Biology subjects

Singh, A.

Publications and source records attributed to Singh, A..

At least 163 records · Page 9Linked to original sources

bioRxiv2025

Evolution of the rate, spectrum, and fitness effects of mutation under minimal selection in Caenorhabditis elegans

The rate, molecular spectrum, and fitness effects of mutations vary at all levels of the biological hierarchy, from within individual genomes to among taxonomic domains. Understanding the evolutionary factors underpinning that variation is of fundamental importance to biology. Accurate quantification of the properties of mutations requires that other evolutionary forces, especially natural selection, be minimized as much as possible. To investigate the evolution of the mutational process in C. elegans, we propagated a set of 100 "first order" mutation accumulation (O1MA) lines under minimal selection for [~]150 generations, divided each O1MA line into two "second order" MA (O2MA) lines and propagated them for another [~]150 generations, at which time the genome of each O2MA line was sequenced, and a subset of 50 O1MA families was assayed for competitive fitness. Over the course of the experiment, the mean nucleotide substitution mutation rate did not change, but the variance increased. In contrast, the indel mutation rate increased significantly. The two types of mutations fulfill the predictions of different theoretical models for the evolution of mutation rate. These results reinforce previous findings that the rate of indels is more sensitive to endogenous stress than the rate of nucleotide substitutions. Several evolutionary quandaries could be resolved if deleterious mutations interact synergistically (negative epistasis). Evidence for synergistic epistasis is famously inconclusive, although there is reason to think it may be more detectable under competitive conditions. However, a model of constant mutational effects on competitive fitness explains the results significantly better than a model including epistasis.

evolutionary biology↗

bioRxiv2025

Regulation of TCR-induced Transcriptional Kinetics by Interleukin 2-inducible T Cell Kinase (ITK) in CD8+ T cells

The initial activation of naive CD8+ T cells induces three major transcription factor pathways, NFAT, NF{kappa}B, and AP-1, predominantly regulated by T-cell receptor (TCR) signaling. Downstream of the TCR, the Tec family tyrosine kinase ITK modulates the transcriptional response in T cells by differentially affecting the kinetics and magnitude of activation of these three transcription factors. How signaling through ITK regulates the duration and/or termination of TCR signaling has not been investigated. To address this, we utilized the "Nr4a3-Tocky" reporter mouse which provides information on the kinetics of TCR signaling over time courses from hours to days. OT-I CD8+ Nr4a3-Tocky T cells were stimulated with peptide ligands of varying affinities, and cells were assayed for a panel of surface and intracellular markers of activation and differentiation at multiple time points post-stimulation. As previously reported, at early time points ITK signaling enhanced the kinetics and magnitude of expression of these markers. At later time points the absence of ITK led to persistent expression of several proteins, indicating an important role for ITK in the termination of TCR-dependent transcriptional responses. Using a dual ITK/ RLK inhibitor (PRN694) at 24h post OVA peptide stimulation it was found that this function of ITK was required in the first 24 hours. These findings reveal a possible key negative regulatory mechanism that is programmed in the first day of activation but impacts CD8+ T cell gene expression patterns days later. One Sentence Summary StatementThe Tec family tyrosine kinase ITK differentially regulates early and late T-cell receptor transcriptional responses.

immunology↗

bioRxiv2025

Binding Affinities of Disheveled PDZ Protein-Ligand Complexes

Wnt/{beta}-catenin signaling is critical for cell growth and development, with its hyperactive dysregulation implicated in the development of cancer. Current therapeutic research on inhibition of Wnt/{beta}-catenin signaling is impeded by the high cost of experimentally determining binding affinities. Consequently, interest has risen in screening potential inhibitors binding affinities with computational tools to reduce costs. Here, we test the validity of a computational molecular dynamics simulator, Binding Free Energy Estimator 2 (BFEE2), for determining peptide ligand affinity for Wnt/{beta}-catenin signaling. We focus on the Dishevelled (DVL) PDZ domain, a key mediator in WNT signaling through its ability to bind to various peptide ligands. We analyze the binding affinities of several DVL PDZ domain-peptide against previously established results to determine the validity of computational analysis. We conclude that computational molecular dynamics simulations were successful for peptide-ligand complexes.

