Biology subjects

Choudhary, S.

Publications and source records attributed to Choudhary, S..

At least 19 recordsLinked to original sources

bioRxiv2026

EIDD-3525 is a broad-spectrum antiviral against alphavirus infection that targets the nsP4 RNA-dependent RNA polymerase

Mosquito-transmitted alphaviruses, including chikungunya (CHIKV), Mayaro, Eastern equine encephalitis, and Venezuelan equine encephalitis viruses, cause severe arthritic or neurologic disease in humans. There are currently no approved antiviral treatments against alphavirus infection. Here, we report that the purine ribonucleoside EIDD-3525 is a potent inhibitor of alphavirus replication in vitro with broad spectrum antiviral activity against the Alphavirus genus. Using a trans-replicase assay, we find that EIDD-3525 blocks alphavirus RNA replication. Mutational profiling suggests that EIDD-3525 targets the alphavirus nsP4 RNA-dependent RNA polymerase that is conserved across medically important alphaviruses. In a pre-clinical mouse model of CHIKV infection and disease, oral treatment with EIDD-3525 reduced viral tissue burdens and the severity of virus-induced disease. Collectively, our findings support the continued development of the purine ribonucleoside EIDD-3525 as an orally available antiviral therapeutic for use against a broad group of alphaviruses.

microbiology↗

bioRxiv2026

Optimizing genome assembly, chromosome synteny, and genetic variant discovery from Oxford Nanopore sequences of Dermacentor reticulatus ticks

Generating high-quality genome assemblies for small animals with large genomes is complex due to their small body size, DNA contamination, and repetitive elements. Ticks exemplify these complexities, while also being a global health threat to humans, domestic animals, and wildlife. Advances in long-read sequencing platforms now make it feasible to obtain large amounts of raw sequence data from individual specimens, but challenges remain. Key genome workflow challenges include error correction, assembly, transposable element annotation, and chromosome assignment and synteny. Here we examine three individual Dermacentor reticulatus ticks using deep Oxford Nanopore sequencing. Comparing and contrasting bioinformatic tools for raw read and assembly manipulation allows us to identify the parameters that provide a high-quality haploid genome assembly among 38 assemblies. We find Dorado error corrected raw read data from approximately two flow cells are needed, but limiting assembly coverage to 40x produces the highest quality assemblies. Then we examined the effects of optimal workflow parameters on downstream analyses of gene synteny and manual transposable element annotation of the three tick assemblies in comparison to an independent assembly of a fourth D. reticulatus individual from a different country. Gene synteny analysis allows chromosome assignment of scaffolds and transposable element identification was improved markedly with manual curation. Finally, we compared the genetic variation of D. reticulatus across two populations and found similar genetic diversity. Our study provides a clear workflow to obtain high-quality assemblies from Oxford Nanopore sequences and genetic characterization of a species with a large, complex, and repetitive genome.

genomics↗

bioRxiv2026

Integrated Transcriptomic and Network-Based Identification of Prognostic Hub Genes in Oral Squamous Cell Carcinoma

BackgroundThe most prevalent kind of oral cancer is oral squamous cell carcinoma (OSCC), which has a poor prognosis because of delayed detection and a lack of molecular indicators. MethodsTranscriptomic data from TCGA were analyzed to identify differentially expressed genes between OSCC and normal samples. Functional enrichment analysis was performed to determine biological pathways. A protein-protein interaction network was constructed using STRING and visualized in Cytoscape to identify hub genes. ResultsA total of 5732 differentially expressed genes were identified, including 2459 upregulated and 3273 downregulated genes. Network analysis revealed several highly connected hub genes such as CDK1, CCNB1, TOP2A, BUB1, and MMP9. Functional enrichment indicated significant involvement of cell cycle regulation and cancer-associated pathways. ConclusionThis integrative analysis identified key regulatory hub genes that may be involved in OSCC progression. These genes may serve as promising biomarkers and therapeutic targets for future studies.

