Biology subjects

Duffy, J.

Publications and source records attributed to Duffy, J..

12 recordsLinked to original sources

bioRxiv2026

Potent reaction hijacking inhibitors of Plasmodium falciparum asparagine tRNA synthetase

Malaria remains one of the major threats to human health. Breakthrough drugs with high potency and low resistance risk are needed to combat the ever-increasing resistance to currently deployed antimalarials. Here, we explore a series of 4-amino-quinazoline-based sulfonamides, with drug-like physicochemical parameters and a synthetically accessible scaffold. Exemplars exhibit nanomolar potency against blood stage Plasmodium cultures, with up to 300-fold selectivity compared with a mammalian cell line. The compounds are also active against transmissible stages of P. falciparum and are refractory to resistance development. Targeted mass spectrometry reveals that the compounds act as reaction hijacking inhibitors targeting P. falciparum aminoacyl tRNA synthetases (aaRSs). Subtle changes to the chemical structure switch the main target from cytoplasmic tRNA threonine synthetase (PfThrRS) to cytoplasmic asparagine synthetase (PfAsnRS), a change that is associated with increased potency and selectivity. The target preference was confirmed by selective knock-down of different P. falciparum aaRSs and by tolerance selection in a mutator line. Consistent with aaRS targets, exemplar compounds activate the amino acid starvation response. Recombinant enzyme inhibition and thermal stabilisation assays confirm the susceptibility of PfAsnRS to reaction hijacking and show that human AsnRS is less susceptible. A molecular model of Asn-tRNA-bound PfAsnRS reveals that a potent hijacker adopts a pose similar to adenosine 5-monophosphate (AMP). An AlphaFold model of the native PfAsnRS dimer helps explain the tolerance-conferring effect of a mutation at the dimer interface.

biochemistry↗

bioRxiv2025

A Novel Method to Simultaneously Estimate Bacterial Respiration and Growth from Oxygen Dynamics

Bacterial growth and respiration are fundamental metabolic processes that affect how energy is used and impact carbon sequestration at the ecosystem scale. However, these traits are usually quantified independently, growth is quantified with endpoint biomass measurements while respiration is quantified by monitoring oxygen or carbon dioxide. Because the two physiological traits are collected at different temporal and volumetric scales (hours-to-days for growth versus minutes-to-hours for respiration), reconciling them is challenging and often introduces scale-mismatch bias, obscuring causal links between metabolism and environmental drivers. In this study, we develop a novel method for quantifying the rates of bacterial growth and respiration concurrently from a single dissolved oxygen time series. Our approach introduces a model that couples exponential biomass growth with biomass-specific respiration, enabling the simultaneous inference of both rates in real time. We applied our high throughput method to 15 bacterial strains isolated from natural environments. Our approach yielded growth estimates in close agreement with measurements based on popular methods using optical density or flow cytometry (R2 > 0.9) with no evidence of taxon-specific bias. We also tested our approach in quantifying the effects of temperature on respiration, growth and carbon use-efficiency in Pseudomonas sp. Our method yielded typical unimodal thermal response curves for growth and respiration where rates were highest at moderate temperatures, while carbon-use efficiency increased from cooler temperatures, peaked around the thermal optimum ([~]30-35 {degrees}C), and declined at high temperature. By quantifying respiration and growth simultaneously and in high throughput, our approach effectively enables measurement of microbial metabolic strategies and adaptations to stress. It offers a non-invasive and scalable tool for high throughput phenotyping and studies of environmental perturbations, enabling a new class of trait-based microbial ecology that links cellular physiology to broader ecosystem function.

microbiology↗

bioRxiv2025

Targeting CHEK2-YBX1&YBX3 regulatory hub to potentiate immune checkpoint blockade response in gliomas

