Biology subjects

Weston, S.

Publications and source records attributed to Weston, S..

17 recordsLinked to original sources

bioRxiv2026

Pan-Coronavirus Protease Engagement and Differentiated Pharmacology of Antiviral Candidate CMX990

COVID-19 highlighted the need for antivirals that can be rapidly deployed against both current and future coronavirus threats. CMX990 is a clinical stage coronavirus main protease (Mpro) inhibitor bearing a unique trifluoromethoxymethyl ketone warhead. Here, we broadly evaluate its pan-coronavirus target engagement, antiviral breadth, and pharmacologic differentiation. Across a reporter panel of 24 Mpro enzymes spanning alpha-, beta-, delta-, and gamma-coronaviruses, CMX990 retained robust potent activity, including against nirmatrelvir-resistant SARS-CoV-2 variants. In direct comparison, nirmatrelvir and ensitrelvir showed diminished potency against non-SARS-CoV-2 coronaviruses. Live-virus assays corroborated this breadth, with potent, selective activity against SARS-CoV-2, SARS-CoV-1, MERS-CoV, and additional human coronaviruses. Structural studies revealed a conserved covalent binding mode centered on the catalytic Cys of coronavirus Mpro. In serial-passage studies, resistance emerged only after approximately twice as many serial passages under CMX990 selection as under nirmatrelvir selection, without selecting for the canonical nirmatrelvir-resistance substitution E166V. Extracellular washout experiments revealed superior persistence of antiviral activity relative to nirmatrelvir, consistent with durable target engagement. In a murine SARS-CoV-2 infection model, once-daily CMX990 protected against weight loss and reduced lung viral titers to near baseline levels. Collectively, these findings identify CMX990 as a structurally and functionally validated pan-coronavirus protease inhibitor supporting its potential as a pandemic-ready antiviral candidate.

pharmacology and toxicology↗

bioRxiv2025

A SARS-CoV-2 entry inhibitor trimerizes to lock the spike protein in a closed conformation

The SARS-CoV-2 spike protein binds its receptor ACE2 to initiate target-cell infection. To engage ACE2, at least one of the three receptor-binding domains (RBDs) of the spike must adopt the up orientation. Here we describe S22, a potent, bioavailable, and non-toxic inhibitor of BA.2 and all subsequent Omicron variants. Cryo-EM analyses showed that S22 assembled as a trimer in a previously uncharacterized pocket of the spike apex, stabilizing all three RBDs in the down orientation, thereby preventing ACE2 association. Binding studies, especially those using mixed S22-sensitive and -resistant spikes, imply a cooperative assembly of three S22 molecules with three RBDs, resulting in an unusually slow S22 off-rate. Consistent with its slow dissociation and favorable pharmacokinetics, S22 suppressed viral replication 100-fold in the lungs of XBB.1.5-infected mice. Thus, S22 potently inhibits Omicron entry through a distinct mechanism whereby a small compound assembles cooperatively as a trimer to stabilize spike in an inactive conformation.

microbiology↗

bioRxiv2024

The mammalian SKI complex is a broad-spectrum antiviral drug target that upregulates cellular cholesterol to inhibit viral replication

