Biology subjects

Natarajan, A.

Publications and source records attributed to Natarajan, A..

At least 19 recordsLinked to original sources

bioRxiv2026

A Multi-Kinase Inhibitor Screen Identifies mTOR Signaling as a Vulnerability in Advanced Prostate Cancer.

The receptor tyrosine kinase AXL has context-dependent roles in prostate cancer (PCa). Although AXL signaling has been implicated in PCa progression, it can also promote disseminated tumor cell dormancy. Paradoxically, AXL expression has been reported to be lost in metastatic PCa. This raises two critical questions: Is AXL loss associated with aggressive disease, and does it create therapeutically exploitable vulnerabilities? In this study, we comprehensively analyzed AXL expression across pan-cancer and PCa cohorts and found that AXL downregulation was significantly associated with treatment resistance, aggressive PCa phenotypes, and shorter overall survival. To investigate the functional consequences of AXL loss, we used CRISPR-Cas9 based gene editing to generate isogenic AXLKO cells and characterized their proliferative and migratory properties. To identify therapeutically exploitable vulnerabilities associated with AXL loss, we screened these cells against a library of 1,814 kinase inhibitors using the isogenic cell lines. We identified Torin-2 as a candidate therapeutic agent and validated its activity using dose-response assays, which demonstrated an ~ 18-fold lower IC50 in AXLKO cells than in AXLWT cells. In parallel, PRISM/DepMap analysis provided orthogonal evidence of drug dependencies associated with low AXL expression and revealed a selective vulnerability of AXLKO cells to PI3K/AKT/mTOR pathway inhibition. Collectively, our findings link AXL downregulation to aggressive PCa phenotypes and identify mTOR inhibition as a potential therapeutic strategy for AXL-deficient PCa.

cancer biology↗

bioRxiv2026

Capsular polysaccharide can sensitize bacteria to non-antibiotic drugs

Medications administered over long durations, such as phenothiazine antipsychotics, accumulate in the gut at concentrations that affect microbial growth. However, the bacterial features influencing sensitivity to these non-antibiotics remain poorly understood. Bacterial capsular polysaccharides (CPSs) typically confer protection against environmental stressors, including chemical, viral, and immunological pressures within the gut. But their roles under non-antibiotic drug pressure are unknown. Here, we show that the K5 CPS of Escherichia coli Nissle 1917 (EcN) sensitizes it to thioridazine (TDZ) and related antipsychotics. Among a panel of E. coli strains grown in minimal medium, EcN exhibited the highest sensitivity to TDZ. Experimentally evolving EcN under gut-relevant TDZ concentrations selected for resistant populations with convergent variants affecting the CPS locus, and TDZ-resistant clones correspondingly had lower CPS expression than their drug-susceptible counterparts. Genetic, transcriptomic, and phenotypic analyses confirmed that the K5 CPS enhances, rather than mitigates, TDZ sensitivity. These findings demonstrate that canonically protective surface structures can become vulnerabilities under non-antibiotic pharmaceutical pressure. Human medications may therefore inadvertently shape the expression and evolution of bacterial surface structures in the gastrointestinal tract, challenging presumptions of CPS-mediated environmental protection.

