Biology subjects

Casey, A.

Publications and source records attributed to Casey, A..

14 recordsLinked to original sources

bioRxiv2025

Inhibition of FicD-mediated AMPylation and deAMPylation by Isoprenoid Diphosphates

FicD regulates Unfolded Protein Response (UPR) through reversible AMPylation and deAMPylation of BiP, an HSP70 chaperone and master regulator of the UPR. FicD activity is regulated by ER-stress, catalyzing BiP AMPylation under low stress conditions to hold inactive chaperone in reserve. In stressed cells, FicD deAMPylates BiP, acutely increasing its active pool to assist in protein folding. Variants in UPR machinery, including those in the FicD gene, are linked to hereditary diseases. Despite the known role of FicD in UPR, in-vivo regulation of its activity remains elusive, and identifying metabolites that alter FicD activity could prove useful pharmaceutically. We applied an unbiased high-throughput screening platform, known as Mass spectrometry Integrated with equilibrium Dialysis for the discovery of Allostery Systematically (MIDAS), to identify novel small molecule metabolites that might regulate FicD activity. MIDAS revealed interactions between FicD and two mavelonate pathway intermediates : geranyl-pyrophosphate and farnesyl-pyrophosphate. Biochemical characterization indicates that both potently inhibit FicD-mediated AMPylation and deAMPylation. The crystal structure of FicD bound to farnesyl-pyrophosphate demonstrates a competitive inhibition mechanism, with the pyrophosphate adopting the alpha and beta phosphate positions of ATP and the hydrocarbon chain filling the nucleoside pocket. FicD variants previously appeared as biochemically indistinguishable, yet lead to different human pathologies. We demonstrate farnesyl-pyrophosphate inhibits FicDR374H and FicDR374C variants implicated in causing hereditary spastic paraplegia, but not the FicDR371S variant associated with neonatal diabetes. This study furthers our understanding of FicD inhibitors and distinguishes disease causing variants, providing insight into pharmacological targeting of UPR activity. Significance StatementFicD regulates UPR signaling in metazoans by fine-tuning BiP chaperone capacity. Therefore, targeting FicD activity may be a tractable method of altering UPR signaling for therapeutic benefit. We identify geranyl- and farnesyl-pyrophosphate as specific FicD inhibitors. Notably, these small molecules differentially inhibit disease-causing variants of FicD. A structure of farnesyl-pyrophosphate bound to the FicD active site helps explain the differential inhibition of pathogenic variants and provides insight into interactions that can be differentially exploited for modifying FicD activity. Their composition provides a novel chemical foundation for future drug development efforts targeting FicD activity.

biochemistry↗

bioRxiv2024

Pan-cancer N-glycoproteomic atlas of patient derived xenografts uncovers FAT2 as a therapeutic target for head and neck cancers

Cell surface proteins offer significant cancer therapeutic potential attributable to their accessible membrane localization and central role in cellular signaling. Despite this, their promise remains largely untapped due to the technical challenges inherent to profiling cell surface proteins. Here, we employed N-glycoproteomics to analyze 85 patient-derived xenografts (PDX), constructing Glyco PDXplorer - an in vivo pan-cancer atlas of cancer-derived cell surface proteins. We developed a target discovery pipeline to prioritize proteins with favorable expression profiles for immunotherapeutic targeting and validated FAT2 as a head and neck squamous cancer (HNSC) enriched surface protein with limited expression in normal tissue. Functional studies revealed that FAT2 is essential for HNSC growth and adhesion through regulation of surface architecture and integrin-PI3K signaling. Chimeric antigen receptor (CAR) T cells targeting FAT2 demonstrated potent anti-tumor activity in HNSC models. This work lays the foundation for developing FAT2-targeted therapies and represents a pivotal resource to inform therapeutic target discovery for multiple cancers. HIGHLIGHTSO_LIPan-cancer landscape of cancer-derived cell surface proteins detected in vivo C_LIO_LIDevelopment of a multi-omic discovery pipeline to prioritize proteins with optimal expression profiles as immunotherapy targets C_LIO_LIIdentification and validation of FAT2 as a head and neck squamous cancer enriched surface protein with minimal expression in normal tissues C_LIO_LIFAT2 coordinates cell surface organization, adhesion, growth and survival through the integrin-PI3K-AKT pathway C_LIO_LIFAT2 CAR T cells demonstrate anti-tumour activity in pre-clinical models C_LI