Although GBMs immunosuppressive environment is well known, the tumors resistance to CD8+ T cell killing is not fully understood. Our previous study identified Checkpoint Kinase 2 (Chek2) as the key driver of CD8+ T cell resistance in mouse glioma through an in vivo CRISPR screen and demonstrated that Chk2 inhibition, combined with PD-1/PD-L1 blockade, significantly enhanced CD8+ T cell-mediated tumor killing and improved survival in preclinical model. Here, we aimed to elucidate the immunosuppressive function of Chek2. Immunoprecipitation (IP) followed by mass spectrometry (MS) and phosphoproteomics identified an association between Chek2 with the DNA/RNA-binding proteins YBX1 and YBX3 that are implicated in transcriptional repression of pro-inflammatory genes. Single-gene knock-out and overexpression studies of CHEK2, YBX1, and YBX3 in multiple glioma cell lines revealed that these proteins positively regulate each others expression. RNA sequencing coupled with chromatin immunoprecipitation-sequencing (ChIP-seq) analysis demonstrated common inflammatory genes repressed by CHK2-YBX1&YBX3 hub. Targeting one of the hub proteins, YBX1, with the YBX1 inhibitor SU056 led to degradation of CHK2-YBX1&YBX3 hub. Targeting of this hub by SU056 led to enhanced antigen presentation and antigen specific CD8+ T cell proliferation. Further, combination of SU056 with ICB significantly improved survival in multiple glioma models. Collectively, these findings reveal an immunosuppressive mechanism mediated by the CHK2-YBX1&YBX3 hub proteins. Therefore, CHK2-YBX1&YBX3 hub targeting in combination with immune checkpoint blockade therapies in gliomas is warranted.

cancer biology↗

bioRxiv2024

Modeling Mountain Pine Beetle Abundance in Novel Hosts

The mountain pine beetle has recently expanded its range into northern and central Alberta, posing an immediate threat to the novel host, jack pine. To date, some experiments suggest that jack pine has limited defensive capabilities despite of the restrictions from the physical environment. In this work, we explore the susceptibility of jack pine compared to the primary host, lodgepole pine, evaluating the risk of potential range expansion into Canadas boreal forest. We employ a hierarchical model, incorporating environmental and ecological covariates, to examine mountain pine beetle dynamics in a pine forest with lodgepole, hybrid and jack pines. Our results show that pine species significantly influence the probability of being killed, with jack pine being less likely to be infested than lodgepole pine, all else being equal. The hierarchical model demonstrates that beetles perform poorer in jack pine, characterized by a reproduction rate 0.16 times that of non-jack pine. Although jack pine is a suitable host, our results indicate that the number of infestations in jack pine could be lower than in lodgepole pine, with a reduced probability of emerging beetles.

ecology↗

bioRxiv2024

MAPK/ERK signaling in gliomas modulates interferon responses, T cell recruitment, enhances tumor-microglia crosstalk, and drives immune checkpoint blockade efficacy.

BackgroundGlioblastoma (GB) remains a formidable challenge in neuro-oncology, with immune checkpoint blockade (ICB) showing limited efficacy in unselected patients. We previously recently established that MAPK/ERK signaling is associated with overall survival following anti-PD-1 and anti-CTLA-4 treatment in recurrent GB. However, the causal relationship between MAPK/ERK signaling and susceptibility to ICB, as well as the mechanisms underlying this association, remain poorly understood. MethodWe conducted in vivo kinome-wide CRISPR/Cas9 screenings in murine gliomas to identify key regulators of susceptibility to anti-PD-1 and CD8+ T cell responses and performed survival studies to validate the most relevant genes. Additionally, paired single cell RNA- sequencing (scRNA-seq) with p-ERK staining, spatial transcriptomics on GB samples, and ex-vivo slice culture of a BRAFV600E mutant GB tumor treated with BRAFi/MEKi were used to determine the causal relationship between MAPK signaling, tumor cell immunogenicity, and modulation of microglia phenotype. ResultsCRISPR/Cas9 screens identified the MAPK pathway, particularly the RAF-MEK-ERK pathway, as the most critical modulator of glioma susceptibility to CD8+ T cells, and anti-PD-1 across all kinases. Experimentally-induced ERK phosphorylation in gliomas enhanced survival with ICB treatment, led to durable anti-tumoral immunity upon re-challenge and memory T cell infiltration in long-term survivors. Elevated p-ERK in glioma cells correlated with increased interferon responses, antigen presentation and T cell infiltration in GB. Moreover, spatial transcriptomics and scRNA-seq analysis revealed the modulation of interferon responses by the MAPK/ERK pathway in BRAFV600E human GB cells with ERK1/2 knockout and in slice cultures of human BRAFV600E GB tissue. Notably, BRAFi/MEKi treatment disrupted the interaction between tumor cells and tumor-associated macrophages/microglia in slice cultures from BRAFV600E mutant GB. ConclusionOur data indicate that the MAPK/ERK pathway is a critical regulator of GB cell susceptibility to anti-tumoral immunity, modulating interferon responses, and antigen-presentation in glioma cells, as well as tumor cell interaction with microglia. These findings not only elucidate the mechanistic underpinnings of immunotherapy resistance in GB but also highlight the MAPK/ERK pathway as a promising target for enhancing immunotherapeutic efficacy in this challenging malignancy.