bioinformatics↗

bioRxiv2025

BRCA2-deficiency Causes Global Transcriptomic Alterations in Endothelial Cells

This study aims to explore the alterations in gene expression following BRCA2 loss in endothelial cells and to investigate the potential contribution of BRCA2 in regulating the endothelial protein-coding transcriptome. Cultured human umbilical vein endothelial cells (HUVECs) were transfected with siBRCA2 or non-targeting scrambled RNA as control. Total RNA was extracted and used for RNA sequencing following purification and quality check. Transcription profiles and differentially expressed genes (DEGs) were identified. Enrichment of functions and signaling pathways analysis were performed based on Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In total, 3196 DEGs with (cut-off 1.5-fold) were identified, of which 1852 genes were upregulated, and 1344 genes were downregulated in BRCA2-deficient HUVECs. Cell cycle exit and neuronal differentiation 1 (CEND1) and mannose receptor C-type 1 (MRC1) were the most significantly up- and downregulated genes, respectively, in BRCA2-deficient endothelial cells. The expression of top DEGs were further validated by RT-qPCR in HUVECs and in human coronary artery endothelial cells. The GO and KEGG analysis suggest that cell adhesion molecules and cytokine-cytokine receptor interaction may play an important role in BRCA2-deficient endothelial cells. Overall, these findings contribute to a greater understanding of the mechanisms involved in BRCA2-mediated endothelial function.

genomics↗

bioRxiv2025

A dominant role of cell death in limiting Chandipura virus propagation at cell-saturating high multiplicity of infection

Viruses transit from a low to high multiplicity of infection (MOI) regime in infected tissues. Type-1 interferons (IFNs) enforce a cellular state refractory to virus multiplication, while the death of infected cells eliminates viral replicative niche. Here, we investigated how these two innate antiviral mechanisms cooperate at various MOIs upon cell infection by Chandipura virus (CHPV), a cytopathic RNA virus implicated in several outbreaks of acute encephalitis in India. We found that as expected, a gradual increase in the input MOI from 0.02 to 2 led to a proportionate surge in the viral yield. Surprisingly, a further rise to MOI 20 caused a reduction in the progeny titer. Our mathematical modeling together with ex vivo infection studies involving mutant cells suggested that cell death - more so than virus-induced type-1 IFNs - restricted CHPV propagation at cell-saturating high MOIs, leading to a net fall in the yield at MOI 20. We argue that the distinct involvement of innate immune pathways at varied MOIs imparts robust cellular defense against cytopathic viruses. Significance sentenceCell death - more so than type-1 interferons - limits Chandipura virus propagation at cell-saturating high multiplicity of infection. Highlights# Type-1 IFNs and cell death cooperate at varied MOI in limiting CHPV multiplication. # At sub-saturating low MOI, type-1 IFNs play a dominant role in controlling CHPV propagation. # At cell-saturating high MOI, cell death determines the progeny yield.