bioinformatics↗

bioRxiv2025

A conserved logic for the development of cortical layering in tetrapods

The cerebral cortex is part of the pallium, a brain region conserved across vertebrates yet remarkably diverse in structure and cellular composition. A defining feature of the cerebral cortex is its organization into neuronal layers with distinct gene expression profiles, input-output connectivity, and function. According to prevailing models, the cerebral cortex emerged in ancestral amniotes (mammals and reptiles) following innovations in pallial development that enabled the generation of diverse neuron types and their laminar organization.1-11 However, little is known about pallial development and architecture in amphibians, the sister group of amniotes. Here we show that in the salamander Pleurodeles waltl, the dorsal pallium is organized in distinct superficial and deep layers with neurons that develop following cellular and molecular principles of mammalian corticogenesis. Using birthdating analysis, barcode-based lineage tracing, and single-cell RNA sequencing, we find that radial glia temporal states and intermediate progenitor cells are conserved across species, while neuronal differentiation trajectories are highly evolvable. Neurons generated at different developmental time points occupy different layers and exhibit distinct molecular and projection identities. Thus, temporally-patterned neurogenesis represents an ancient organizing principle of layered pallia, although mammals display an inverted layer order along the radial axis. Together, these findings demonstrate that the core developmental principles underlying cortical layering - including temporal patterning, intermediate progenitors, and laminar organization - predate the origin of amniotes. Our results suggest that the evolutionary expansion of the mammalian neocortex built upon a deeply conserved developmental framework already present in early tetrapods.

developmental biology↗

bioRxiv2025

Increased Quadriceps Intermuscular Adipose Tissue in Chronic Liver Disease is Associated with an Altered Muscle Transcriptome compared with Healthy Age Matched Controls.

Muscle fat infiltration (i.e., myosteatosis) is an important index of muscle health and pathology in disease states, such as chronic liver disease (CLD). However, it is unknown whether myosteatosis differs due to anatomical location. Further, the mechanistic implications of divergent intermuscular adipose tissue (IMAT) deposition remains unknown in patients with CLD. 33 patients with CLD (55.0{+/-}10.5years) and 17 healthy controls (HC) (49.6{+/-}15.4years) were recruited. Quadriceps IMAT was estimated between 20-80% of muscle length and psoas IMAT at L3 via MRI. Body composition, muscle strength and habitual activity were assessed. Fasted vastus lateralis muscle biopsies were collected and subjected to RNA sequencing analysis. https://ClinicalTrials.gov identifier: NCT04734496. IMAT was greater in CLD compared with HC in both quadriceps and psoas (P<0.0001). Quadriceps IMAT positively correlated with BMI (r=0.62), body fat (r=0.65), age (r=0.36) and negatively correlated with maximal knee extensor strength (r=-0.45) and habitual physical activity (r=-0.50) in CLD and HC. 169 differentially expressed genes (DEGs) were identified in low IMAT CLD vs HC, and 178 DEGs were identified in high IMAT CLD vs HC. CLD patients with high and low IMAT exhibited defined expression profiles, with only 39 DEGs (representing 12.7%) in common. Pathway analysis of DEGs revealed enrichment of atrophic and pro-inflammatory pathways in the high IMAT group. IMAT is greater in patients with CLD compared to HC, irrespective of anatomical location and is associated with reductions in muscle function and may be exacerbated by physical inactivity. IMAT appears to alter the muscle transcriptome in COLD, potentially exacerbating muscle loss.

physiology↗

bioRxiv2025

Opposing Copy Number Variation Dynamics Shape Adaptation to Glucose and Galactose in Diploid Yeast