microbiology↗

bioRxiv2026

A First-In-Class Broad Spectrum Inhibitor of Copper Exporting P1B-type ATPases

Copper (Cu) transporting ATPases represent a highly conserved subclass of P-type ATPases with critical roles in Cu export and metalloenzyme synthesis. Despite their important biological roles and association with a wide range of human diseases, no high-affinity small-molecule inhibitors have been described. Here, we identify MKV3 as a first-in-class inhibitor of Cu-transporting P-type ATPases that targets a conserved Cu+ entry site to the translocation pathway. In silico docking against the Xenopus ATP7B structure revealed a highly conserved pocket suitable for pharmacological inhibition. MKV3 bound human ATP7A and ATP7B with nanomolar affinity, competed with N-terminal metal-binding domains for access to the Cu+ entry site, and selectively inhibited Escherichia coli CopA ATPase activity and Cu+ transport. Mechanistically, MKV3 blocked chaperone-mediated Cu+ delivery to the intramembranous CPC site of CopA that is essential for its transport function. We further identified a single charged P-domain residue that governed MKV3 affinity and potency across species. Functionally, MKV3 phenocopied the genetic loss of Cu+-ATPases in bacteria, fungi, plants, zebrafish, and mammals, impairing copper-dependent enzymes, transporter trafficking, and copper tolerance. These findings establish a conserved, druggable vulnerability in Cu+-ATPases and introduce MKV3 as a broadly active chemical tool to modulate copper homeostasis across biological kingdoms. Significance StatementCopper-transporting P1B-type ATPases are essential for copper homeostasis in all domains of life, yet have lacked pharmacological inhibitors. This work identifies MKV3 as the first small-molecule inhibitor of Cu+-ATPases in bacteria, fungi, plants and animals, and defines a conserved, druggable Cu+ entry pocket that governs metal delivery to the transmembrane pathway. MKV3s ability to potentiate copper-mediated killing in multidrug-resistant bacterial pathogens highlights its potential as an antimicrobial adjuvant, while its attenuation of mammalian ATP7A/B function offers promise in oncology and copper-related diseases. Collectively, these findings establish a new tool for targeting of Cu+-ATPases with wide-ranging applications across biological systems.

cell biology↗

bioRxiv2025

A conserved sRNA regulates mucin adhesion and gut colonization across the Enterococcaceae

Enterococci, particularly E. faecalis, can survive in diverse settings within and outside human hosts. The capacity of E. faecalis to colonize these locations relies on its ability to adapt by altering gene expression in response to environmental exposures. One mechanism for quickly altering gene expression is through regulation by small noncoding RNAs (sRNAs); sRNAs can regulate one or many target genes and either up- or down-regulate transcript stability and protein expression. While many sRNAs have been predicted in E. faecalis, few have experimentally established target mRNAs or physiological functions. Here, we investigate the targets, function, and mechanism of Enterococcus sRNA 84. We found that sRNA 84 is conserved within the family Enterococcaceae, suggesting that it plays a role in the regulation of core genes and functions. RNA sequencing and proteomic analysis revealed that the absence of sRNA 84 led to downregulation of many cell surface proteins, including mucin-binding proteins. Consistent with these findings, an sRNA 84 knockout strain had reduced binding to mucin in vitro and impaired intestinal colonization of specific-pathogen-free mice. Taken together, these data support a model whereby sRNA 84 upregulates cell surface adhesins, which subsequently facilitate host colonization through binding to mucin. sRNA 84 is one of the first sRNAs in enterococci with demonstrated targets and function. This finding establishes the conserved sRNA 84 as a potential key regulator of enterococcal host adaptation, providing insight into how these organisms adapt their gene expression to survive both within and outside animal hosts.

microbiology↗

bioRxiv2025

Age-related microbiome metabolites modulate splicing and chromatin accessibility in the brain

The gut microbiome generates diverse metabolites that can enter the bloodstream and alter host biology, including brain function. Hundreds of physiologically relevant, gut-brain signaling molecules likely exist; however, there has been no systematic, high-throughput effort to identify and validate them. Here, we integrate computational, in vitro, and in vivo approaches to pinpoint microbiome-derived metabolites whose blood levels change during aging, and that induce molecular changes in the mouse brain. First, we mine large-scale metabolomics datasets from human cohorts (each n [≥] 1200) to identify 30 microbiome-associated metabolites whose blood levels change with age. We then screen this panel in an in vitro transcriptomic assay to identify metabolites that perturb genes linked to age-related neurodegeneration. To assess in vivo relevance, we then test four metabolites in male mice by acute exposure, using multi-omic approaches to evaluate the metabolites impact on cellular functions in the brain. With RNA-seq, we confirm known effects of trimethylamine N-oxide (TMAO), including changes in mitochondrial pathways, and further discover its effects on the pathways of glycolysis, GABAergic signaling, and RNA splicing. Additionally, using both RNA- and ATAC-seq, we identify glycodeoxycholate (GDCA), a microbiome-derived secondary bile acid, as a potent regulator of chromatin accessibility and of genes involved in protecting the brain from age-related stressors. GDCA also acutely reduces locomotion in male but not female mice. In summary, we present a generalizable framework for identifying microbiome metabolites that impact host biology, and apply it to identify age-related microbial metabolites that affect processes related to brain aging and neurodegeneration.