There is a need for the development of broad-spectrum antiviral compounds that can act as first line therapeutic countermeasures to emerging viral infections. Host-directed approaches present a promising avenue of development and carry the benefit of mitigating risks of viral escape mutants. We have previously found the SKI (super killer) complex to be a broad-spectrum, host-target with our lead compound ("UMB18") showing activity against influenza A virus, coronaviruses, and filoviruses. The SKI complex is a cytosolic RNA helicase and we previously found that UMB18 inhibited viral RNA production but did not further define the mechanism. Here, we demonstrate that UMB18 directly binds to SKIC8 of the SKI complex and through transcriptomic analysis of UMB18 treated A549 cells revealed an upregulation of genes in the mevalonate pathway which drives cholesterol synthesis. Further investigation validated the genetic upregulation and confirmed an increase in total cellular cholesterol. This upregulation was dependent on the sterol regulatory element binding proteins (SREBPs) and their regulator SCAP, the major regulators for cholesterol and fatty acid synthesis. Depletion of the SREBPs or SCAP with siRNA, or extraction of cholesterol with methyl {beta}-cyclodextrin attenuated UMB18 antiviral activity, emphasizing the role of increased cholesterol synthesis in this mechanism of action. Our findings further define the antiviral mechanism of a developmental host-directed therapeutic approach with broad applicability against emerging viral pathogens. Author SummaryThe COVID-19 pandemic has underscored the urgent need for effective countermeasures to novel and emerging viral pathogens. Our research presented here builds upon our previously published data on an experimental novel antiviral compound termed UMB18. We have found this compound capable of inhibiting replication of influenza A virus, coronaviruses and the filoviruses Marburg and Ebola virus, but did not fully define a mechanism of action. In this work, we demonstrate that UMB18 exerts antiviral activity by modulating cellular cholesterol levels. By targeting the SKI complex, UMB18 triggers an increase in endogenous cellular cholesterol which disrupts the fine balance viruses rely on for efficient infection. We demonstrate that this mechanism inhibits replication of SARS-CoV-2, revealing a previously undescribed host-directed strategy for antiviral intervention. These findings highlight UMB18s potential as a broad-spectrum antiviral agent and pave the way for further research into its mechanism and therapeutic applications, offering a promising avenue for development of antiviral countermeasures to current, novel and emerging pathogens.

microbiology↗

bioRxiv2024

Expression, purification, and characterization of diacylated Lipo-YcjN from Escherichia coli

YcjN is a putative substrate-binding protein expressed from a cluster of genes involved in carbohydrate import and metabolism in Escherichia coli. Here, we determine the crystal structure of YcjN to a resolution of 1.95 [A], revealing that its three-dimensional structure is similar to substrate binding proteins in subcluster D-I, which includes the well-characterized maltose binding protein (MBP). Furthermore, we found that recombinant overexpression of YcjN results in the formation of a lipidated form of YcjN that is posttranslationally diacylated at cysteine 21. Comparisons of size-exclusion chromatography profiles and dynamic light scattering measurements of lipidated and non-lipidated YcjN proteins suggest that lipidated YcjN aggregates in solution via its lipid moiety. Additionally, bioinformatic analysis indicates that YcjN-like proteins may exist in both Bacteria and Archaea, potentially in both lipidated and non-lipidated forms. Together, our results provide a better understanding of the aggregation properties of recombinantly expressed bacterial lipoproteins in solution and establish a foundation for future studies that aim to elucidate the role of these proteins in bacterial physiology.

biophysics↗

bioRxiv2024

Global siRNA Screen Reveals Critical Human Host Factors of SARS-CoV-2 Multicycle Replication

Defining the subset of cellular factors governing SARS-CoV-2 replication can provide critical insights into viral pathogenesis and identify targets for host-directed antiviral therapies. While a number of genetic screens have previously reported SARS-CoV-2 host dependency factors, these approaches relied on utilizing pooled genome-scale CRISPR libraries, which are biased towards the discovery of host proteins impacting early stages of viral replication. To identify host factors involved throughout the SARS-CoV-2 infectious cycle, we conducted an arrayed genome-scale siRNA screen. Resulting data were integrated with published datasets to reveal pathways supported by orthogonal datasets, including transcriptional regulation, epigenetic modifications, and MAPK signalling. The identified proviral host factors were mapped into the SARS-CoV-2 infectious cycle, including 27 proteins that were determined to impact assembly and release. Additionally, a subset of proteins were tested across other coronaviruses revealing 17 potential pan-coronavirus targets. Further studies illuminated a role for the heparan sulfate proteoglycan perlecan in SARS-CoV-2 viral entry, and found that inhibition of the non-canonical NF-kB pathway through targeting of BIRC2 restricts SARS-CoV-2 replication both in vitro and in vivo. These studies provide critical insight into the landscape of virus-host interactions driving SARS-CoV-2 replication as well as valuable targets for host-directed antivirals.