systems biology↗

bioRxiv2025

CerS2 is a druggable target in triple-negative breast cancer

Triple-negative breast cancer (TNBC) remains a significant clinical challenge due to its aggressive nature and lack of effective targeted therapies. The enzyme ceramide synthase 2 (CerS2), which synthesizes pro-apoptotic very long-chain ceramides (VLCCs), represents a promising therapeutic target. Here, we identify and characterize DH20931, a novel, first-in-class small-molecule agonist of CerS2. We demonstrate that DH20931 directly activates CerS2 with nanomolar potency, leading to significant VLCC accumulation in breast cancer cells. This lipotoxic event induces endoplasmic reticulum (ER) stress and triggers apoptosis via the canonical ATF4/CHOP/PUMA signaling pathway. Mechanistically, we uncover a novel interaction between CerS2 and the ER calcium channel, Inositol 1,4,5-trisphosphate receptor 1 (IP3R1). We demonstrate that DH20931 promotes this interaction, enhancing ER-mitochondria proximity and facilitating a CerS2-dependent flux of calcium (Ca{superscript 2}) from the ER into mitochondria. This subsequent mitochondrial Ca{superscript 2} overload serves as a critical trigger for apoptosis. In preclinical evaluations, DH20931 potently inhibited the growth of TNBC cells in 2D and 3D cultures and significantly suppressed tumor progression in orthotopic and patient-derived xenograft (PDX) models, all while exhibiting a favorable safety profile. Our findings validate CerS2 as a druggable target in TNBC and establish a novel therapeutic strategy that leverages a coordinated attack on cancer cells through ER stress and calcium-mediated mitochondrial dysfunction. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/670525v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@2db377org.highwire.dtl.DTLVardef@6175d4org.highwire.dtl.DTLVardef@7edb8corg.highwire.dtl.DTLVardef@80042e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDH20931 is a first-in-class, potent agonist of Ceramide Synthase 2 (CerS2). C_LIO_LICerS2 activation induces ER stress and engages the ATF4/CHOP/PUMA apoptotic pathway. C_LIO_LIDH20931 promotes a novel CerS2-IP3R1 interaction, causing lethal mitochondrial calcium overload. C_LIO_LITargeting CerS2 shows significant preclinical efficacy against triple-negative breast cancer. C_LI

cancer biology↗

bioRxiv2025

Cell cycle dynamics regulate H3K27 and H3K9 histone modifications

Cell cycle progression presents a fundamental challenge to genome integrity, particularly due to the need to reestablish post-translational histone modifications (PTMs) following DNA replication. Although proliferative and differentiating tissues exhibit markedly different cell cycle dynamics, how these differences shape the histone modification landscape in vivo remains largely unexplored. Here, we show that levels of H3K27ac, H3K27me3, and H3K9me3 are tightly linked to cell cycle dynamics in the Drosophila wing imaginal disc. We demonstrate that both physiological and pathological elongation of the cell cycle led to an accumulation of H3K9me3 and H3K27me3, whereas cell cycle acceleration reduces their levels. In contrast, H3K27ac exhibits the opposite pattern: levels decrease in arrested cells and increase with faster cycling. Genome-wide CUT&Tag analysis reveals that these changes predominantly affect genomic loci already modified in normally proliferating tissue. Importantly, the regulation of methylation levels at H3K9 and H3K27 is not solely mediated by the cell cycle machinery but reflects a metabolically guided process in which the rate of methylation is coupled to the rate of cell proliferation through metabolic activity, including signaling via the Insulin/PI3K/Akt pathway. Our study thus reveals key principles for understanding histone methylation in proliferating, senescent, and differentiating cells. In contrast, H3K27 acetylation is regulated through a distinct, cell cycle-coupled mechanism. We find that CBP/Nejire-mediated acetylation of H3K27 peaks during S-phase and is reversed by HDAC1, as cells exit replication. Disruption of this acetylation cycle leads to replication stress and a G2 cell cycle arrest via a DNA damage checkpoint. Notably, this genome-protective function of CBP/Nejire depends specifically on acetylation of the H3K27 residue itself, revealing a novel role for H3K27ac beyond its well-established function in transcriptional activation. Together, our findings establish a robust link between cell cycle progression and histone modification dynamics, highlighting the necessity of maintaining balanced PTM levels under varying proliferative states. These insights have broad implications for our understanding of development, aging, and tumor growth.