neuroscience↗

bioRxiv2025

Phosphorylation by the PknB serine/threonine kinase stimulates dimer formation by WalR

WalKR is a two-component system that regulates cell wall homeostasis and other processes in low-GC Gram-positive bacteria. It is essential for viability in Staphylococcus aureus and regulates genes that encode the autolysins and other critical proteins. The sensor kinase WalK phosphorylates WalR at aspartic acid residue 53 (D53) in the receiver domain, stimulating promoter DNA binding. Unlike other response regulators, WalR is thought to have a second kinase, the serine/threonine kinase PknB, which phosphorylates the receiver domain at threonine residue 101. We previously reported that a walR mutation that changed T101 to a methionine conferred low level resistance to vancomycin and greatly increased susceptibility to tunicamycin along with several other phenotypic traits. In this work we demonstrate similarities between pknB null and the WalRT101M mutants that support a regulatory role for PknB-mediated phosphorylation of WalR. Using a combination of in vitro and in vivo approaches, we confirm the specificity of this posttranslational modification and confirm that phosphorylation at both D53 and T101 is important for dimer formation. Importantly, using an in silico approach, we have discovered that phosphorylation at T101 creates an intermolecular hydrogen bond between the phosphate group and E108, that strengthens contacts along the WalR dimerization interface. This is the first time that a clear molecular role has been assigned to this posttranslational modification and it is the strongest molecular evidence to date for a direct functional relationship between PknB and WalR. We propose that, together with primary activation by WalK, PknB serves as a second potentiating input into the WalKR system, stimulating WalR dimer formation, and target DNA binding and tethering this transcriptional event to important extracellular stimuli.

microbiology↗

bioRxiv2025

Design and Optimization of Multifunctional Peptide Candidates for Cosmeceutical Applications: Combining Anti-Inflammatory and Collagen-Boosting Properties

Skin aging is multifactorial and involves inflammation, oxidative stress, collagen degradation, and impaired metal ion homeostasis, which requires holistic approaches for dermal rejuvenation. Conventional cosmeceutical peptides often target singular pathways, limiting their efficacy. In this study, we present a computational strategy for designing and evaluating multifunctional peptides that can simultaneously modulate collagen remodelling, exhibit anti-inflammatory responses, and maintain extracellular matrix (ECM) stability. We focused on three important skin-associated proteins: TGF-{beta} receptor II, Integrin 5{beta}1, and MMP-1, and generated peptide sequences specific to them through a de novo sequence generation method that utilized protein-specific profiles alongside machine learning-based property screening. Over 15,000 peptides were generated and filtered using physicochemical and synthesis-based criteria (charge, hydrophobicity, pI, solubility, and synthesis rules). A multi-parametric composite score was developed to visualize peptide favourability in a 3D physicochemical space. The top peptides from each target set were subjected to structure prediction and docking, resulting in binding affinities that ranged from -19.8 to -34.5 kcal/mol. Cross-target docking analysis demonstrated a significant potential for dual or pan-target applications, indicating the broad-spectrum utility of the cosmeceutical. This study firmly establishes a foundation for multifunctional peptide candidates that combine collagen-boosting, anti-inflammatory, and MMP-inhibitory effects. These candidates are poised to lead the way for innovative skincare formulations, paving the path for future validation and experimental evidence that will undeniably strengthen our findings.

