Biology subjects

Fernig, D.

Publications and source records attributed to Fernig, D..

12 recordsLinked to original sources

bioRxiv2025

Topical Formulation of Repurposed FDA-Approved Compounds Inhibits Pseudomonas aeruginosa ExoU and Improves Corneal Infection Outcomes

Microbial keratitis, infection of the cornea, cause by ExotoxinU (ExoU) strains of Pseudomonas aeruginosa have a poor clinical outcome and response to antimicrobials. ExoU, a phospholipase, is secreted directly into host cells, causing their lysis. A screen of 3,034 FDA-approved compounds identified zinc pyrithione (Zp), bismuth subcitrate (Bis), and polymyxin B (Pol) as lead inhibitors of ExoU selectively inhibiting it without affecting human PLA2s or bacterial viability. The compounds have distinct inhibitory mechanisms: Zp disrupted ExoU oligomerization and protein stability; Bis impaired phosphatidylinositol 4,5-bisphosphate-dependent membrane association; Pol directly inhibited catalysis via its lipid-peptide architecture. Compound combinations enhanced ExoU inhibition in vitro to nanomolar concentrations. In mammalian cells, Bis and Pol promoted lysosomal trafficking and degradation of ExoU. Using a high-throughput microscopy platform, we screened 53 P. aeruginosa keratitis isolates, confirming broad efficacy of these inhibitors in exoU strains. Therapeutic efficacy was evaluated in ex vivo porcine corneas, Galleria mellonella, and in vivo mouse keratitis models. Topical delivery of ExoU inhibitors, particularly in combination, significantly reduced corneal opacity, ulceration, and stromal damage in porcine corneas without affecting bacterial load. In Galleria, compound combinations significantly maintained larval health, improved larval survival and delayed mortality. In a mouse eye infection model, combinatorial treatment reduced disease severity and preserved tissue viability without altering bacterial burden. These findings validate ExoU as a druggable virulence factor and support the repurposing of these compounds as an anti-virulence strategy for the treatment of P. aeruginosa infections in humans and veterinary medicine.

microbiology↗

bioRxiv2022

A phospholipase assay screen identifies synergistic inhibitors of the P. aeruginosa toxin ExoU

The opportunistic pathogen Pseudomonas aeruginosa is a leading cause of disability and mortality worldwide and the World Health Organisation has listed it with the highest priority for the need of new therapies. P. aeruginosa strains that express ExoU are implicated in the worst clinical outcomes. ExoU is phospholipase that is secreted by P. aeruginosa directly into the cytoplasm of target host cells, where its catalytic activity, directed towards plasma membranes, causes rapid cell lysis. Inhibition of ExoU may be a novel strategy to combat acutely cytotoxic ExoU expressing P. aeruginosa infections. Using an in vitro phospholipase assay, we performed a high throughput screen to identify compounds that might be repurposed as therapeutic ExoU inhibitors. We discovered a panel of compounds that appeared to inhibit ExoU through distinct mechanisms. Compound C prevented ExoU membrane localisation in HEK293T cells and caused colocalization with lysosomes, whereas compound D prevented PIP2 dependent oligomerisation of ExoU in vitro suggestive of synergistic action. Indeed, the concentrations required by compounds C and D to inhibit in vitro ExoU catalytic activity, when used in combination, was in the nanomolar region. In corneal scratch and infection assays, these compounds reduced ExoU mediated cytotoxicity, as assessed by fluorescence microscopy and lactate dehydrogenase release assays.

biochemistry↗

bioRxiv2021

Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity

Infection from the opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments and lysosomal degradation. ExoU is a potent phospholipase which causes rapid destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitro than other antimicrobial classes. However, their overuse over the past decade has caused alarming rates of antibiotic resistance to emerge. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered tobramycin disrupted T3SS expression and inhibited both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-T cells even at concentrations below the minimal inhibitory concentrations (MIC). Fluoroquinolones moxifloxacin and ciprofloxacin, however, upregulated the T3SS and in particular did not subvert the cytotoxic effects of ExoS and ExoU.

biochemistry↗

bioRxiv2020

Evaluation of ExoU inhibitors in a Pseudomonas aeruginosa scratch infection assay