cancer biology↗

bioRxiv2024

Longitudinal genomic surveillance of a UK intensive care unit shows a lack of patient colonisation by multi-drug resistant Gram-negative pathogens

Vulnerable patients in an intensive care unit (ICU) setting are at high risk of infection from bacteria including gut-colonising Escherichia coli and Klebsiella species. Complex ICU procedures often depend on successful antimicrobial treatment, underscoring the importance of understanding the extent of patient colonisation by multi-drug resistant organisms (MDRO) in large UK ICUs. Previous work on ICUs globally uncovered high rates of colonisation by and transmission of MDRO, but the situation in UK ICUs is less understood. Here, we investigated the diversity and antibiotic resistance gene (ARG) carriage of bacteria present in one of the largest UK ICUs at the Queen Elizabeth Hospital Birmingham (QEHB), focussing primarily on E. coli as both a widespread commensal and a globally disseminated multidrug resistant pathogen. Samples were taken during highly restrictive COVID-19 control measures from May - December 2021. Whole-genome and metagenomic sequencing were used to detect and report strain level colonisation of patients, focussing on E. coli sequence types (STs), their colonisation dynamics, and antimicrobial resistance (AMR) gene carriage. We found a lack of multidrug resistance (MDR) in the QEHB. Only one carbapenemase-producing organism was isolated, a Citrobacter carrying blaKPC-2. There was no evidence supporting the spread of this strain, and there was little evidence overall of nosocomial acquisition or circulation of colonising E. coli. Whilst 22 different E. coli STs were identified, only one strain of the pandemic ST131 lineage was isolated. This ST131 strain was non-MDR and was found to be a clade A strain, associated with low levels of antibiotic resistance. Overall, the QEHB ICU had very low levels of pandemic or MDR strains, a result which may be influenced in part by the strict COVID-19 control measures in place at the time. Employing some of these infection prevention and control measures where reasonable in all ICUs might therefore assist in maintaining low levels of nosocomial MDR. Impact statementThis study contributes to current literature on the potential routes for AMR spread in a healthcare setting. This study used whole genome sequencing (WGS) to investigate at strain-level bacterial species (including E. coli) colonising the gut of long-stay patients in the ICU. WGS in combination with patient ward movement and prescribing information was used to assess any links or driving factors in strain acquisition and AMR spread in the ICU. Our study gives an insight at a point in time where infection and prevention control restrictions and awareness were high due to the COVID-19 pandemic, combined with local and national travel restrictions and isolation criteria. Consequently, it provides a novel longitudinal dataset that gives a picture of colonising E. coli in a sheltered ICU patient population. Trends seen in this E. coli population are likely linked to the United Kingdom in 2021 rather than the global picture that may have been seen prior to the COVID-19 pandemic. Data summaryAll supporting data, code and protocols have been provided within the article or through supplementary data files. All genomic and metagenomic data are available from NCBI under BioProject accession PRJNA1136496. All relevant metadata is provided in supplementary data files.

microbiology↗

bioRxiv2023

Small molecule nitroalkenes inhibit RAD51-mediated homologous recombination and amplify triple-negative breast cancer cell killing by DNA-directed therapies.

