observation, this study investigated the underlying regulatory mechanisms contributing to the low MIC values and evaluated the efficacy of meropenem as a therapeutic preference for such isolates. Six clinical blaNDM-positive Enterobacterales exhibited meropenem MICs between 0.5-2.0 mg/L. Successful transmission of blaNDM into recipient E. coli J53 indicated its presence on plasmids. Conventional Sanger and whole-genome sequencing revealed that no truncation or deletion was observed within blaNDM or its core promoter region up to 120 bp upstream. Quantitative real-time PCR of blaNDM showed significantly lower expression levels of blaNDM, i.e., 0.2-0.5 in study isolates, compared to a clinical blaNDM-possessing isolate (expression=1) exhibiting a high meropenem MIC value (64 mg/L). Droplet digital PCR (ddPCR) further demonstrated that these isolates carried a lower copy number of the blaNDM gene, compared to the blaNDM-possessing isolate with high meropenem MIC (64 mg/L). Furthermore, the in vitro time-kill assay exhibited bactericidal efficacy of meropenem >3 log10 decrease in cfu/mL over the 24 hours from the starting inoculum. Administration of meropenem in in vivo neutropenic mice produced a 4-log cfu reduction compared to untreated controls at the start of dosing. Our data suggest that a reduction in blaNDM gene dosage, reflecting lower expression of blaNDM, correlates with lower meropenem MIC values. Meropenem is effective against these isolates when the meropenem MICs for the isolates are 0.5-2.0 mg/L. ImportanceThe production of NDM by Enterobacterales poses one of the most urgent global public health threats due to its plasmid-mediated spread, leading to treatment failure by exhibiting high MICs against a broad-spectrum of antibiotics. However, in rare instances, blaNDM-1-positive clinical Enterobacterales with unexpectedly low meropenem MICs (0.5-2.0 mg/L) raise a further question of reconsidering meropenem as a potential therapeutic option in these specific cases. This study identified that low MICs were associated with reduced expression and lower copy number of the plasmid-mediated blaNDM gene, despite intact promoter regions. Both in vitro and in vivo assays validated the bactericidal activity of meropenem against these isolates. The study raised an argument about the potential limitations of relying solely on genotypic detection of resistance genes for therapeutic decision-making, which emphasises the need for further investigation. As a safe medication, repurposing meropenem for neonatal treatment may eliminate the need for other toxic drugs.

microbiology↗

bioRxiv2025

Pathogenic mutations in Clathrin Heavy Chain associated with intellectual disability impair synaptic architecture and learning in Drosophila

Clathrin-mediated endocytosis is essential for neural development and function. Recent studies have linked de novo mutations in the clathrin heavy chain (Chc) gene to a range of neurodevelopmental disorders. In this study we have modelled two pathogenic mutations: L1047P and W1108R in Drosophila melanogaster and examined their effects on vesicle dynamics, ligand uptake, neuronal development and memory formation. Our data shows that expression of these mutant forms of Chc result in reduced survival and defective learning when expressed ubiquitously or exclusively in neurons. Our analysis also reveals that these mutations have the ability to disrupt vesicle dynamics and reduce ligand uptake in cells. Although we do not see a defect in neuronal morphology and function at the larval neuro-muscular junction, we see an increase in the number of Dlg-negative boutons, and a significant reduction in Spectrin expression, indicative of disruptions in the process of synapse maturation. Overall, this study provides mechanistic insights into the cellular and molecular basis of Chc-related neurodevelopmental disorders.

cell biology↗

bioRxiv2025

Molecular Insights Into PGPR-Biochar-Mediated Arsenic Detoxification and Nutritional Enhancement in Rice Rhizospheres