Pseudomonas aeruginosa has recently been highlighted by the World Health Organisation (WHO) as a major threat with high priority for the development of new therapies. The type III secretion system of P. aeruginosa delivers the toxin ExoU into the cytosol of target host cells, where its plasma membrane directed phospholipase activity induces rapid cell lysis. Therefore, inhibition of the phospholipase activity of ExoU would be an important treatment strategy in P. aeruginosa infections. We evaluated a panel of ExoU small molecule inhibitors, previously identified from high throughput cellular based assays, and analysed their inhibition of ExoU phospholipase activity in vitro. A corneal epithelial (HCE-T) scratch and infection model using florescence microscopy, and cell viability assays, were used to test the efficacy of compounds to inhibit ExoU from P. aeruginosa. Compounds Pseudolipasin A, compound A and compound B were effective at mitigating ExoU mediated cytotoxicity after infection at concentrations as low as 0.5 M. Importantly, by using the antimicrobials moxifloxacin and tobramycin to control bacterial load, these assays were extended from 6 h to 24 h. P. aeruginosa remained cytotoxic to HCE-T cells with moxifloxacin, present at the minimal inhibitory concentration (MIC) for 24 h, but, when used in combination with either PSA, compound A or compound B, partial scratch healing was observed. These results provide evidence that ExoU inhibitors could be used in combination with certain antimicrobials as a novel means to treat clinical infections of ExoU producing P. aeruginosa.

biochemistry↗

bioRxiv2020

SARS-CoV-2 Spike S1 Receptor Binding Domain undergoes Conformational Change upon Interaction with Low Molecular Weight Heparins.

The dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses when added exogenously, including S-associated coronavirus strain HSR1 and inhibits cellular invasion by SARS-CoV-2. We have previously demonstrated that unfractionated heparin binds to the Spike (S1) protein receptor binding domain, induces a conformational change and have reported the structural features of heparin on which this interaction depends. Furthermore, we have demonstrated that enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. Here we expand upon these studies, to a wide range of low molecular weight heparins and demonstrate that they induce a variety of conformational changes in the SARS-CoV-2 RBD. These findings may have further implications for the rapid development of a first-line therapeutic by repurposing low molecular weight heparins, as well as for next-generation, tailor-made, GAG-based antiviral agents, against SARS-CoV-2 and other members of the Coronaviridae.

biochemistry↗

bioRxiv2020

Glycosaminoglycans induce conformational change in the SARS-CoV-2 Spike S1 Receptor Binding Domain.

The glycosaminoglycan (GAG) class of polysaccharides are utilised by a plethora of microbial pathogens as receptors for adherence and invasion. The GAG heparin prevents infection by a range of viruses when added exogenously, including the S-associated coronavirus strain HSR1 and more recently we have demonstrated that heparin can block cellular invasion by SARS-CoV-2. Heparin has found widespread clinical use as anticoagulant drug and this molecule is routinely used as a proxy for the GAG, heparan sulphate (HS), a structural analogue located on the cell surface, which is a known receptor for viral invasion. Previous work has demonstrated that unfractionated heparin and low molecular weight heparins binds to the Spike (S1) protein receptor binding domain, inducing distinct conformational change and we have further explored the structural features of heparin with regard to these interactions. In this article, previous research is expanded to now include a broader range of GAG family members, including heparan sulphate. This research demonstrates that GAGs, other than those of heparin (or its derivatives), can also interact with the SARS-CoV-2 Spike S1 receptor binding domain and induce distinct conformational changes within this region. These findings pave the way for future research into next-generation, tailor-made, GAG-based antiviral agents, against SARS-CoV-2 and other members of the Coronaviridae.

biochemistry↗

bioRxiv2020

Heparin inhibits cellular invasion by SARS-CoV-2: structural dependence of the interaction of the surface protein (spike) S1 receptor binding domain with heparin.

The dependence of the host on the interaction of hundreds of extracellular proteins with the cell surface glycosaminoglycan heparan sulphate (HS) for the regulation of homeostasis is exploited by many microbial pathogens as a means of adherence and invasion. The closely related polysaccharide heparin, the widely used anticoagulant drug, which is structurally similar to HS and is a common experimental proxy, can be expected to mimic the properties of HS. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Heparin prevents infection by a range of viruses if added exogenously, including S-associated coronavirus strain HSR1. Here, we show that the addition of heparin to Vero cells between 6.25 - 200 g.ml-1, which spans the concentration of heparin in therapeutic use, and inhibits invasion by SARS-CoV-2 at between 44 and 80%. We also demonstrate that heparin binds to the Spike (S1) protein receptor binding domain and induces a conformational change, illustrated by surface plasmon resonance and circular dichroism spectroscopy studies. The structural features of heparin on which this interaction depends were investigated using a library of heparin derivatives and size-defined fragments. Binding is more strongly dependent on the presence of 2-O or 6-O sulphation, and the consequent conformational consequences in the heparin structure, than on N-sulphation. A hexasaccharide is required for conformational changes to be induced in the secondary structure that are comparable to those that arise from heparin binding. Enoxaparin, a low molecular weight clinical anticoagulant, also binds the S1 RBD protein and induces conformational change. These findings have implications for the rapid development of a first-line therapeutic by repurposing heparin as well as for next-generation, tailor-made, GAG-based antiviral agents against SARS-CoV-2 and other members of the Coronaviridae.