systems biology↗

bioRxiv2023

The most remarkable migrants - systematic analysis of the Western European insect flyway at a Pyrenean mountain pass

In 1950 David and Elizabeth Lack chanced upon a huge migration of insects and birds flying through the Pyrenean Pass of Bujaruelo, later describing the spectacle as combining both grandeur with novelty. The intervening years have seen many changes to land use and climate, posing the question as to the current status of this migratory phenomenon, while a lack of quantitative data has prevented insights into the ecological impact of this mass insect migration and into the factors affecting it. To address this, we revisited the site in autumn over a 4-year period and systematically monitored diurnal insect species and numbers. We document an annual mean of 17.1 million day-flying insects from 5 orders moving south, with mass migration events associated with warmer temperatures, the presence of a headwind, sunlight, low windspeed, and low rainfall. Diptera dominated the migratory assemblage and annual numbers varied by more than fourfold with larger annual migration flows associated with higher autumn temperatures in Northwest Europe. Finally, using observed environmental thresholds for migration, we estimate an annual bioflow of at least 14.6 billion day-flying insects migrating south over the whole Pyrenean Mountain range, highlighting the importance of this route for seasonal insect migrants.

zoology↗

bioRxiv2022

SIRT-1 connects autophagy and release of virus-containing vesicles during picornavirus infection

Enterovirus D68 is a re-emerging enterovirus that causes acute respiratory illness in infants and has recently been linked to Acute Flaccid Myelitis. Here, we show that the histone deacetylase, SIRT-1, is essential for autophagy and EV-D68 infection. Knockdown of SIRT-1 inhibits autophagy and reduces EV-D68 extracellular titers. The proviral activity of SIRT-1 does not require its deacetylase activity or functional autophagy. SIRT-1s proviral activity is, we demonstrate, mediated through the repression of ER stress. Inducing ER stress through thapsigargin treatment or SERCA2A knockdown in SIRT-1 knockdown cells had no additional effect on EV-D68 extracellular titers. Knockdown of SIRT-1 also decreases poliovirus and SARS-CoV-2 titers but not coxsackievirus B3. In non-lytic conditions, EV-D68 is primarily released in an enveloped form, and SIRT-1 is required for this process. Our data show that SIRT-1, through its translocation to the cytosol, is critical to promote the release of enveloped EV-D68 viral particles.

microbiology↗

bioRxiv2022

Migratory hoverflies orientate north during spring migration

Migratory hoverflies are long-range migrants that, in the northern hemisphere, move seasonally to higher latitudes in the spring and lower latitudes in the autumn. The preferred migratory direction of hoverflies in the autumn has been the subject of radar and flight simulator studies while spring migration has proved to be more difficult to characterise due to a lack of ground observations. Consequently, the preferred migratory direction during spring has only been inferred from entomological radar studies and patterns of local abundance and currently lacks ground confirmation. Here, during a springtime arrival of migratory insects onto the Isles of Scilly and mainland Cornwall, UK, we provide ground proof that spring hoverfly migrants have an innate northward preference. Captured migratory hoverflies displayed northward vanishing bearings when released under sunny conditions under both favourable wind and zero-wind conditions. In addition, and unlike autumn migrants, spring individuals were also able to orientate when the sun was obscured. Analysis of winds suggests an origin for insects arriving on the Isles of Scilly in from western France. These findings of spring migration routes and preferred migration directions are likely to extend to the diverse set of insects found within the western European migratory assemblage.

zoology↗

bioRxiv2022

Targeted delivery of interferon-gamma by synNotch T cells sensitizes neuroblastoma cells to T cell-mediated killing