bioinformatics↗

bioRxiv2025

A New Class of Precision Therapeutics that Inhibit Prostate Cancer Mediated Bone Destruction

Cancer mediated bone destruction remains a common cause of morbidity and mortality. Here we couple an inhibitor of cell movement to bone trophic bisphosphonates, forming heterobifunctional compounds termed Dual Acting Bone Defender agents (DABDs). DABDs inhibit prostate cancer cell motility and osteoclast mediated bone destruction in vitro. Our lead compound, BTE-EN1, inhibits circulating human prostate cancer cells from forming bone destructive lesions in mice and prolongs life in a dose-dependent fashion. In mice with established metastatic lesions, BTE-EN1 significantly decreases bone destruction by over 60% compared to the control. Mechanistically, BTE-EN1 binds hydroxyapatite, inhibits prenylation of Rap1A, induces apoptosis in osteoclasts, and inhibits Raf1 activation and formation of bone resorptive cavities in osteoclasts, thus retaining the function of its constituent chemicals. BTE-EN1 is not toxic to mice at doses 4,000-fold over those required for efficacy, exhibits anti-cell motility activity across other bone-destructive cancer cell types, and does not interfere with standard-of-care hormone therapy or chemotherapy. BTE-EN1 is a new class of highly active therapeutics that inhibit cancer mediated bone destruction and holds high potential for translation into humans.

cancer biology↗

bioRxiv2025

A pan-cancer atlas of T cell targets

T-cell-based immunotherapies have revolutionized cancer treatment, yet only a minority of patients are eligible for these approaches, significantly constrained by the limited knowledge of tumor-specific antigens. Here we present ImmunoVerse, a comprehensive map of T cell targets across 21 cancer types, revealing actionable tumor-specific targets in 89% of tumors analyzed. To define the repertoire of actionable T cell targets, we conducted an exhaustive pan-cancer analysis, integrating data from 7,188 RNA-Seq, 1,771 immunopeptidomes from 512 biological samples and 208 single-cell cancer datasets using novel AI methods, and compared these against 17,384 normal samples covering 51 tissues. Our analysis uncovered 62 viable surface protein targets and 28,446 tumor-specific HLA-presented antigens, deriving from 11 distinct molecular events, across 21 tumor types. Among these, we identified 5,928 previously uncharacterized neoantigens, new tumor self-antigens, peptides derived from tumor-specific cryptic ORFs, tumor-associated microbial targets and a novel splicing-derived PMEL peptide (sPMEL) with enhanced abundance and safety compared to the canonical clinical targets. We successfully expanded sPMEL-specific T cells, validating the therapeutic potential of these targets in functional assays. We highlight 153 promising new tumor targets and experimentally validate 19 targets representing six antigen classes. In addition to being the most comprehensive atlas of targets in scope, ImmunoVerse offers the most extensively annotated resource with key parameters for target selection, providing critical insights for therapeutic prioritization and clinical translation. To catalyze therapeutic development, we released our pan-cancer target atlas through an interactive web portal (https://www.immuno-verse.com) and made the accompanying toolkits available to the scientific community. This work redefines the landscape of therapeutic T cell targets and provides a foundational resource to unlock immunotherapy development across multiple cancers previously considered intractable.

cancer biology↗

bioRxiv2024

Small molecule targeting of FBXO21 mediated p85αubiquitylation in acute myeloid leukemia