Nitro fatty acids (NO2-FAs) are endogenously generated lipid signaling mediators from metabolic and inflammatory reactions between conjugated diene fatty acids and nitric oxide or nitrite-derived reactive species. NO2-FAs undergo reversible Michael addition with hyperreactive protein cysteine thiolates to induce posttranslational protein modifications that can impact protein function. Herein, we report a novel mechanism of action of natural and non-natural nitroalkenes structurally similar to (E) 10-nitro-octadec-9-enoic acid (CP-6), recently de-risked by preclinical Investigational New Drug-enabling studies and Phase 1 and Phase 2 clinical trials and found to induce DNA damage in a TNBC xenograft by inhibiting homologous-recombination (HR)-mediated repair of DNA double-strand breaks (DSB). CP-6 specifically targets Cys319, essential in RAD51-controlled HR-mediated DNA DSB repair in cells. A nitroalkene library screen identified two structurally different nitroalkenes, a non-natural fatty acid [(E) 8-nitro- nonadec-7-enoic acid (CP-8)] and a dicarboxylate ester [dimethyl (E)nitro-oct-4-enedioate (CP- 23)] superior to CP-6 in TNBC cells killing, synergism with three different inhibitors of the poly ADP-ribose polymerase (PARP) and {gamma}-IR. CP-8 and CP-23 effectively inhibited {gamma}-IR-induced RAD51 foci formation and HR in a GFP-reported assay but did not affect benign human epithelial cells or cell cycle phases. In vivo, CP-8 and CP-23s efficacies diverged as only CP-8 showed promising anticancer activities alone and combined with the PARP inhibitor talazoparib in an HR-proficient TNBC mouse model. As preliminary preclinical toxicology analysis also suggests CP-8 as safe, our data endorse CP-8 as a novel anticancer molecule for treating cancers sensitive to homologous recombination-mediated DNA repair inhibitors. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=172 SRC="FIGDIR/small/552990v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1dcec56org.highwire.dtl.DTLVardef@1202a09org.highwire.dtl.DTLVardef@a7e7b6org.highwire.dtl.DTLVardef@15d8925_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2023

Integrative transcriptomic and metabolic analyses of the mammalian hibernating brain identifies a key role for succinate dehydrogenase in ischemic tolerance

Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites1. Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus, provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage2. Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and -ketoglutarate-KG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo. Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.

neuroscience↗

bioRxiv2022

Transcriptome changes in chlorsulfuron-treated plants are caused by acetolactate synthase inhibition and not induction of a herbicide detoxification system in Marchantia polymorpha

A sensing mechanism in mammals perceives xenobiotics and induces the transcription of genes encoding proteins that detoxify these molecules. However, it is unclear if plants sense xenobiotics, and activate an analogous signalling system leading to their detoxification. Using the liverwort Marchantia polymorpha, we tested the hypothesis that there is a sensing system in plants that perceives herbicides resulting in the increased transcription of genes encoding proteins that detoxify these herbicides. Consistent with the hypothesis, we show that chlorsulfuron-treatment induces changes in the M. polymorpha transcriptome. However, these transcriptome changes do not occur in chlorsulfuron (CS)-treated target site resistant mutants, where the gene encoding the target carries a mutation that confers resistance to chlorsulfuron. Instead, we show that inactivation of the chlorsulfuron target, acetolactate synthase (ALS) (also known as acetohydroxyacid synthase (AHAS)), is required for the transcriptome response. These data are consistent with the changes in the transcriptome of chlorsulfuron-treated plants being caused by disrupted amino acid synthesis and metabolism resulting from acetolactate synthase inhibition. These conclusions suggest that chlorsulfuron is not sensed in M. polymorpha leading to induce a detoxification system. Author SummaryHerbicide use is increasing throughout the world, however we know little about how plants respond to herbicide treatment and regulate their metabolism. Some plants have evolved resistance to herbicides such as chlorsulfuron by increasing the detoxification of the herbicide compared to sensitive plants. It has been suggested that plants can directly sense the herbicide chemical which activates a detoxification response, in a similar way to the detoxification of foreign chemicals in mammalian cells. The liverwort Marchantia polymorpha is an excellent system to study plant herbicide responses due to its short generation time, ease of propagation and low genetic redundancy. We show that chlorsulfuron treatment alters the expression of many genes in M. polymorpha, however plants with a resistance-conferring mutation in the molecular target of chlorsulfuron do not show any changes in gene expression in response to chlorsulfuron treatment. This result indicates that transcriptome changes caused by chlorsulfuron depend on the inhibition of the target by chlorsulfuron. This suggests that plants do not sense chlorsulfuron and activate a detoxification system. This finding has implications for herbicide use and discovery.