Adaptation to new environments arises from multiple forms of genetic variation, including single-nucleotide polymorphisms (SNPs) and structural variants such as copy number variations (CNVs). Although both mutation classes have been implicated in evolutionary change, their relative contributions during adaptation remain incompletely understood and may depend strongly on environmental context. Here we investigated genome evolution during experimental adaptation of diploid Saccharomyces cerevisiae populations propagated for 1200 generations in two alternative carbon sources, glucose or galactose. Whole-genome sequencing of replicate populations at multiple evolutionary time points revealed relatively few SNPs but widespread copy number variation across the genome. Strikingly, CNV dynamics differed markedly between environments. Populations evolved in glucose exhibited extensive early deletions followed by later genomic remodeling that reduced the overall deletion burden. In contrast, populations evolved in galactose accumulated persistent subtelomeric duplications throughout the experiment. Many loci displayed opposing copy number changes in the two environments, with genes deleted during evolution in glucose frequently duplicated during evolution in galactose. These contrasting trajectories indicate that structural genomic changes repeatedly arise during adaptation and that their direction and persistence depend strongly on environmental conditions. Together, our results illustrate how distinct genomic routes can emerge during adaptation of genetically identical populations and highlight the importance of considering multiple classes of mutations when studying evolutionary responses to environmental change. Impact statementThis study demonstrates that copy number variations (CNVs), rather than single-nucleotide polymorphisms, dominate genome evolution during adaptation of diploid Saccharomyces cerevisiae to alternative carbon sources. By evolving genetically identical populations in glucose and galactose, the work reveals that environmental context drives strikingly opposing CNV trajectories, widespread deletions in glucose and persistent duplications in galactose, often targeting the same genomic loci in opposite directions. These findings provide direct evidence that structurally mediated gene dosage changes are a primary and environment-dependent mechanism of adaptation, reshaping metabolic networks and specialization strategies. More broadly, the study challenges the prevailing SNP-centric view of adaptive evolution and highlights CNVs as a predictable and major axis of genome remodeling in microbial systems.

evolutionary biology↗

bioRxiv2025

Molecular docking of Pyocyanin from Pseudomonas aeruginosa with NADPH oxidase and Toll-like receptor 4 : Exploring the Link Between Oral Microbiome and Oral Cancer Pathogenesis

Oral cancer is among the leading cancers in India, with its progression influenced not only by genetic and environmental factors but also by dysbiosis in the oral microbiome. Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, has been implicated in oral carcinogenesis through the action of its virulence factor, pyocyanin--a redox-active phenazine that induces reactive oxygen species (ROS), promoting oxidative stress, immune dysregulation, and inflammation. Despite evidence linking pyocyanin to ROS-related pathways, its molecular interaction with key host proteins like NADPH oxidase and Toll-like receptor 4 (TLR4) remains unexplored. The present study aims to investigate the molecular interactions of pyocyanin with NADPH oxidase and TLR4 through molecular docking, aiming to elucidate its role in oxidative and inflammatory mechanisms associated with oral cancer pathogenesis. The structures of pyocyanin and the NADPH oxidase (NOX2) and TLR4 target proteins were retrieved from PubChem and RCSB PDB, respectively. CASTp was used to identify potential binding pockets. Molecular docking was performed using the HADDOCK server. The docked complexes were analyzed based on HADDOCK scores, binding energies, RMSD, and buried surface area. Interaction profiles were visualized using BIOVIA Discovery Studio. Among the docking clusters, Cluster 6 for the NOX2-pyocyanin complex showed a favorable HADDOCK score (84.4 {+/-} 2.9), low RMSD (0.2 {+/-} 0.0), and substantial buried surface area (456.1 {+/-} 13.2 [A]2). For the TLR4-pyocyanin complex, Cluster 1 exhibited the most favorable interaction profile with a HADDOCK score of 25.0 {+/-} 5.5 and a Z-score of -2.3. Binding site prediction and hydropathy analysis supported the structural feasibility of pyocyanin interaction with both targets. This study provides structural insights into the potential interaction of pyocyanin with NADPH oxidase and TLR4, supporting its role in modulating oxidative stress and inflammatory signaling pathways. These findings enhance our understanding of oral microbiome-host interactions and their contribution to oral cancer pathogenesis.

bioinformatics↗

bioRxiv2025

Investigating active dynamics of contractile actomyosin gels with Micro Particle Image Velocimetry (Micro-PIV) analysis