PI3K inhibitors that target the catalytic sub-unit p110 are used in cancer therapy to inhibit overactive PI3K signaling pathway. However, their clinical use is limited by severe adverse effects and development of resistance, highlighting the need for further research on regulation of the PI3K pathway. We have identified that FBXO21 ubiquitinates regulatory PI3K subunit p85, and silencing of FBXO21 inhibits canonical PI3K signaling. To target FBXO21, we developed a small molecule designed to interfere with substrate:ligase interaction. Our novel small molecule effectively blocks p85 ubiquitination leading to decreased PI3K pathway activation and cell death in acute myeloid leukemia (AML). Moreover, our studies demonstrate selectivity for AML cells over healthy counterparts, and elimination of AML in vivo, emphasizing FBXO21s potential as a promising therapeutic target for AML. Targeting substrate:ligase interactions provide new avenues for drug discovery that may enhance the efficacy of current therapies and benefits for improving patient outcomes. STATEMENT OF SIGNIFICANCEOur studies highlight the potential of the ubiquitin E3 ligase FBXO21 as an alternative therapeutic target for the PI3K signaling pathway, not only in AML but in cancers with aberrant PI3K signaling.

cancer biology↗

bioRxiv2024

Fluorescence based live cell imaging identifies exon 14 skipped hepatocyte growth factor receptor (MET) degraders

Despite ongoing efforts to employ structure-based methods to discover targeted protein degraders (TPD), the prevailing strategy continues to be the synthesis of a focused set of heterobifunctional compounds and screen them for target protein degradation. Here we used a fluorescence based live cell imaging screen to identify degraders that target exon 14 skipped hepatocyte growth factor receptor (MET). MET is a known oncogenic driver. MET exon 14 skipping mutations (METex14{Delta}) are found in lung cancers and result in the loss of a degron that is required for E3-ligase recognition and subsequent ubiquitination, prolonging the half-life and oncogenicity of MET. Since proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules that promote target degradation by the proteosome, we sought to restore degradation of MET lost with METex14{Delta} using a MET-targeting PROTAC. We generated a library of sixty PROTACs of which 37 used the MET inhibitor capmatinib as the protein of interest targeting ligand. We screened this PROTAC library for targeted degradation of METex14{Delta}-GFP using live cell imaging. We benchmarked out MET-targeting PROTACs to that of a previously reported MET-targeting PROTAC, SJF8240. Curve fitting live cell imaging data affords determination of time required to degrade 50% of the target protein (DT50), which was used in determining structure activity relationships. A promising candidate, 48-284, identified from the screen, exhibited classic PROTAC characteristics, was > 15-fold more potent than SJF8240, had fewer off targets compared to SJF8240, and degraded MET in multiple cell lines.

cancer biology↗

bioRxiv2024

The role of Tyr34 in proton-coupled electron transfer of human manganese superoxide dismutase

Human manganese superoxide dismutase (MnSOD) plays a crucial role in controlling levels of reactive oxygen species (ROS) by converting superoxide (O2*-) to molecular oxygen (O2) and hydrogen peroxide (H2O2) with proton-coupled electron transfers (PCETs). The reactivity of human MnSOD is determined by the state of a key catalytic residue, Tyr34, that becomes post-translationally inactivated by nitration in various diseases associated with mitochondrial dysfunction. We previously reported that Tyr34 has an unusual pKa due to its proximity to the Mn metal and undergoes cyclic deprotonation and protonation events to promote the electron transfers of MnSOD. To shed light on the role of Tyr34 MnSOD catalysis, we performed neutron diffraction, X-ray spectroscopy, and quantum chemistry calculations of Tyr34Phe MnSOD in various enzymatic states. The data identifies the contributions of Tyr34 in MnSOD activity that support mitochondrial function and presents a thorough characterization of how a single tyrosine modulates PCET catalysis.