Downregulation of HLA is one of the most common tumor escape mechanisms by enabling tumors to persist in the presence of tumor-reactive T cells. HLA loss is particularly common in children with high-risk neuroblastoma, who have a 50% long-term survival despite dose-intensive regimens. We have now developed an approach for the targeted induction of HLA to restore sensitivity of neuroblastoma cells to T cell-mediated killing. Using synNotch technology, we have generated T cells that, upon binding of the neuroblastoma surface antigen GD2, secrete IFN-{gamma} without conferring direct cytotoxic activity (snGD2i). Treatment with snGD2i cells induces high and durable expression of HLA on neuroblastoma cells in vitro and in vivo and restores sensitivity to TCR-transgenic T cells targeting neuroblastoma-specific antigens. In contrast, treatment does not lead to upregulation of immune checkpoints or systemically increased levels of IFN-{gamma}. Targeted delivery of IFN-{gamma} using snGD2i cells is a promising new strategy to address immune escape in neuroblastoma. STATEMENT OF SIGNIFICANCEHLA loss remains one of the most common and unsolved immune escape mechanisms in cancer cells. We have now developed a cell-based approach for the targeted upregulation of HLA on tumor cells, which efficiently sensitizes the malignant cells to killing by tumor-reactive T cells.

bioengineering↗

bioRxiv2021

Pyrimidine biosynthesis inhibitors synergize with nucleoside analogs to block SARS-CoV-2 infection

The ongoing COVID-19 pandemic has highlighted the dearth of approved drugs to treat viral infections, with only [~]90 FDA approved drugs against human viral pathogens. To identify drugs that can block SARS-CoV-2 replication, extensive drug screening to repurpose approved drugs is underway. Here, we screened [~]18,000 drugs for antiviral activity using live virus infection in human respiratory cells. Dose-response studies validate 122 drugs with antiviral activity and selectivity against SARS-CoV-2. Amongst these drug candidates are 16 nucleoside analogs, the largest category of clinically used antivirals. This included the antiviral Remdesivir approved for use in COVID-19, and the nucleoside Molnupirivir, which is undergoing clinical trials. RNA viruses rely on a high supply of nucleoside triphosphates from the host to efficiently replicate, and we identified a panel of host nucleoside biosynthesis inhibitors as antiviral, and we found that combining pyrimidine biosynthesis inhibitors with antiviral nucleoside analogs synergistically inhibits SARS-CoV-2 infection in vitro and in vivo suggesting a clinical path forward.

microbiology↗

bioRxiv2021

Immunogenicity and In vivo protection of a variant nanoparticle vaccine that confers broad protection against emerging SARS-CoV-2 variants

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to spread globally. As SARS-CoV-2 has transmitted from person to person, variant viruses have emerged with elevated transmission rates and higher risk of infection for vaccinees. We present data showing that a recombinant prefusion-stabilized Spike (rS) protein based on the B.1.351 sequence (rS-B.1.351) was highly immunogenic in mice and produced neutralizing antibodies against SARS-CoV-2/WA1, B.1.1.7, and B.1.351. Mice vaccinated with our prototype vaccine NVX-CoV2373 (rS-WU1) or rS-B.1.351 alone, in combination, or as a heterologous prime boost, were protected when challenged with live SARS-CoV-2/B.1.1.7 or SARS-CoV-2/B.1.351. Virus titer was reduced to undetectable levels in the lungs post-challenge in all vaccinated mice, and Th1-skewed cellular responses were observed. A strong anamnestic response was demonstrated in baboons boosted with rS-B.1.351 approximately one year after immunization with NVX-CoV2373 (rS-WU1). An rS-B.1.351 vaccine alone or in combination with prototype rS-WU1 induced protective antibody- and cell-mediated responses that were protective against challenge with SARS-CoV-2 variant viruses.