Micro Particle Image Velocimetry (Micro-PIV), an advanced imaging technique, enables high-resolution velocity field measurements by tracking fluorescent tracers in microscopic environments. Here, we adapt conventional micro-PIV to study the rapidly contractile dynamics of active poroelastic gels. We demonstrate how frame-to-frame correlation improves signal-to-noise ratios and how the elastic nature of the solid phase of the gel can be included in the analysis. To do this, we average the gel displacement data under an axisymmetric assumption to extract radial strain profiles that reliably reveal local deformations of the gel. By analyzing gels of varying shapes, we further show that our method extends robustly to gels that are not completely circular or that do not displace symmetrically towards their geometric center. The analysis reveals common underlying features in the radial profiles of gel deformation. These strain profiles will allow the inference of the spatial and orientational distribution of motor-generated active stresses with appropriate constitutive models for the gel mechanics. Our findings emphasize the importance of tailored micro-PIV methodologies for analyzing complex fluids, particularly autonomously contracting poroelastic materials. This approach significantly enhances understanding of cytoskeletal dynamics and self-organization processes, with broad implications for cell motility, morphogenesis, and active matter physics.

biophysics↗

bioRxiv2025

Huntingtin preserves mitochondrial genome integrity in neurons, which is impaired in Huntingtons disease

Huntingtin (HTT) function is enigmatic, as the native protein plays critical roles in neuronal health, while mutant HTT (mHTT), carrying an expanded polyglutamine stretch, triggers neurotoxicity and contributes to the pathogenesis of Huntingtons disease (HD). We recently found that HTT is part of a nuclear transcription-coupled DNA repair (TCR) complex with DNA repair enzymes including polynucleotide-kinase-3-phosphatase (PNKP). This complex resolves DNA lesions during transcription to maintain genome integrity, while in HD, mHTT impairs the activity of this complex, resulting in accumulation of DNA lesions. Using molecular, cellular biology and computational methods, we find that HTT has a role in assembling a functional DNA repair complex in mitochondria. Together with mitochondrial RNA polymerase and transcription factors, HTT resolves mitochondrial DNA lesions to preserve mitochondrial genome integrity and function. Pathogenic mHTT impairs this activity, resulting in persistent DNA lesions and reduced mitochondrial function in HD. Importantly, restoring activity of this complex in a Drosophila HD model through ectopic HTT or PNKP expression significantly improves mitochondrial genome integrity and ameliorates motor deficits. HIGHLIGHTSO_LIHTT organizes a functional, multifactorial mitochondrial DNA repair complex C_LIO_LIMutant HTT impairs the mitochondrial DNA repair complex causing DNA damage accumulation C_LIO_LIHTT-associated repair complex resolves mitochondrial DNA lesions and DNA integrity C_LIO_LIRestoring repair activity in HD flies rescues mitochondrial DNA integrity and motor defects C_LI

neuroscience↗

bioRxiv2025

UFMylation orchestrates chromatin engagement of core NHEJ components to promote DNA double-strand break repair

DNA double-strand breaks (DSBs) are highly cytotoxic lesions whose misrepair can lead to genomic instability, cancer and developmental disorders. Through systematic screening of understudied ubiquitin-like modifiers (UBLs), we identify UFM1 as a previously unrecognised regulator of non-homologous end-joining (NHEJ). Using a structure-guided chemical biology strategy, we develop a photo-crosslinkable UFM1 probe and, together with high-resolution NMR, uncover non-canonical UFM1-binding regions in core NHEJ components, including XRCC4. Mechanistically, proximity-dependent proteomics reveals Ku70 as a key UFMylation substrate, establishing a functional axis in which XRCC4 engages UFMylated Ku70 to promote the chromatin assembly of NHEJ factors. Perturbation of UFM1 signalling, via UFSP2 depletion or a hypomorphic UBA5 allele in patient-derived fibroblasts, impairs these processes, linking UFMylation defects to altered regulation of DSB repair. Our findings define a complete UFM1 signalling module in genome maintenance and uncover a molecular connection between hereditary UFMylation disorders and dysregulated DSB repair pathways.