biochemistry↗

bioRxiv2024

Protein Structure Inspired Drug Discovery

Drug discovery starts with known function, either of a compound or a protein, in-turn prompting investigations to probe 3D structure of the compound-protein interface. As protein structure determines function, we hypothesized that unique 3D structural motifs represent primary information denoting unique function that can drive discovery of novel agents. Using a physics-based protein structure analysis platform developed by us, designed to conduct computationally intensive analysis at supercomputing speeds, we probed a high-resolution protein x-ray crystallographic library developed by us. We selected 3D structural motifs whose function was not otherwise established, that offered environments supporting binding of drug-like chemicals and were present on proteins that were not established therapeutic targets. For each of eight potential binding pockets on six different proteins we accessed a 60 million compound library and used our analysis platform to evaluate binding. Using eight-day colony formation assays acquired compounds were screened for efficacy against human breast, prostate, colon and lung cancer cells and toxicity against human bone marrow stem cells. Compounds selectively inhibiting cancer growth segregated to two pockets on separate proteins. The compound, Dxr2-017, exhibited selective activity against human melanoma cells in the NCI-60 cell line screen, had an IC50 of 19 nM against human melanoma M14 cells in our eight-day assay, while over 2100-fold higher concentrations inhibited stem cells by less than 30%. We show that Dxr2-017 induces anoikis, a unique form of programmed cell death in need of targeted therapeutics. The predicted target protein for Dxr2-017 is expressed in bacteria, not in humans. This supports our strategy of focusing on unique 3D structural motifs. It is known that functionally important 3D structures are evolutionarily conserved. Here we demonstrate proof-of-concept that protein structure represents high value primary data to support discovery of novel therapeutics. This approach is widely applicable. Author summaryWe introduce the concept that protein 3D structure represents primary information which can support downstream investigations, in this instance leading to the discovery of novel anticancer therapeutics.

cancer biology↗

bioRxiv2024

Novel spirocyclic dimer, SpiD3, targets chronic lymphocytic leukemia survival pathways with potent preclinical effects

Chronic lymphocytic leukemia (CLL) cell survival and growth is fueled by the induction of B-cell receptor (BCR) signaling within the tumor microenvironment (TME) driving activation of NF- {kappa}B signaling and the unfolded protein response (UPR). Malignant cells have higher basal levels of UPR posing a unique therapeutic window to combat CLL cell growth using pharmacological agents that induce accumulation of misfolded proteins. Frontline CLL therapeutics that directly target BCR signaling such as Bruton-tyrosine kinase (BTK) inhibitors (e.g., ibrutinib) have enhanced patient survival. However, resistance mechanisms wherein tumor cells bypass BTK inhibition through acquired BTK mutations, and/or activation of alternative survival mechanisms have rendered ibrutinib ineffective, imposing the need for novel therapeutics. We evaluated SpiD3, a novel spirocyclic dimer, in CLL cell lines, patient-derived CLL samples, ibrutinib-resistant CLL cells, and in the E{micro}-TCL1 mouse model. Our integrated multi-omics and functional analyses revealed BCR signaling, NF-{kappa}B signaling, and endoplasmic reticulum stress among the top pathways modulated by SpiD3. This was accompanied by marked upregulation of the UPR and inhibition of global protein synthesis in CLL cell lines and patient-derived CLL cells. In ibrutinib-resistant CLL cells, SpiD3 retained its anti-leukemic effects, mirrored in reduced activation of key proliferative pathways (e.g., PRAS, ERK, MYC). Translationally, we observed reduced tumor burden in SpiD3-treated E{micro}-TCL1 mice. Our findings reveal that SpiD3 exploits critical vulnerabilities in CLL cells including NF-{kappa}B signaling and the UPR, culminating in profound anti-tumor properties independent of TME stimuli. STATEMENT OF SIGNIFICANCESpiD3 demonstrates cytotoxicity in CLL partially through inhibition of NF-{kappa}B signaling independent of tumor-supportive stimuli. By inducing the accumulation of unfolded proteins, SpiD3 activates the UPR and hinders protein synthesis in CLL cells. Overall, SpiD3 exploits critical CLL vulnerabilities (i.e., the NF-{kappa}B pathway and UPR) highlighting its use in drug-resistant CLL. VISUAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=137 HEIGHT=200 SRC="FIGDIR/small/578283v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1dd7a38org.highwire.dtl.DTLVardef@1831c85org.highwire.dtl.DTLVardef@c571f9org.highwire.dtl.DTLVardef@bdce53_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