cancer biology↗

bioRxiv2024

Identification of mCMQ069, a novel antimalarial with potential for a single-dose cure and/or 28-day chemoprevention

In efforts towards eliminating malaria, a discovery program was initiated to identify a novel antimalarial using KAF156 as a starting point. Following the most recent TCP/TPP guidelines, we have identified mCMQ069 with a predicted single oral dose for treatment ([~]40-106 mg) and one-month chemoprevention ([~]96-216 mg). We have improved unbound MPC and predicted human clearance by 18-fold and 10-fold respectively when compared to KAF156.

microbiology↗

bioRxiv2024

A Prodrug Strategy to Reposition Atovaquone as a Long-Acting Injectable for Malaria Chemoprotection

Recent malaria drug discovery approaches have been extensively focused on the development of oral, smallmolecule inhibitors for disease treatment whereas parenteral routes of administration have been avoided due to limitations in deploying a shelf-stable injectable even though it could be dosed less frequently. However, an updated target candidate profile from Medicines for Malaria Venture (MMV) and stakeholders have advocated for long-acting injectable chemopreventive agents as an important interventive tool to improve malaria prevention. Here, we present strategies for the development of a long-acting, intramuscular, injectable atovaquone prophylactic therapy. We have generated three prodrug approaches that are contrasted by their differential physiochemical properties and pharmacokinetic profiles: mCBK068, a docosahexaenoic acid ester of atovaquone formulated in sesame oil, mCKX352, a heptanoic acid ester of atovaquone formulated as a solution in sesame oil, and mCBE161, an acetic acid ester of atovaquone formulated as an aqueous suspension. As a result, from a single 20 mg/kg intramuscular injection, mCKX352 and mCBE161 maintain blood plasma exposure of atovaquone above the minimal efficacious concentration for >70 days and >30 days, respectively, in cynomolgus monkeys. The differences in plasma exposure are reflective of the prodrug strategy, which imparts altered chemical properties that ultimately influence aqueous solubility and depot release kinetics. On the strength of the pharmacokinetic and safety profiles, mCBE161 is being advanced as a first-in-class clinical candidate for first-in-human trials.

pharmacology and toxicology↗

bioRxiv2022

Replenishing the malaria drug discovery pipeline: Screening and hit evaluation of the MMV Hit Generation Library 1 (HGL1) against asexual blood stage Plasmodium falciparum, using a nano luciferase reporter read-out