molecular biology↗

bioRxiv2025

A Robust Fluorogenic Substrate for Chikungunya Virus Protease (nsP2) Activity

Chikungunya virus (CHIKV) is an emerging pathogen with pandemic potential. CHIKV infection in humans is transmitted by mosquitoes and induces common symptoms of high fever, arthralgia and myalgia. Because no specific antiviral drugs for treatment of CHIKV infection are available, drug development remains a central goal. The chikungunya virus protease nsP2 (CHIKVP) has emerged as a key drug target due to its indispensable role in viral replication via cleavage of the viral polyprotein. To date, effective tools for screening for CHIKVP inhibitors that reflect the most critical polyprotein cleavage sites have been lacking, hampering drug-development efforts. We found that the recognition ability of CHIKVP is sensitive to the length of peptide substrates. In this study, we report a robust fluorogenic substrate comprising a 15-mer peptide derived from the nsP3/4 junction from the CHIKV polyprotein. This peptide is flanked by an ACC-Lys(dnp) donor-quencher pair. Our new substrate acc-CHIK15-dnp shows a 20-fold improved signal-to-noise ratio as compared to the previously reported edab8 substrate, which is also based on the nsP3/4 junction. We found acc-CHIK15-dnp is recognized only by CHIKVP but not by other alphavirus proteases. This is surprising due to the high level of sequence conservation in the alpha virus polyprotein junctions and indicates that the P-side residues are more important than the P-side sequence for effective CHIKVP cleavage. The robust signal-to-noise ratio obtained using acc-CHIK15-dnp derived from the nsP3/4 cleavage site enabled much improved small molecule HTS on CHIKV relative to other fluorogenic reporters.

biochemistry↗

bioRxiv2025

Exploring the Role of Kv1.3 and MAPK14 in Mediating Microglial Oxidative Stress and Neuroinflammation Following Organic Dust Exposure

BackgroundAgricultural contaminants, including inhaled organic dust (OD) and gases, are known to cause inflammation in the lungs and the brain. We investigated the role of the potassium channel Kv1.3 in organic dust (OD)-induced neuroinflammation models. Kv1.3 channels play a multifaceted role in microglial immune modulation, cancer, neurodegenerative diseases, and constitute a potential therapeutic target. MethodsWe used in vivo (C57BL/6 mice), in vitro (microglial cell line, primary microglia), and ex vivo (brain slice culture) models of OD-induced neuroinflammation. A sterile OD extract (ODE) was prepared, and mice were exposed to either normal saline or ODE intra-nasally for 5 weeks (5 days/week) to simulate an occupational exposure scenario. Primary microglia were isolated from neonatal mice for total RNA sequencing (RNA-seq). The ODE-induced expression of Kv1.3 was quantified using in vitro and ex vivo models with and without PAP-1 treatments. Exposure-induced changes in cytokines and reactive species markers were measured. Using western blot, we quantified phosphorylated p38 MAPK14 (p-p38 MAPK) and NOX2. We measured the currents through Kv1.3 channels using a microglial patch-clamp assay. ResultsExposure to ODE increased the expression of Kv1.3 and p-p38 MAPK in mouse microglia without affecting the Kv1.3 currents at the cell surface. Exposure increased the levels of inflammatory cytokines and NOX2. Kv1.3 inhibition with PAP-1 decreased inflammatory markers (TNF- and IL-6), levels of Kv1.3, p-p38 MAPK, NOX2, and nitrites. ConclusionOur study revealed that pharmacological inhibition of Kv1.3 potassium channels reduces ODE-induced neuroinflammation by decreasing inflammatory and oxidative stress markers.

pharmacology and toxicology↗

bioRxiv2024

Glucose alters the evolutionary response to gentamicin in uropathogenic Escherichia coli