Design, synthesis, and evaluation of a mitoxantrone probe (MXP) for biological studies

Mitoxantrone (MX) is a robust chemotherapeutic with well-characterized applications in treating certain leukemias and advanced breast and prostate cancers. The canonical mechanism of action associated with MX is its ability to intercalate DNA and inhibit topoisomerase II, giving it the designation of a topoisomerase II poison. Years after FDA approval, investigations have unveiled novel protein-binding partners, such as methyl-CpG-binding domain protein (MBD2), PIM1 serine/threonine kinase, RAD52, and others that may contribute to the therapeutic profile of MX. Moreover, recent proteomic studies have revealed MXs ability to modulate protein expression, illuminating the complex cellular interactions of MX. Although mechanistically relevant, the differential expression across the proteome does not address the direct interaction with potential binding partners. Identification and characterization of these MX-binding cellular partners will provide the molecular basis for the alternate mechanisms that influence MXs cytotoxicity. Here, we describe the design and synthesis of a MX-biotin probe (MXP) and negative control (MXP-NC) that can be used to define MXs cellular targets and expand our understanding of the proteome-wide profile for MX. In proof of concept studies, we used MXP to successfully isolate a recently identified protein-binding partner of MX, RAD52, in a cell lysate pulldown with streptavidin beads and western blotting. Graphical abstract (Draft) O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY HighlightsO_LIAn 8-step synthesis was used to generate a biotinylated-mitoxantrone probe (MXP). C_LIO_LIA pulldown of MXP demonstrated selectivity for RAD52, but not Replication Protein A. C_LIO_LIWestern blot confirmed the identity of the isolated protein, RAD52. C_LI

biochemistry↗

bioRxiv2023

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is 1) strictly dependent on pyrimidine nucleotide depletion, 2) independent of canonical antigen presentation pathway transcriptional regulators, and 3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

cancer biology↗

bioRxiv2023

Tomato brown rugose fruit virus Mo gene is a novel microbial source tracking marker

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using fecal host-associated markers. While there are numerous bacterial MST markers, there are few viral markers. Here we design and test novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States of America. Next, we developed two novel probe-based RT-PCR assays based on conserved regions of the ToBRFV genome, and tested the markers sensitivities and specificities using human and non-human animal stool as well as wastewater. TheToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a currently used marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We applied the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, ToBRFV is a promising viral human-associated MST marker. ImportanceHuman exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of fecal host-associated MST markers. Here we design and test novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool, and highly abundant in human stool and wastewater samples.

microbiology↗

bioRxiv2022

EPEK: creation and analysis of an Ectopic Pregnancy Expression Knowledgebase

Ectopic pregnancy (EP) is one of the leading causes of maternal mortality, where the fertilized embryo grows outside of the uterus. Recent experiments on mice have uncovered the importance of genetic factors in the transport of embryos inside the uterus. In the past, efforts have been made to identify possible gene or protein markers in EP in humans through multiple expression studies. Although there exist comprehensive gene resources for other maternal health disorders, there is no specific resource that compiles the genes associated with EP from such expression studies. Here, we address that knowledge gap by creating a computational resource, Ectopic Pregnancy Expression Knowledgebase (EPEK), that involves manual compilation and curation of expression profiles of EP in humans from published articles. In EPEK, we compiled information on 314 differentially expressed genes, 17 metabolites, and 3 SNPs associated with EP. Computational analyses on the gene set from EPEK showed the implication of cellular signaling processes in EP. We also identified possible exosome markers that could be clinically relevant in the diagnosis of EP. In a nutshell, EPEK is the first and only dedicated resource on the expression profile of EP in humans. EPEK is accessible at https://cb.imsc.res.in/epek.

bioinformatics↗