A central challenge of antimalarial therapy is the emergence of resistance to the components of artemisinin-based combination therapies (ACTs) and the urgent need for new drugs acting through novel mechanism of action. Over the last decade, compounds identified in phenotypic high throughput screens (HTS) have provided the starting point for six candidate drugs currently in the Medicines for Malaria Venture (MMV) clinical development portfolio. However, the published screening data which provided much of the new chemical matter for malaria drug discovery projects have been extensively mined. Here we present a new screening and selection cascade for generation of hit compounds active against the blood stage of Plasmodium falciparum. In addition, we validate our approach by testing a library of 141,786 compounds not reported earlier as being tested against malaria. The Hit Generation Library 1 (HGL1) was designed to maximise the chemical diversity and novelty of compounds with physicochemical properties associated with potential for further development. A robust HTS cascade containing orthogonal efficacy and cytotoxicity assays, including a newly developed and validated nanoluciferase-based assay was used to profile the compounds. 75 compounds (Screening Active hit rate of 0.05%) were identified meeting our stringent selection criteria of potency in drug sensitive (NF54) and drug resistant (Dd2) parasite strains (IC50 [≤] 2 {micro}M), rapid speed of action and cell viability in HepG2 cells (IC50 [≥] 10 {micro}M). Following further profiling, 33 compounds were identified that meet the MMV Confirmed Active profile and are high quality starting points for new antimalarial drug discovery projects.

microbiology↗

bioRxiv2022

Rudimentary Structure-Activity-Relationship study of the MMV Pathogen Box compound MMV675968 (2,4-diaminoquinazoline) unveils novel inhibitors of Trypanosoma brucei brucei DHFR enzyme

New drugs are urgently needed for the treatment of human African trypanosomiasis (HAT). In line with our quest for dihydrofolate reductase (DHFR) inhibitors in trypanosomes, a small library of analogs of the antitrypanosomal hit (MMV675968) available at MMV as solid materials was screened for antitrypanosomal activity. In silico confirmation of two potent antitrypanosomal analogs as inhibitors of DHFR was achieved, together with elucidation of other antitrypanosomal modes of action. Overall, two analogs of the diaminoquinazoline derivative, MMV675968 reputed to inhibit the enzyme DHFR, displayed approximately 40-fold and 60-fold more potency and selectivity than the parent hit, respectively (MMV1578445 (10): IC50 = 0.045 {micro}M, SI=1737; MMV1578467 (7): IC50 = 0.06 {micro}M; SI=412). Analogs 7 and 10 were also strong binders of the DHFR enzyme, in all their accessible protonation states, and interacted with key DHFR ligand recognition residues Val32, Asp54, and Ile160. MMV1578445 (10) portrayed fast and irreversible trypanosomes growth arrest after 72 h and 4 h at IC99. Analogs 7 and 10 induced ferric iron reduction and DNA fragmentation or apoptosis induction, respectively. The two potent analogs endowed with suitable physicochemical properties are good candidates for further deciphering their potential as starting points for new drug development for HAT. Author summaryAfrican trypanosomiasis is a disease caused by parasites of the Trypanosoma brucei species which affect both humans and animals. We have adopted the fast-track drug repurposing approach to determine the antitrypanosomal activity of the open access MMV Pathogen Box compound library. The compound 2,4-diaminoquinazoline (MMV675998), known as a dihydrofolate reductase (DHFR) inhibitor was identified. Rudimentary structure-activity relationship investigation of this compound unveiled two highly active and selective derivatives MMV1578445 and MMV1578467 which were found to strongly inhibit the Trypanosoma brucei dihydrofolate reductase enzyme in silico. Besides, we demonstrated that analogues MMV1578445 and MMV1578467 could respectively reduce intrinsic ferric iron and induce apoptosis in trypanosomes. The two identified inhibitors of trypanosomes qualify as potential drug candidates that could be useful for future development as novel antitrypanosomal drugs.