Urinary tract infections (UTI) are a major health and economic concern. Uropathogenic Escherichia coli (UPEC) are the leading cause of UTI, and antibiotic resistant UPEC are increasingly common. The microenvironment of the urinary tract is metabolically distinct, and there is growing interest in understanding the extent to which metabolism may influence UPEC infection and response to antibiotics, and how this varies between individuals. Diabetes, characterised in part by glycosuria, is a known risk factor for UTI and is associated with more severe infections. The role that glucose plays in driving UPEC evolution is unclear. Here, we found that a pathologically-relevant glucose concentration reduced the efficacy of the antibiotic gentamicin against a UPEC strain. Through experimental evolution, we identified mutations in the RNA polymerase sigma factor rpoS associated with long-term glucose exposure. We found that the presence of gentamicin resulted in mutations in genes including trkH, encoding a potassium ion uptake system and linked previously to aminoglycoside resistance, and in the autotransporter hyxB. Strikingly, these mutations were not present in populations exposed to a combination of both glucose and gentamicin. Together, this suggests that whilst glucose may reduce growth inhibition by gentamicin, it may also influence mutation acquisition, providing new avenues for understanding the evolution and treatment of UPEC-mediated UTI in high-risk individuals.

microbiology↗

bioRxiv2024

Designing and bioengineering of CDRs with higher affinity against receptor-binding domain (RBD) of SARS-CoV-2 Omicron variant

The emergence of the SARS-CoV-2 Omicron variant highlights the need for innovative strategies to address evolving viral threats. This study bioengineered three nanobodies H11-H4, C5, and H3 originally targeting the Wuhan RBD, to bind more effectively to the Omicron RBD. A structure-based in silico affinity maturation pipeline was developed to enhance their binding affinities. The pipeline consists of three key steps: high-throughput in silico mutagenesis of complementarity-determining regions (CDRs), protein-protein docking for screening, and molecular dynamics (MD) simulations for assessment of the complex stability. A total of 741, 551, and 684 mutations were introduced in H11-H4, C5, and H3 nanobodies, respectively. Protein-protein docking and MD simulations shortlisted high-affinity mutants for H11-H4(6), C5(5), and H3(6). Further, recombinant production of H11-H4 mutants and Omicron RBD enabled experimental validation through Isothermal Titration Calorimetry (ITC). The H11-H4 mutants R27E, S57D, S107K, D108W, and A110I exhibited improved binding affinities with dissociation constant (KD) values ranging from [~]8.8 to [~]27 {micro}M, compared to the H11-H4 nanobody KD of [~]32 {micro}M, representing a three-fold enhancement. This study demonstrates the potential of the developed in silico affinity maturation pipeline as a rapid, cost-effective method for repurposing nanobodies, aiding the development of robust prophylactic strategies against evolving SARS-CoV-2 variants and other pathogens. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/613860v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1ea8ff7org.highwire.dtl.DTLVardef@91d567org.highwire.dtl.DTLVardef@51629corg.highwire.dtl.DTLVardef@1212752_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Chromatin remodeler BRG1 recruits huntingtin to repair DNA double-strand breaks in neurons

Persistent DNA double-strand breaks (DSBs) are enigmatically implicated in neurodegenerative diseases including Huntingtons disease (HD), the inherited late-onset disorder caused by CAG repeat elongations in Huntingtin (HTT). Here we combine biochemistry, computation and molecular cell biology to unveil a mechanism whereby HTT coordinates a Transcription-Coupled Non-Homologous End-Joining (TC-NHEJ) complex. HTT joins TC-NHEJ proteins PNKP, Ku70/80, and XRCC4 with chromatin remodeler Brahma-related Gene 1 (BRG1) to resolve transcription-associated DSBs in brain. HTT recruitment to DSBs in transcriptionally active gene- rich regions is BRG1-dependent while efficient TC-NHEJ protein recruitment is HTT-dependent. Notably, mHTT compromises TC-NHEJ interactions and repair activity, promoting DSB accumulation in HD tissues. Importantly, HTT or PNKP overexpression restores TC-NHEJ in a Drosophila HD model dramatically improving genome integrity, motor defects, and lifespan. Collective results uncover HTT stimulation of DSB repair by organizing a TC-NHEJ complex that is impaired by mHTT thereby implicating dysregulation of transcription-coupled DSB repair in mHTT pathophysiology. Highlights* BRG1 recruits HTT and NHEJ components to transcriptionally active DSBs. * HTT joins BRG1 and PNKP to efficiently repair transcription related DSBs in brain. * Mutant HTT impairs the functional integrity of TC-NHEJ complex for DSB repair. * HTT expression improves DSB repair, genome integrity and phenotypes in HD flies.