immunology↗

bioRxiv2021

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail

The SARS-CoV-2 pandemic has led to an urgent need to understand the molecular basis for immune recognition of SARS-CoV-2 spike (S) glycoprotein antigenic sites. To define the genetic and structural basis for SARS-CoV-2 neutralization, we determined the structures of two human monoclonal antibodies COV2-2196 and COV2-21301, which form the basis of the investigational antibody cocktail AZD7442, in complex with the receptor binding domain (RBD) of SARS-CoV-2. COV2-2196 forms an "aromatic cage" at the heavy/light chain interface using germline-encoded residues in complementarity determining regions (CDRs) 2 and 3 of the heavy chain and CDRs 1 and 3 of the light chain. These structural features explain why highly similar antibodies (public clonotypes) have been isolated from multiple individuals1-4. The structure of COV2-2130 reveals that an unusually long LCDR1 and HCDR3 make interactions with the opposite face of the RBD from that of COV2-2196. Using deep mutational scanning and neutralization escape selection experiments, we comprehensively mapped the critical residues of both antibodies and identified positions of concern for possible viral escape. Nonetheless, both COV2-2196 and COV2-2130 showed strong neutralizing activity against SARS-CoV-2 strain with recent variations of concern including E484K, N501Y, and D614G substitutions. These studies reveal germline-encoded antibody features enabling recognition of the RBD and demonstrate the activity of a cocktail like AZD7442 in preventing escape from emerging variant viruses.

immunology↗

bioRxiv2021

Proteomic characterization of GSK3β knockout shows altered cell adhesion and metabolic pathway utilisation in colorectal cancer cells

Glycogen-specific kinase (GSK3{beta}) is an integral regulator of the Wnt signalling pathway as well as many other diverse signalling pathways and processes. Dys-regulation of GSK3{beta} is implicated in many different pathologies, including neurodegenerative disorders as well as many different tumour types. In the context of tumour development, GSK3{beta} has been shown to play both oncogenic and tumour suppressor roles, depending upon tissue, signalling environment or disease progression. Although multiple substrates of the GSK3{beta} kinase have been identified, the wider protein networks within which GSK3{beta} participates are not well known, and the consequences of these interactions not well understood. In this study, LC-MS/MS expression analysis was performed using knockout GSK3{beta} colorectal cancer cells and isogenic controls in colorectal cancer cell lines carrying dominant stabilizing mutations of {beta}-Catenin. Consistent with the role GSK3{beta}, we found that {beta}-Catenin levels and canonical Wnt activity are unaffected by knockout of GSK3{beta} and therefore use this knockout cell model to identify other processes in which GSK3{beta} is implicated. Quantitative proteomic analysis revealed perturbation of proteins involved in cell-cell adhesion, and we characterize the phenotype and altered proteomic profiles associated with this. We also characterize the perturbation of metabolic pathways resulting from GSK3{beta} knockout and identify defects in glycogen metabolism. In summary, using a precision colorectal cancer cell-line knockout model with constitutively activated {beta}-Catenin we are able to identify several of the diverse pathways and processes associated with GSK3{beta} function.

cancer biology↗

bioRxiv2020

Repurposing the Ebola and Marburg Virus Inhibitors Tilorone, Quinacrine and Pyronaridine: In vitro Activity Against SARS-CoV-2 and Potential Mechanisms

SARS-CoV-2 is a newly identified virus that has resulted in over 1.3 M deaths globally and over 59 M cases globally to date. Small molecule inhibitors that reverse disease severity have proven difficult to discover. One of the key approaches that has been widely applied in an effort to speed up the translation of drugs is drug repurposing. A few drugs have shown in vitro activity against Ebola virus and demonstrated activity against SARS-CoV-2 in vivo. Most notably the RNA polymerase targeting remdesivir demonstrated activity in vitro and efficacy in the early stage of the disease in humans. Testing other small molecule drugs that are active against Ebola virus would seem a reasonable strategy to evaluate their potential for SARS-CoV-2. We have previously repurposed pyronaridine, tilorone and quinacrine (from malaria, influenza, and antiprotozoal uses, respectively) as inhibitors of Ebola and Marburg virus in vitro in HeLa cells and of mouse adapted Ebola virus in mouse in vivo. We have now tested these three drugs in various cell lines (VeroE6, Vero76, Caco-2, Calu-3, A549-ACE2, HUH-7 and monocytes) infected with SARS-CoV-2 as well as other viruses (including MHV and HCoV 229E). The compilation of these results indicated considerable variability in antiviral activity observed across cell lines. We found that tilorone and pyronaridine inhibited the virus replication in A549-ACE2 cells with IC50 values of 180 nM and IC50 198 nM, respectively. We have also tested them in a pseudovirus assay and used microscale thermophoresis to test the binding of these molecules to the spike protein. They bind to spike RBD protein with Kd values of 339 nM and 647 nM, respectively. Human Cmax for pyronaridine and quinacrine is greater than the IC50 hence justifying in vivo evaluation. We also provide novel insights into their mechanism which is likely lysosomotropic.