cell biology↗

bioRxiv2025

Enhancer Placement Impacts Transcriptional Dynamics in Drosophila Embryos

The functional role of enhancer-promoter configurations in transcription regulation remains poorly understood, despite the wide range of linear genomic distance and relative enhancer positioning observed in endogenous contexts. While canonical models suggest that enhancers operate independently of genomic position, technical limitations have obscured insights on transcriptional kinetics. Here, we employ single-cell MS2/MCP-based live imaging in Drosophila embryos to systematically analyze transcriptional dynamics driven by sub-10kb enhancer-promoter arrangements. Kinetic analyses reveal that while linear enhancer-promoter distance moderately tunes transcriptional output, downstream enhancer positioning reduces mRNA output by 70%. Each configuration modulates distinct transcriptional parameters: linear distance governs initiation kinetics, while relative enhancer positioning dictates transcriptional stability. These effects are consistent across varied enhancer and reporter sequences, revealing configuration-dependent cis-regulatory element arrangement as an intrinsic mechanism for transcriptional fine-tuning. This work challenges the paradigm of configuration-independent enhancer function and establishes a framework to dissect the interplay between genome architecture and trans-acting factors.

molecular biology↗

bioRxiv2025

Tumor-draining lymph node derived CD8⁺ T cells sustain durable systemic immunity after neoadjuvant IL-15 and PD-1 blockade.

The tumor-draining lymph node (tdLN) serves as a critical reservoir of tumor-reactive T cells, yet its contribution to preventing metastatic recurrence remains poorly understood. In this study, we investigate CD8 T cell clonal dynamics and memory responses induced by neoadjuvant anti-PD-1 therapy. We demonstrate that PD-1 blockade promotes the emergence of protective CD8 T cell memory, enriched with tumor-relevant clones that persist in the lymph nodes of long-term survivors. Furthermore, we show that combination of a neoadjuvant IL-15 superagonist with PD-1 blockade significantly increases clonal diversity, cytotoxicity, and clonal persistence in non-draining lymph nodes. These changes result in enhanced systemic antitumor immunity and durable memory that prevents tumor relapse. Our findings establish the tdLN as a critical immunologic hub that can be therapeutically targeted by IL-15 to augment PD-1 blockade efficacy and support a rationale for a phase II neoadjuvant combination immunotherapy trial in early-stage NSCLC. One sentence summaryNeoadjuvant IL-15 with PD-1 blockade expands lymph node clonal diversity and preserves stem-like CD8 T cell features, yielding superior antitumor efficacy, durable memory, and reduced metastatic recurrence.

immunology↗

bioRxiv2025

Commensal taxa in gut microbiota limit antibiotic resistance during extended oral antibiotic use

Certain bacterial infections, such as those involving prosthetics, can require antimicrobial therapy over months to years, potentially increasing the burden of antimicrobial resistance. Here we longitudinally track the antimicrobial resistome in mice during continuous antibiotic dosing over 21 months. The burden of antibiotic resistance genes (ARGs) initially increases, but, surprisingly, declines in later months, approaching levels observed in untreated animals. ARG burden is regulated by taxonomy and declines as ARG-harboring taxa that initially bloom are replaced by commensals. Furthermore, we find that the dynamics of antibiotic-induced ARG burden are influenced by age-related differences in microbial taxonomy and can be removed by fecal microbiota transplantation. We show that commensals may regulate the resistome by limiting the growth of ARG-harboring taxa, thereby providing antimicrobial expansion resistance.

microbiology↗

bioRxiv2025

Discovery of Prognostic Biomarkers in Gastric Cancer Through Machine Learning and Bioinformatics Analysis of Gene Expression Data

Gastric cancer (GC) often gets diagnosed in its advanced stages, resulting in poorer prognoses. To identify potential biomarkers in GC, we used fifteen datasets from NCBI-GEO and integrated them into a complete dataset. From the complete dataset, we extracted a subset of only the known cancer driver genes. Using Recursive Feature Elimination (RFE), mutual information (MI), and tree-based (TB) method (SelectFromModel), we extracted top gene features using RFE (10, 20, 30, 40, and 50) and 50 features each from MI and TB method. Subsequently, we applied machine learning classifiers to these selected gene features to classify cancer and normal samples. The SVC classifiers demonstrated better performance when utilizing the top 50 gene features using RFE and MI, while the AB classifier achieved the highest performance using TB for the complete dataset and for driver datasets, RF performed well using 40 features using RFE and SVC, and ET showed up better performance using 50 features using MI and TB feature selection for the test dataset. After combining all the genes from both datasets from and from all three feature selection methods, only 115 showed differentially expressed genes. A Lasso-penalized Cox regression model was applied to narrow down the gene selection to fourteen. This study highlights the effectiveness of integrating machine learning and bioinformatics analysis to identify new biomarkers for GC.