neuroscience↗

bioRxiv2024

NMR structures and magnetic force spectroscopy studies of small molecules binding to models of an RNA CAG repeat expansion

RNA repeat expansions fold into stable structures and cause microsatellite diseases such as Huntingtons disease (HD), myotonic dystrophy type 1 (DM1), and spinocerebellar ataxias (SCAs). The trinucleotide expansion of r(CAG), or r(CAG)exp, causes both HD and SCA3, and the RNAs toxicity has been traced to its translation into polyglutamine (polyQ; HD) as well as aberrant pre-mRNA alternative splicing (SCA3 and HD). Previously, a small molecule, 1, was discovered that binds to r(CAG)exp and rescues aberrant pre-mRNA splicing in patient-derived fibroblasts by freeing proteins bound to the repeats. Here, we report the structures of single r(CAG) repeat motif (5CAG/3GAC where the underlined adenosines form a 1x1 nucleotide internal loop) in complex with 1 and two other small molecules via nuclear magnetic resonance (NMR) spectroscopy combined with simulated annealing. Compound 2 was designed based on the structure of 1 bound to the RNA while 3 was selected as a diverse chemical scaffold. The three complexes, although adopting different 3D binding pockets upon ligand binding, are stabilized by a combination of stacking interactions with the internal loops closing GC base pairs, hydrogen bonds, and van der Waals interactions. Molecular dynamics (MD) simulations performed with NMR-derived restraints show that the RNA is stretched and bent upon ligand binding with significant changes in propeller-twist and opening. Compound 3 has a distinct mode of binding by insertion into the helix, displacing one of the loop nucleotides into the major groove and affording a rod-like shape binding pocket. In contrast, 1 and 2 are groove binders. A series of single molecule magnetic force spectroscopy studies provide a mechanistic explanation for how bioactive compounds might rescue disease-associated cellular phenotypes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/608150v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@145398org.highwire.dtl.DTLVardef@7c37caorg.highwire.dtl.DTLVardef@132cbbeorg.highwire.dtl.DTLVardef@1de59d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Unveiling Mechanistic and Structural Insights of EstS1 Esterase: A Potent Broad-Spectrum Phthalate Diester Degrading Enzyme

The ubiquitous presence of plastics and plasticizers around the globe has raised an alarming condition. Phthalate diesters are high-priority pollutants that mimic natural hormones and act as endocrine disruptors upon entering living systems. While certain bacterial esterases have been identified for their role in phthalate diester degradation, their structural and mechanistic characteristics remain largely unexplored. A thermostable and pH-tolerant EstS1 esterase from Sulfobacillus acidophilus catalyzes the conversion of low molecular weight phthalate diesters to monoesters. This study highlights the unique potential of EstS1 to degrade high molecular weight bis(2-ethylhexyl) phthalate (DEHP) by employing biophysical and biochemical approaches along with in-depth structural analysis utilizing high-resolution crystal structures in both apo and complex forms, with various substrates, products, and their analogs to elucidate mechanistic details. The catalytic tunnel mediating entry and exit of the substrate and product, respectively, centralized the Ser-His-Asp triad performing catalysis by bi-bi ping-pong mechanism, forming a tetrahedral intermediate. Additionally, structural analysis of the polypropylene analog jeffamine with EstS1 revealed effective covalent binding, demonstrating its multifunctional capability. Mutation analysis showed that the Met207Ala mutation abolished DEHP binding at the active site, confirming its essential role in supporting catalysis. These findings underscore the potential of EstS1 as a key tool for advancing technologies aimed at phthalate diesters biodegradation.

biophysics↗

bioRxiv2024

NMR structures of small molecules bound to a model of an RNA CUG repeat expansion

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntingtons disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntingtons disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1x1 nucleotide UU loops (5CUG/3GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5CUG/3GUC motifs in complex with three different small molecules, a diguandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5CUG/3GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.

biochemistry↗