plant biology↗

bioRxiv2022

Genes encoding cytochrome P450 monooxygenases and glutathione S-transferases associated with herbicide resistance evolved before the origin of land plants

Cytochrome P450 (CYP) monooxygenases and glutathione S-transferases (GST) are enzymes that catalyse chemical modifications of a range of organic compounds. Herbicide tolerance is associated with higher levels of CYP and GST gene expression in some herbicide-resistant weed populations compared to sensitive populations of the same species. By comparing the protein sequences of 9 representative species of the Archaeplastida - the lineage which includes red algae, glaucophyte algae, chlorophyte algae, and streptophytes - and generating phylogenetic trees, we identified the CYP and GST proteins that existed in the common ancestor of the Archaeplastida. All CYP clans and all but one land plant GST classes present in land plants evolved before the divergence of streptophyte algae and land plants from their last common ancestor. We also demonstrate that there are more genes encoding CYP and GST proteins in land plants than in algae. The larger numbers of genes among land plants largely results from gene duplications in CYP clans 71, 72, and 85 and in the GST Phi and Tau classes. Enzymes that either chemically alter herbicides or confer herbicide resistance belong to CYP clans 71 and 72 and the GST Phi and Tau classes. These results demonstrate that the clan and class diversity in extant plant CYP and GST proteins evolved in the Proterozoic before the divergence of land plants and streptophyte algae from a last common ancestor. Then, early in embryophyte evolution during the Palaeozoic, gene duplication in four of the twelve CYP clans, and in two of the fourteen GST classes, led to the large numbers of CYP and GST proteins found in extant land plants. It is among the genes of CYP clans 71 and 72 and GST classes Phi and Tau that alleles conferring herbicide resistance evolved in the last fifty years.

plant biology↗

bioRxiv2022

Transcriptional targets of senataxin and E2 promoter binding factors are associated with neuro-degenerative pathways during increased autophagic flux

Autophagy is an intracellular recycling process that degrades harmful molecules, maintains optimal composition of cellular organelles and enables survival during starvation. Previous studies have demonstrated how transcription factors (TFs) can increase autophagy with therapeutic potential (impaired autophagy in the ageing brain, in particular, may be an important risk factor for dementia). To investigate the transcriptional regulation of autophagy from a systems perspective, we induced autophagy by amino acid starvation and mTOR inhibition in HeLa, HEK 293 and SH-SY5Y cells and used RNA-seq to measure gene expression at three time points. We observed 453 differentially expressed (DE) genes due to starvation and 284 genes due to mTOR inhibition (PFDR < 0.05 in every cell line). Pathway analyses confirmed enrichment of genes implicated in Alzheimers (PFDR < 0.001 in SH-SY5Y and HeLa) and Parkinsons (PFDR [≤] 0.024 in SH-SY5Y and HeLa) diseases and amyotrophic lateral sclerosis (ALS, PFDR < 0.05 in 4 of 6 experiments). We then integrated Signaling Pathway Impact Analysis and TF target enrichment testing to predict which TF target genes were contributing to pathway perturbation. Differential expression of the Senataxin (SETX) target gene set was predicted to activate multiple neurodegenerative pathways (PFDR [≤] 0.04). Notably, SETX is a causal gene for a rare form of ALS. In the SH-SY5Y cells of neuronal origin, the E2F transcription family was predicted to activate Alzheimers disease pathway (PFDR [≤] 0.0065). SETX and E2F may be important mediators of transcriptional regulation of autophagy and may provide new therapeutic opportunities for neuro-degenerative conditions.

genomics↗

bioRxiv2021

Nrf2 is a central regulator of the metabolic landscape in macrophages and finetunes their inflammatory response