bioinformatics↗

bioRxiv2025

LuxS-mediated AI-2 signaling regulates chemotaxis, flagellar motility, and virulence genes in Salmonella Typhimurium and contributes to colonization in the murine gut

The cooperative response through AI-2 allows Salmonella to coordinate and regulate its community behaviours crucial for its survival and infection. Understanding these mechanisms is essential for developing strategies to interfere with Salmonellas ability to cause disease. In this study, we report that Salmonella Typhimurium uses AI-2 signaling to modulate the expression of chemotaxis and motility-associated genes and the formation of flagella on the bacterial cell surface. The AI-2 acts as a chemoattractant, and the cascade mediates the chemotactic response of Salmonella. Such regulations assist in the attachment and invasion of Salmonella into intestinal epithelial cells both in vitro and in a murine model. Furthermore, we determined that HilD, known to regulate motility by induction of a chemoreceptor and SPI-1 in Salmonella, along with key SPI-1 genes, is underexpressed in the absence of LuxS/AI-2 signaling, which unravels a complex regulatory network by AI-2 in Salmonella. Beyond the initial phase, this signaling facilitates survival in the presence of polymyxin B and supports intracellular survival and persistence within epithelial cells by regulating the pmrD/AB system, thereby evading the antimicrobial peptide immune response. Additionally, our findings show that Salmonella activates its LuxS/AI-2 signalling cascade within the Caco-2 epithelial cells. Complimentary observation through transcriptomic profiling revealed that LuxS/AI-2 signalling governs the coordinated regulation of genes implicated in distinct phases of Salmonella pathogenesis. Lastly using a mouse model, we show that AI-2 signaling is critical for organ colonization and virulence of Salmonella, and inhibition of LuxS/AI-2 signaling in combination with antibiotics can be an alternative therapeutic approach. Thus, our study dissects an integrated regulatory mechanism by which AI-2 signaling mediates critical processes during multiple stages of Salmonella Typhimurium pathogenesis.

microbiology↗

bioRxiv2025

RUNX2 inhibition disrupts a PAX3::FOXO1-RUNX2 feed-forward loop and dismantles oncogenic gene programs in fusion-positive rhabdomyosarcoma

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer of skeletal muscle lineage, with a 5-year overall survival of <30% for high-risk disease, and <8% when metastatic. The PAX3::FOXO1 fusion gene, resulting from t(2:13), is a signature driver of fusion-positive rhabdomyosarcoma, but similar to other transcription-factor based fusion genes in other cancers, not currently pharmacologically tractable. To identify novel druggable proteins in fusion-positive rhabdomyosarcoma tumor tissue and cell lines, we performed mRNA-seq of RMS patient tumors and utilizing the human FP-RMS cell lines Rh30 and Rh4, found that the RUNX2 transcription factor was the top druggable dependency. In vitro loss of function studies using genetic (RNAi) or pharmacologic (small molecule CADD522) inhibition showed that RUNX2 suppression inhibited FP-RMS cell growth, induced myogenic differentiation and apoptosis, and phenocopied PAX3::FOXO1 suppression. In vivo loss of function studies using conditional (dox-inducible) or pharmacologic (small molecule CADD522) blockade of tumor growth in a xenograft model system showed that RUNX2 suppression inhibited tumor growth. Mechanistically, we identify a PAX3::FOXO1 feed-forward loop whereby PAX3::FOXO1 binds a RUNX2 enhancer to upregulate gene expression alongside MYOD1, while RUNX2 expression supports the expression of PAX3::FOXO1 at the mRNA and protein level. SignificanceRUNX2 inhibition reduces PAX3::FOXO1 expression and signaling, which impairs fusion-positive rhabdomyosarcoma oncogenic phenotypes. In vivo treatment with CADD522 decreased tumor growth and increased survival, indicating that RUNX2 is a promising therapeutic target.