Arsenic (As) contamination in paddy soils threatens rice productivity and food safety through enhanced As(III) mobility under anaerobic conditions. This study evaluated the synergistic effects of rice biochar (RBC) and plant growth-promoting rhizobacteria (PGPR) on arsenic bioavailability and rice physiological responses under controlled and field conditions. Rice straw biochar pyrolyzed at 400 {degrees}C exhibited high porosity and surface functionality, while Metabacillus indicus AMB4 demonstrated arsenic tolerance ([≤]3500 mg kg-1) and multiple PGPR traits (IAA: 45.2 g mL-1, phosphate solubilization index: 2.8, ACC deaminase: 18.7 nmol -ketobutyrate mg-1 h-1). Combined RBC+PGPR treatment reduced soil arsenic bioavailability by 85% (p<0.001) and plant arsenic accumulation by 78% compared to controls, while significantly enhancing antioxidant enzyme activities (CAT: 4.2-fold, SOD: 3.7-fold, APX: 3.1-fold increase). Metagenomics revealed restructured microbial communities with increased Proteobacteria abundance and upregulated arsenic detoxification genes (arsC, arsB, aioA) coupled with enhanced nutrient cycling pathways (nifH, phoD, soxA). Network analysis demonstrated functional integration between As-transformation and nutrient metabolism, with coordinated expression of 47 arsenic-responsive and 126 nutrient-cycling genes. Field trials confirmed superior plant performance with RBC+PGPR treatment yielding 56% more tillers and 73% more panicles than controls, while maintaining enhanced soil nutrient status. This integrated biochar-PGPR system provides a mechanistically robust approach for simultaneous arsenic remediation and crop productivity enhancement in contaminated rice ecosystems, demonstrating practical applicability for sustainable paddy agriculture.

microbiology↗

bioRxiv2025

Furrow tillage reduces soil carbon loss and enhances microbial metabolism

With increasing global carbon loss from agricultural fields, agricultural practices should be monitored with proper regulatory measures. To address three problems of minimising carbon loss, optimising soil fertility and enhancing soil microbiota together, this study has developed a new innovative "Furrow tillage" approach that affords a balance between conventional and conservative tillage. Based on two years of trials in rice fields at multiple sites, this practice has justifications from agronomy, geochemistry, crop physiology, and molecular microbiology. Furrow tillage illustrated low carbon loss, similar to no-tillage, while retaining high nutrient bioavailability, similar to deep tillage. Microbial diversity, molecular responses and metabolic activities showed that furrow tillage induced microbial interactions while allowing a better entrapment of carbon dioxide in the soil. The study used CO2 flux, spatial distribution, total and available elemental analysis, plant ultra-structural observations, microbial metagenomics, high-throughput sequencing and molecular modelling to establish an optimal soil-crop quality, microbial maintenance and reduce carbon loss through furrow tillage practice.

ecology↗

bioRxiv2025

Loss of CTLH component MAEA impairs DNA repair and replication and leads to developmental delay

Ubiquitin E3 ligases play crucial roles in the DNA damage response (DDR) by modulating the turnover, localization, activation, and interactions of DDR and DNA replication proteins. To gain further insight into how the ubiquitin system regulates the DDR, we performed a CRISPR-Cas9 knockout screen focused on E3 ligases and related proteins with the DNA topoisomerase I inhibitor, camptothecin. This uncovered the CTLH ubiquitin E3 ligase complex -- and particularly one of its core subunits, MAEA -- as a critical regulator of the cellular response to single-ended DNA double-strand breaks (seDSBs) and replication stress. In tandem, we identified patients with variants in MAEA who present with neurodevelopmental deficits including global developmental delay, dysmorphic facial features, brain abnormalities, intellectual disability, and abnormal movement. Analysis of patient-derived cell lines and mutation modeling reveal an underlying defect in HR-dependent DNA repair and replication fork restart as a likely cause of disease. We propose that MAEA dysfunction hinders DNA repair by reducing the efficiency of RAD51 loading at sites of DNA damage, which compromises genome integrity and cell division during development.

genetics↗

bioRxiv2025

12-Hydroxystearic acid induces epidermal keratinocytes to secrete antimicrobial peptides that are potent inhibitors of viral infection.