biochemistry↗

bioRxiv2019

A glycosaminoglycan extract from Portunus pelagicus inhibits BACE1, the β secretase implicated in Alzheimer’s disease

Therapeutic options for Alzheimers disease, the most common form of dementia, are currently restricted to palliative treatments. The glycosaminoglycan heparin, widely used as a clinical anticoagulant, has previously been shown to inhibit the Alzheimers disease-relevant {beta}-secretase 1 (BACE1). Despite this, the deployment of pharmaceutical heparin for the treatment of Alzheimers disease is largely precluded by its potent anticoagulant activity. Furthermore, ongoing concerns regarding the use of mammalian sourced heparins, primarily due to prion diseases and religious beliefs, hinder the deployment of alternative heparin based therapeutics. A marine-derived, heparan sulphate-containing glycosaminoglycan extract isolated from the crab Portunus pelagicus, was identified to inhibit human BACE1 with comparable bioactivity to that of mammalian heparin (IC50 = 1.85 g.mL-1 (R2 = 0.94) and 2.43 g.mL-1 (R2 = 0.93), respectively) possessing highly attenuated anticoagulant activities. The results from several structural techniques suggest that the interactions between BACE1 and the extract from P. pelagicus are complex and distinct from those of heparin.

biochemistry↗

bioRxiv2019

Selective labelling of arginine residues engaged in binding sulfatedglycosaminoglycans

IThe activities of hundreds of proteins in the extracellular space are regulated by binding to the glycosaminoglycan heparan sulfate (HS). These interactions are driven by ionic bonds between sulfate and carboxylate groups on the polysaccharide and the side chains of basic residues in the protein. Here we develop a method to selectively label the guanidino side chains of arginine residues in proteins that engage the anionic groups in the sugar. The protein is bound to heparin (a common experimental proxy for HS) on an affinity column. Arginine side chains that are exposed to solvent, and thus involved in binding, are protected by reaction with the dicarbonyl phenylgyoxal (PGO). Elution of the bound proteins then exposes arginine side chains that had directly engaged with anionic groups on the polysaccharide. These are reacted with hydroxyl-phenylglyoxal (HPG). PGO was found to generate three products: a 1:1 product, the 1:1 water condensed product and a 2:1 PGO:arginine product. These three reaction products and that of HPG had distinct masses. Scripts were written to analyse the mass spectra and so identify HPG labelled arginine residues. Proof of principle was acquired on model peptides. The method was then applied to the identification of heparin binding arginine residues in fibroblast growth factors (FGF) 1 and 2. The data demonstrate that four out of eleven arginine residues on FGF2 and five out of six arginine residues of FGF1 engage heparin. Our approach provides a rapid and reliable means to identify arginines involved in functional complexes such as those of proteins with heparin

biochemistry↗

bioRxiv2018

Enhanced cell-cell contact stability upon Fibroblast Growth factor Receptor/N-cadherin cross-talk

N-cadherin adhesion has been reported to enhance cancer and neuronal cell migration either by mediating actomyosin-based force transduction or initiating Fibroblast Growth Factor Receptor (FGFR)-dependent biochemical signalling. Here we show that FGFR1 reduces N-cadherin-mediated cell migration. Both proteins are co-stabilised at cell-cell contacts through direct interaction. As a consequence, cell adhesion is strengthened, limiting the migration of cells on N-cadherin. Both the inhibition of migration and the stabilisation of cell adhesions require the FGFR activity stimulated by N-cadherin engagement. FGFR1 stabilises N-cadherin at the cell membrane through a pathway involving Src and p120. Moreover, FGFR1 stimulates the anchoring of N-cadherin to actin. We found that the migratory behaviour of cells depends on an optimum balance between FGFR-regulated N-cadherin adhesion and actin dynamics. Based on these findings we propose a positive feedback loop between N-cadherin and FGFR at adhesion sites limiting N-cadherin-based single cell migration.