cancer biology↗

bioRxiv2025

Spatial dynamics of IFITM1: a core component of the interferon-stimulated gene-resistance signature in glioma

The IFITM1 protein is a key component of the Interferon-Stimulated Gene (ISG) network, which has been linked to treatment-resistant signatures in various cancers, including glioblastoma (GBM). Despite its impact, the mechanisms underlying IFITM1s role in cancer remain poorly understood. Here, we demonstrate that the spatial dynamics of IFITM1 localisation are highly context-dependent, particularly in GBM tissue. In the vasculature, IFITM1 is predominantly localised to the plasma membrane of endothelial cells. However, when present in a subset of cancer stem cells, IFITM1 adopts a distinct perinuclear location, where it co-localises with IFITM3 and is notably absent from the membrane. The spatial dynamics of IFITM1 localisation was investigated in patient-derived glioma stem cells (GSCs), primary endothelial cells and engineered cell lines. Endogenous IFITM1 localised primarily to assemblies in the perinuclear space, however loss of functional IFITM3 led to a shift in the distribution of the protein to a predominantly membrane location. In contrast, IFITM3 localisation was unaffected by the loss of IFITM1. The results were recapitulated by transient expression of IFITM1 or -3 into double knockout (DKO) GSCs and by using an engineered system where IFITM1 expression was IFN-independent. Co-expression studies demonstrate that IFITM3 is sufficient to localise IFITM1 to vesicle structures in the perinuclear space and that mutant forms of IFITM3 lead to retention of IFITM1 primarily at the plasma membrane. Our data highlights dynamic changes in the subcellular localisation of IFITM1 suggesting that distinct functions of this resistance factor may present a specific target for therapeutic intervention.

cancer biology↗

bioRxiv2025

Multi-omic mapping of Drosophila protein secretomes reveals tissue-specific origins and inter-organ trafficking

Secreted proteins regulate many aspects of animal biology and are attractive targets for biomarkers and therapeutics. However, comprehensively identifying the "secretome", along with their tissues of origin, remains extremely challenging. To address this, we employed multiple omics methods to define a tissue-secretome map of 535 blood plasma proteins derived from specific cell-types and organs in Drosophila melanogaster. This map was enabled by methodological improvements including a collection of transgenic flies to label endogenous secreted proteins in 10 major tissue types, large-scale blood isolation, whole animal snRNA-seq, and a collection of 40 knock-in strains. Using this map, we discover surprising findings about circulating proteins: most originate from specific tissues including unusual sources (e.g. glia), many are uncharacterized, and some are shed ectodomains of transmembrane proteins. In addition, in vivo experiments revealed circulating proteins with remarkably tissue-specific expression, as well as proteins that are deposited in a different tissue from where they are synthesized, suggesting potential inter-organ functions. Our secretome map will serve as a resource to investigate blood protein function, discover novel tissue-tissue communication signals, and mine for homologues of human biomarkers.

genomics↗

bioRxiv2025

CloneCoordinate: Open-source software for collaborative DNA construction

Custom DNA constructs have never been more common or important in the life sciences. Many researchers therefore devote substantial time and effort to molecular cloning, aided by abundant computer-aided design tools. However, support for managing and documenting the construction process, and for effectively handling and reducing the frequency of setbacks, is lacking. To address this need, we developed CloneCoordinate, a free, open-source electronic laboratory notebook specifically designed for cloning and fully implemented in Google Sheets. By maintaining a real-time, automatically prioritized task list, a uniform physical sample inventory, and standardized data structures, CloneCoordinate enables productive, collaborative cloning for individuals or teams. We demonstrate how the information captured by CloneCoordinate can be leveraged to troubleshoot assembly problems and provide data-driven insights into cloning efficiency. CloneCoordinate offers a new and uniquely accessible model for how to carry out, and iteratively improve on, real-world DNA assembly.