pharmacology and toxicology↗

bioRxiv2020

SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits immunogenicity in baboons and protection in mice

The COVID-19 pandemic continues to spread throughout the world with an urgent need for a safe and protective vaccine to effectuate herd immunity to control the spread of SARS-CoV-2. Here, we report the development of a SARS-CoV-2 subunit vaccine (NVX-CoV2373) produced from the full-length spike (S) protein, stabilized in the prefusion conformation. Purified NVX-CoV2373 S form 27.2nm nanoparticles that are thermostable and bind with high affinity to the human angiotensin-converting enzyme 2 (hACE2) receptor. In mice and baboons, low-dose NVX-CoV2373 with saponin-based Matrix-M adjuvant elicits high titer anti-S IgG that is associated with blockade of hACE2 receptor binding, virus neutralization, and protection against SARS-CoV-2 challenge in mice with no evidence of vaccine-associated enhanced respiratory disease (VAERD). NVX-CoV2373 vaccine also elicits multifunctional CD4+ and CD8+ T cells, CD4+ T follicular helper T cells (Tfh), and the generation of antigen-specific germinal center (GC) B cells in the spleen. These results support the ongoing phase 1/2 clinical evaluation of the safety and immunogenicity of NVX-CoV2327 with Matrix-M (NCT04368988).

microbiology↗

bioRxiv2020

FDA approved drugs with broad anti-coronaviral activity inhibit SARS-CoV-2 in vitro

SARS-CoV-2 emerged in China at the end of 2019 and has rapidly become a pandemic with roughly 2.7 million recorded COVID-19 cases and greater than 189,000 recorded deaths by April 23rd, 2020 (www.WHO.org). There are no FDA approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA approved drugs. Rapid development and human testing of potential antivirals is greatly needed. A quick way to test compounds with potential antiviral activity is through drug repurposing. Numerous drugs are already approved for human use and subsequently there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV. We found that 17 of these inhibit SARS-CoV-2 at a range of IC50 values at non-cytotoxic concentrations. We directly follow up with seven of these to demonstrate all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we have evaluated two of these, chloroquine and chlorpromazine, in vivo using a mouse-adapted SARS-CoV model and found both drugs protect mice from clinical disease.

microbiology↗

bioRxiv2019

Comparative analysis reveals adaptive evolution of bat IFITMs and a novel antiviral determinant

Host interferon-induced transmembrane proteins (IFITMs) are broad-spectrum antiviral restriction factors. Of these, IFITM3 potently inhibits viruses that enter cells through acidic endosomes, many of which are zoonotic and emerging viruses with bats (order Chiroptera) as natural hosts. We previously demonstrated that microbat IFITM3 is antiviral. Here we show that bat IFITMs are characterized by strong adaptive evolution and identify a highly variable and functionally important site - codon 70 - within the conserved CD225 domain of IFITMs. Mutation of this residue in microbat IFITM3 impairs restriction of four different virus families that enter cells via endosomes. This mutant shows altered subcellular localization and reduced S-palmitoylation, a phenotype copied by mutation of conserved cysteine residues in microbat IFITM3. Furthermore, we show that microbat IFITM3 is S-palmitoylated on cysteine residues C71, C72 and C105, mutation of each cysteine residue individually impairs virus restriction, and a triple C71-C72-C105 mutant loses all restriction, concomitant with subcellular re-localization of microbat IFITM3 to Golgi-associated sites. Thus, we propose that S-palmitoylation is critical for Chiropteran IFITM3 function and identify a key molecular determinant of IFITM3 S-palmitoylation.

immunology↗