To overcome oxidative, inflammatory, and metabolic stress, cells have evolved networks of cytoprotective proteins controlled by nuclear factor erythroid 2 p45-related factor 2 (Nrf2) and its main negative regulator the Kelch-like ECH associated protein 1 (Keap1). Here, we used high-resolution mass-spectrometry to characterize the proteomes of macrophages with genetically altered Nrf2 status. Our analysis revealed significant differences among the genotypes in cellular metabolism and redox homeostasis, which we validated with respirometry and metabolomics, as well as in anti-viral immune pathways and the cell cycle. Nrf2 status significantly affected the proteome following lipopolysaccharide (LPS) stimulation, with alterations in redox, carbohydrate and lipid metabolism, and innate immunity observed. Of note, Nrf2 activation was found to promote mitochondrial fusion in inflammatory macrophages. The Keap1 inhibitor, 4-octyl itaconate (4-OI), a derivative of the mitochondrial immunometabolite itaconate, remodeled the inflammatory macrophage proteome, increasing redox and suppressing anti-viral immune effectors in a Nrf2-dependent manner. These data suggest that Nrf2 activation facilitates metabolic reprogramming and mitochondrial adaptation, and finetunes the innate immune response in macrophages. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/456204v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1f5bbb8org.highwire.dtl.DTLVardef@1f7311dorg.highwire.dtl.DTLVardef@1b9722forg.highwire.dtl.DTLVardef@1f777d4_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIFirst high-resolution proteome of macrophages with genetically altered Nrf2 status C_LIO_LINrf2 is key regulator of macrophage redox and intermediary metabolism C_LIO_LINrf2 finetunes the inflammatory response suppressing anti-viral immune and cytokine effectors, whilst promoting T cell activation factors C_LIO_LINrf2 regulates mitochondrial adaptation in inflammatory macrophages promoting the formation of a fused network C_LIO_LI4-octyl itaconate (4-OI) suppresses anti-viral immune effectors in inflammatory macrophages in a Nrf2-dependent manner C_LI

immunology↗

bioRxiv2020

Structures of an intact yeast V-ATPase alone and in complex with bacterial effector VopQ

Vacuolar-type ATPase (V-ATPase) is a rotary protein pump involved in proton translocation across various cellular membranes using the energy of ATP hydrolysis. Despite previous studies on bacterial and eukaryotic V-ATPases, information on the intact structure of a eukaryotic V-ATPase is missing. Here we report cryo-EM structures of the intact yeast V-ATPase and this complex bound to a bacterial effector. We reveal the interaction of the elusive regulatory subunit H with its neighboring subunits. Insight for the catalysis mechanism is gained by determining conformations of the catalytic subunits either empty or bound with nucleotides.

biophysics↗

bioRxiv2020

Cold Atmospheric Plasma induces silver nanoparticle uptake, oxidative dissolution and enhanced cytotoxicity in Glioblastoma multiforme cells

Silver nanoparticles (AgNP) emerged as a promising reagent for cancer therapy with oxidative stress implicated in the toxicity. Meanwhile, studies reported cold atmospheric plasma (CAP) generation of reactive oxygen and nitrogen species has selectivity towards cancer cells. Gold nanoparticles display synergistic cytotoxicity when combined with CAP against cancer cells but there is a paucity of information using AgNP, prompting to investigate the combined effects of CAP using dielectric barrier discharge system (voltage of 75 kV, current is 62.5mA, duty cycle of 7.5kVA and input frequency of 50-60Hz) and 10nm PVA-coated AgNP using U373MG Glioblastoma Multiforme cells. Cytotoxicity in U373MG cells was >100-fold greater when treated with both CAP and PVA-AgNP compared with either therapy alone (IC50 of 4.30 g/mL with PVA-AgNP alone compared with 0.07 g/mL after 25s CAP and 0.01 g/mL 40s CAP). Combined cytotoxicity was ROS-dependent and was prevented using N-Acetyl Cysteine. A novel darkfield spectral imaging method investigated and quantified AgNP uptake in cells determining significantly enhanced uptake, aggregation and subcellular accumulation following CAP treatment, which was confirmed and quantified using atomic absorption spectroscopy. The results indicate that CAP decreases nanoparticle size, decreases surface charge distribution of AgNP and induces uptake, aggregation and enhanced cytotoxicity in vitro. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=65 SRC="FIGDIR/small/969758v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@6cacdcorg.highwire.dtl.DTLVardef@c8c9caorg.highwire.dtl.DTLVardef@381c5dorg.highwire.dtl.DTLVardef@1ce1ece_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2019