cell biology↗

bioRxiv2018

New tools for carbohydrate sulphation analysis: Heparan Sulphate 2-O-sulphotranserase (HS2ST) is a target for small molecule protein kinase inhibitors

Sulphation of carbohydrate residues occurs on a variety of glycans destined for secretion, and this modification is essential for efficient matrix-based signal transduction. Heparan sulphate (HS) glycosaminoglycans control physiological functions ranging from blood coagulation to cell proliferation. HS biosynthesis involves membrane-bound Golgi sulphotransferases, including heparan sulphate 2-O-sulphotransferase (HS2ST), which transfers sulphate from the co-factor PAPS (3-phosphoadenosine 5-phosphosulphate) to the 2-O position of -L-iduronate in the maturing oligosaccharide chain. The current lack of simple non-radioactive enzyme assays that can be used to quantify the levels of carbohydrate sulphation hampers kinetic analysis of this process and the discovery of HS2ST inhibitors. In this paper, we describe a new procedure for thermal shift analysis of purified HS2ST. Using this approach, we quantify HS2ST-catalyzed oligosaccharide sulphation using a novel synthetic fluorescent substrate and screen the Published Kinase Inhibitor Set (PKIS), to evaluate compounds that inhibit catalysis. We report the susceptibility of HS2ST to a variety of cell permeable compounds in vitro, including polyanionic polar molecules, the protein kinase inhibitor rottlerin and oxindole-based RAF kinase inhibitors. In a related study, published back-to-back with this article, we demonstrate that Tyrosyl Protein Sulpho Tranferases (TPSTs) are also inhibited by a variety of protein kinase inhibitors. We propose that appropriately validated small molecule compounds could become new tools for rapid inhibition of glycan (and protein) sulphation in cells, and that protein kinase inhibitors might be repurposed or redesigned for the specific inhibition of HS2ST.\n\nSUMMARY STATEMENTWe report that HS2ST, which is a PAPS-dependent glycan sulphotransferase, can be assayed using a variety of novel biochemical procedures, including a non-radioactive enzyme-based assay that detects glycan substrate sulphation in real time. HS2ST activity can be inhibited by different classes of compounds, including known protein kinase inhibitors, suggesting new approaches to evaluate the roles of HS2ST-dependent sulphation with small molecules in cells.

biochemistry↗

bioRxiv2018

New tools for evaluating protein tyrosine sulphation: Tyrosyl Protein Sulphotransferases (TPSTs) are novel targets for RAF protein kinase inhibitors

Protein tyrosine sulphation is a post-translational modification (PTM) best known for regulating extracellular protein-protein interactions. Tyrosine sulphation is catalysed by two Golgi-resident enzymes termed Tyrosyl Protein Sulpho Transferases (TPSTs) 1 and 2, which transfer sulphate from the co-factor PAPS (3-phosphoadenosine 5-phosphosulphate) to a context-dependent tyrosine in a protein substrate. A lack of quantitative tyrosine sulphation assays has hampered the development of chemical biology approaches for the identification of small molecule inhibitors of tyrosine sulphation. In this paper, we describe the development of a non-radioactive mobility-based enzymatic assay for TPST1 and TPST2, through which the tyrosine sulphation of synthetic fluorescent peptides can be rapidly quantified. We exploit ligand binding and inhibitor screens to uncover a susceptibility of TPST1 and 2 to different classes of small molecules, including the anti-angiogenic compound suramin and the kinase inhibitor rottlerin. By screening the Published Kinase Inhibitor Set (PKIS), we identified oxindole-based inhibitors of the Ser/Thr kinase RAF as low micromolar inhibitors of TPST1/2. Interestingly, unrelated RAF inhibitors, exemplified by the dual BRAF/VEGFR2 inhibitor RAF265, were also TPST inhibitors in vitro. We propose that target-validated protein kinase inhibitors could be repurposed, or redesigned, as more-specific TPST inhibitors to help evaluate the sulphotyrosyl proteome. Finally, we speculate that mechanistic inhibition of cellular tyrosine sulphation might be relevant to some of the phenotypes observed in cells exposed to anionic TPST ligands and RAF protein kinase inhibitors.\n\nSUMMARY STATEMENTWe develop new assays to quantify tyrosine sulphation by the human tyrosine sulphotransferases TPST1 and 2. TPST1 and 2 catalytic activities are inhibited by protein kinase inhibitors, suggesting new starting points to synthesise (or repurpose) small molecule compounds to evaluate biological TPST using chemical biology.

biochemistry↗