synthetic biology↗

bioRxiv2025

Transformer-based Deep Learning for Glycan Structure Inference from Tandem Mass Spectrometry

Glycans play critical roles in diverse biological processes, but their structural analysis by tandem mass spectrometry (MS/MS) remains a major challenge due to their branched structure and stereochemistry. Traditional computational methods, such as database searching, are constrained by the scope of existing libraries and can be computationally intensive. While recent deep learning models have advanced the field, they often struggle to capture the complex, long-range dependencies within MS/MS spectra required for accurate inference. To address these challenges, we present GlycoBERT and GlycoBART, novel transformer-based models for glycan structure prediction from MS/MS data. GlycoBERT, a sequence classifier, achieves 95.1% structural accuracy on test data, surpassing the current state-of-the-art deep learning model, CandyCrunch. However, classification-based methods are inherently limited to predicting structures present in the training data. To overcome this, we developed GlycoBART, a generative sequence-to-sequence model capable of de novo glycan inference. On independent validation datasets, both models demonstrate robust performance. Critically, when applied to an MS/MS dataset from human embryonic kidney cells, GlycoBART generated two de novo glycan structures absent from the training set, one of which is a novel structure not catalogued in major glycan databases. Together, GlycoBERT and GlycoBART establish a new benchmark for glycan analysis, offering a powerful framework that enables more accurate and comprehensive exploration of glycan diversity and discovery.

bioinformatics↗

bioRxiv2025

Molecular insights into inhibitor action on a key bacterial metabolic enzyme Cystathionine β-Synthase

Tuberculosis (TB) remains a major global health threat, with Mycobacterium tuberculosis (Mtb) infecting nearly a quarter of the global population. Drug-resistant TB and HIV-TB co- infections emphasize the need for novel therapeutic approaches targeting essential metabolic pathways. Here, we investigated Mtb cystathionine {beta}-synthase (MtbCBS), a PLP-dependent enzyme critical for sulfur metabolism and redox regulation, owing its potential as a therapeutic target. We present the first high-resolution cryo-EM structure of MtbCBS bound to aminooxyacetic acid (AOAA), and employed molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) calculations, and comparative inhibition studies to reveal the molecular basis and determinants governing irreversible PLP-enzyme inhibition. Our Cryo-EM structural analysis revealed two highly conserved active-site residues, T75 and Q147, critically stabilizing the inhibitor complex. Through molecular mimic studies, we demonstrated the precise structural factors and electronic features critical for inhibition efficiency. These findings provide the first mechanistic rationale for PLP enzyme inhibition and offer a generalizable framework for designing covalent inhibitors targeting PLP-dependent enzymes implicated in infectious diseases, cancer, and neurological disorders. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/662948v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@195c7c7org.highwire.dtl.DTLVardef@6ee0c8org.highwire.dtl.DTLVardef@1679dfborg.highwire.dtl.DTLVardef@1b4e000_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO Proposed mechanism of MtbCBS inhibition by AOAA. Proposed mechanism of action of AOAA illustrated through a graphical representation. The cartoon shows the domains of MtbCBS. Tetrameric structure of MtbCBS with active site highlighted (in red dashes). Enlarged view indicating the PLP-AOAA blocked intermediate interacting with residue Q147. Reaction scheme depicting the previous missing understanding of active site residue role in -elimination. Schematic representation of AOAA inhibition mechanism in the catalytic cycle of MtbCBS. Reactions were drawn using ChemDraw. The proposed scheme highlights that AOAA (highlighted Red) binding is stabilized by nearby residues Q147 and T75, leading to the formation of PLP-AOAA covalent adduct, and substrates (highlighted green) were incapable of reverting the suicidal inhibition. The directionality and reversibility were mentioned for each step. C_FIG

biophysics↗