Loss of microbial diversity and pathogen domination of the gut microbiota in critically ill patients

BackgroundFor long-stay patients on the adult intensive care unit, the gut microbiota plays a key role in determining the balance between health and disease. However, it remains unclear which ICU patients might benefit from interventions targeting the gut microbiota or the pathogens therein.\n\nMethodsWe undertook a prospective observational study of twenty-four ICU patients, in which serial faecal samples were subjected to shotgun metagenomic sequencing, phylogenetic profiling and microbial genome analyses.\n\nResultsTwo-thirds of patients experienced a marked drop in gut microbial diversity (to an inverse Simpsons index of <4) at some stage during their stay in ICU, often accompanied by absence or loss of beneficial commensal bacteria. Intravenous administration of the broad-spectrum antimicrobial agent meropenem was significantly associated with loss of gut microbial diversity, but administration of other antibiotics, including piperacillin-tazobactam, failed to trigger statistically detectable changes in microbial diversity. In three quarters of ICU patients, we documented episodes of gut domination by pathogenic strains, with evidence of cryptic nosocomial transmission of Enterococcus faecium. In some patients we also saw domination of the gut microbiota by commensal organisms, such as Methanobrevibacter smithii.\n\nConclusionsOur results support a role for metagenomic surveillance of the gut microbiota and pave the way for patient-specific interventions that maintain or restore gut microbial diversity in the ICU.

microbiology↗

bioRxiv2019

GCNA preserves genome integrity and fertility across species

The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While these cells employ a number of known repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ Cell Nuclear Acidic Peptidase (GCNA) as a highly conserved regulator of genome stability in flies, worms, zebrafish, and humans. GCNA contains a long acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, GCNA mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, most of GCNAs function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.\n\nHighlightsGCNA ensures genomic stability in germ cells and early embryos across species\n\nGCNA limits replication stress and DNA double stranded breaks\n\nGCNA restricts DNA-Protein Crosslinks within germ cells and early embryos\n\nThe IDR and SprT domains of GCNA govern distinct aspects of genome integrity\n\nGraphic Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC=\"FIGDIR/small/570804_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (54K):\norg.highwire.dtl.DTLVardef@1928d82org.highwire.dtl.DTLVardef@885ff2org.highwire.dtl.DTLVardef@15322c9org.highwire.dtl.DTLVardef@110c615_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2018

A high-throughput whole cell screen to identify inhibitors of Mycobacterium tuberculosis

Tuberculosis is a disease of global importance for which novel drugs are urgently required. We developed a whole-cell phenotypic screen which can be used to identify inhibitors of Mycobacterium tuberculosis growth. We used recombinant strains of virulent M. tuberculosis which express far-red fluorescent reporters and used fluorescence to monitor growth in vitro. We optimized our high throughput assays using both 96-well and 384-well plates; both formats gave assays which met stringent reproducibility and robustness tests. We screened a compound set of 1105 chemically diverse compounds previously shown to be active against M. tuberculosis and identified primary hits which showed [≥] 90% growth inhibition. We ranked hits and identified three chemical classes of interest - the phenoxyalkylbenzamidazoles, the benzothiophene 1-1 dioxides, and the piperidinamines. These new compound classes may serve as starting points for the development of new series of inhibitors that prevent the growth of M. tuberculosis. This assay can be used for further screening, or could easily be adapted to other strains of M. tuberculosis.

microbiology↗