Epidermal keratinocytes produce antimicrobial peptides (AMPs) that serve as a crucial component of the skins innate immune barrier. These peptides effectively target a broad spectrum of pathogenic microorganisms while preserving commensal microbiota essential for skin barrier homeostasis and overall skin health. Regulating the release of these AMPs presents a promising approach to enhancing the skin barriers defense mechanisms with minimal side effects. We have identified 12-hydroxystearic acid (12-HSA) as a potent stimulator of AMP secretion from primary epidermal keratinocytes. Mechanistic investigations revealed that, akin to bacterial stimulation, 12-HSA induces AMP release through the downregulation of caspase-8, which subsequently activates the inflammasome. Notably, we discovered that 12-HSA mediates caspase-8 downregulation via the acute activation of DNA methyltransferase 3A (DNMT3A), leading to transcriptional silencing of the caspase-8 locus. Importantly, 12-HSA is widely utilized in the cosmetic industry for its several skin beneficial properties including hydration and emolliency. Our findings suggest that this compound can be leveraged to enhance innate immune defenses in the skin, effectively mobilizing stored AMPs from keratinocytes to counteract microbial threats. This discovery highlights the potential for 12-HSA as a novel agent in dermatological applications aimed at fortifying skin barrier immunity.

cell biology↗

bioRxiv2025

Bacterial ubiquitin ligase engineered for small molecule and protein target identification

The Legionella SidE effectors ubiquitinate host proteins independently of the canonical E1-E2 cascade. Here we engineer the SidE ligases to develop a modular proximity ligation approach for the identification of targets of small molecules and proteins, which we call SidBait. We validate the method with known small molecule-protein interactions and use it to identify CaMKII as an off-target interactor of the breast cancer drug ribociclib. Structural analysis and activity assays confirm that ribociclib binds the CaMKII active site and inhibits its activity. We further customize SidBait to identify protein-protein interactions, including substrates for enzymes, and discover the F-actin capping protein (CapZ) as a target of the Legionella effector RavB during infection. Structural and biochemical studies indicate that RavB allosterically binds CapZ and decaps actin, thus functionally mimicking eukaryotic CapZ interacting proteins. Collectively, our results establish SidBait as a reliable tool for identifying targets of small molecules and proteins.

molecular biology↗

bioRxiv2025

Trihydroxybenzaldoximes are redox cycling inhibitors of ThDP-dependent DXP synthase

Pathogenic bacteria must swiftly adapt to dynamic infection environments in order to survive and colonize in the host. 1-Deoxy-D-xylulose-5-phosphate synthase (DXPS) is thought to play a critical role in bacterial adaptation during infection and is a promising drug target. DXPS utilizes a thiamine diphosphate (ThDP) cofactor to catalyze the decarboxylative condensation of pyruvate and D-glyceraldehyde-3-phosphate (D-GAP) to form DXP, a precursor to isoprenoids and B vitamins. DXPS follows a ligand-gated mechanism in which pyruvate reacts with ThDP to form a long-lived lactyl-ThDP (LThDP) adduct which is coordinated by an active-site network of residues. D-GAP binding ostensibly disrupts this network to activate LThDP for decarboxylation. Our lab previously reported trihydroxybenzaldoximes inhibitors which are competitive with respect to D-GAP, and uncompetitive with respect to pyruvate, suggesting they bind after E-LThDP complex formation. Here, we conducted mechanistic studies to determine if these compounds inhibit DXPS by preventing LThDP activation or if they act as inducers of LThDP activation. We discovered that the catechol moiety of the trihydroxybenzaldoxime scaffold undergoes oxidation under alkaline aerobic conditions, and inhibitory potency is reduced under oxygen restriction. Leveraging long range 1H-15N HSQC NMR and electrochemical measurements, we demonstrated that the oxidized form of the trihydroxybenzaldoxime induces LThDP decarboxylation. The oxime moiety accepts electrons from the resulting carbanion, resulting in formation of acetyl-ThDP which hydrolyzes to form acetate. SAR studies revealed that the catechol attenuates the redox activity of the oxime moiety, and under aerobic conditions these compounds are oxidized and thus act as redox cycling inhibitors of DXPS. Further exploration of redox active DXPS probes may provide new insights for inhibition strategies and selective probe development.

biochemistry↗