pharmacology and toxicology↗

bioRxiv2021

A New Mouse Model of Diffuse Midline Glioma to Test Targeted Immunotherapies

BACKGROUNDDiffuse midline gliomas remain incurable, with consistently poor outcomes in children despite radiotherapy. Immunotherapeutic approaches hold promise, with the integration of the hosts immune system fundamental to their design. Here, we describe a new, genetically engineered immunocompetent model that incorporates interleukin 13 receptor alpha 2 (IL13R2), a tumor-associated antigen, which is suitable for further evaluation of the antitumor activity of IL13R2-targeted immunotherapeutics in preclinical studies. METHODSThe RCAS-Tv-a delivery system was used to induce gliomagenesis through overexpression of PDGFB and p53 deletion with and without human IL13R2 in Nestin-Tva; p53fl/fl mice. Hindbrain or cerebral cortex nestin progenitors of neonatal pups were infected with Cre recombinase and PDGFB+IL13R2 or Cre recombinase and PDGFB to model diffuse midline glioma and supratentorial high-grade glioma, respectively. Immunoblotting and flow cytometry was used to confirm target expression. Kaplan-Meier survival curves were established to compare tumor latency in both models. Tumor tissue was analyzed through immunohistochemistry and H&E staining. Cell lines generated from tumor-bearing mice were used for in vitro studies and orthotopic injections. RESULTSThe protein expression of PDGFB and IL13R2 was confirmed by flow cytometry and western blot. In both groups, de novo tumors developed without significant difference in median survival between PDGFB and p53 loss (n=25, 40 days) and PDGFB, IL13R2, and p53 loss (n=33, 38 days, p=0.62). Tumors demonstrated characteristics of high-grade glioma such as infiltration, palisading necrosis, microvascular proliferation, high Ki-67 index, heterogeneous IL13R2 expression, and CD11b+ macrophages, along with a low proportion of CD3+ T cells. Orthotopic tumors developed from cell lines retained histopathological characteristics of de novo tumors. Mice orthotopically implanted with cells in the hindbrain or right cortex showed a median survival of 42 days and 41 (p=0.56) days, respectively. CONCLUSIONGeneration of de novo tumors using the RCAS-Tv-a delivery system was successful, with tumors possessing histopathologic features common to pediatric diffuse gliomas. The development of these models opens the opportunity for preclinical assessment of IL13R2-directed immunotherapies with the potential for clinical translation.

cancer biology↗

bioRxiv2021

Lipid chaperone LBP-8 coordinates with nuclear factors to promote longevity in Caenorhabditis elegans

Eukaryotic cells are composed of a variety of organelles. Their coordination plays crucial roles in cellular homeostasis and organism longevity and is mediated by proteins with the ability to transport between different organelles. In Caenorhabditis elegans, LBP-8 is a pro-longevity lipid chaperone that can localize to both lysosomes and the nucleus. Here we profiled LBP-8s binding partners using immunoprecipitation and mass spectrometry. From the 45 identified candidates, we discovered four nuclear factors that are required for the LBP-8-induced longevity. Among them, RPC-2, an RNA Polymerase III core subunit, is also necessary for the nuclear localization of LBP-8. Moreover, we have screened nuclear transport machinery components, and revealed the requirement of the nuclear import, not export, for the LBP-8 longevity effects. Together, these results suggest that the lipid chaperone LBP-8 relies on specific nuclear factors to retain in the nucleus and regulate longevity.

cell biology↗

bioRxiv2020

Multistage and transmission-blocking targeted antimalarials discovered from the open-source MMV Pandemic Response Box

New chemical matter is needed to target the divergent biology associated with the different life cycle stages of Plasmodium. Here, we report the parallel screening of the Medicines for Malaria Venture Pandemic Response Box to identify multistage-active and stage-specific compounds against various life cycle stages of Plasmodium parasites (asexual parasites, stage IV/V gametocytes, gametes, oocysts and liver stages) and for endectocidal activity. Hits displayed unique chemotypes and included two multistage-active compounds, 16 asexual-targeted, six with prophylactic potential and ten gametocyte-targeted compounds. Notably, four structurally diverse gametocyte-targeted compounds with potent transmission-blocking activity were identified: the JmjC inhibitor ML324, two azole antifungals including eberconazole, and the antitubercular clinical candidate SQ109. Besides ML324, none of these have previously attributed antiplasmodial activity, emphasizing the success of de novo parallel screening against different Plasmodium stages to deliver leads with novel modes-of-action. Importantly, the discovery of such transmission-blocking targeted compounds covers a previously unexplored base for delivery of compounds required for malaria elimination strategies.

microbiology↗