ABSTRACT
Human norovirus was discovered more than five decades ago and is a widespread cause of outbreaks of acute gastroenteritis. There are no approved vaccines or antivirals currently available. However, norovirus inhibitors, including capsid-specific monoclonal antibodies (Mabs) and nanobodies, have recently shown promising results. Several Mabs and nanobodies were found to inhibit norovirus replication using a human intestinal enteroid (HIE) culture system and/or could block norovirus attachment to histo-blood group antigen (HBGA) co-factors. In our pursuit to develop a single broad-spectrum norovirus therapeutic, we continued our analysis and development of a cross-reactive and HBGA interfering nanobody (NB26). To improve NB26 binding capacity and therapeutic potential, we conjugated NB26 onto a human IgG Fc domain (Fc-NB26). We confirmed that Fc-NB26 cross-reacts with genetically diverse GII genotype capsid protruding (P) domains (GII.8, GII.14, GII.17, GII.24, GII.26, and GII.NA1) using a direct enzyme-linked immunosorbent assay. Furthermore, X-ray crystallography structures of these P domains and structures of other GII genotypes reveal that the NB26 binding site is largely conserved, validating its broad reactivity. We showed that Fc-NB26 has ~100-fold higher affinity toward the norovirus P domain compared to native NB26. We also found that both NB26 and Fc-NB26 neutralize human norovirus replication in the HIE culture system. Furthermore, the mode of inhibition confirmed that like NB26, Fc-NB26 caused norovirus particle disassembly and aggregation. Overall, these new findings demonstrate that structural modifications to nanobodies can improve their therapeutic potential.IMPORTANCEDeveloping vaccines and antivirals against norovirus remains a challenge, mainly due to the constant genetic and antigenic evolution. Moreover, re-infection with genetically related and/or antigenic variants is not uncommon. We further developed our leading norovirus nanobody (NB26) that indirectly interfered with norovirus binding to HBGAs, by converting NB26 into a dimeric Fc-linked Nanobody (Fc-NB26). We found that Fc-NB26 had improved binding affinity and neutralization capacity compared with native NB26. Using X-ray crystallography, we showed this nanobody engaged highly conserved capsid residues among genetically diverse noroviruses. Development of such broadly reactive potent therapeutic nanobodies delivered as a slow-releasing prophylactic could be of exceptional value for norovirus outbreaks, especially for the prevention or treatment of severe acute gastroenteritis in high-risk groups such as the young, elderly, and immunocompromised.
Subject(s)
Caliciviridae Infections , Capsid Proteins , Norovirus , Single-Domain Antibodies , Norovirus/genetics , Norovirus/drug effects , Norovirus/immunology , Humans , Single-Domain Antibodies/immunology , Single-Domain Antibodies/pharmacology , Single-Domain Antibodies/chemistry , Capsid Proteins/immunology , Capsid Proteins/metabolism , Capsid Proteins/chemistry , Capsid Proteins/genetics , Caliciviridae Infections/immunology , Caliciviridae Infections/virology , Caliciviridae Infections/therapy , Antiviral Agents/pharmacology , Immunoglobulin Fc Fragments/immunology , Immunoglobulin Fc Fragments/chemistry , Antibodies, Viral/immunology , Cross Reactions , Capsid/metabolism , Capsid/immunology , Blood Group Antigens/metabolism , Virus Replication/drug effects , Gastroenteritis/virology , Immunoglobulin G/immunology , Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunologyABSTRACT
Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.
Subject(s)
Erythrocytes , Plasmodium falciparum , Polysaccharides , Protozoan Proteins , Humans , Antigens, Protozoan/metabolism , Antigens, Protozoan/immunology , Antigens, Protozoan/genetics , Erythrocytes/parasitology , Erythrocytes/metabolism , Lectins/metabolism , Lectins/genetics , Malaria, Falciparum/parasitology , Plasmodium falciparum/metabolism , Polysaccharides/metabolism , Protein Binding , Protozoan Proteins/metabolism , Protozoan Proteins/geneticsABSTRACT
Toll-like receptor (TLR) innate immunity signalling protects against pathogens, but excessive or prolonged signalling contributes to a range of inflammatory conditions. Structural information on the TLR cytoplasmic TIR (Toll/interleukin-1 receptor) domains and the downstream adaptor proteins can help us develop inhibitors targeting this pathway. The small molecule o-vanillin has previously been reported as an inhibitor of TLR2 signalling. To study its mechanism of action, we tested its binding to the TIR domain of the TLR adaptor MAL/TIRAP (MALTIR). We show that o-vanillin binds to MALTIR and inhibits its higher-order assembly in vitro. Using NMR approaches, we show that o-vanillin forms a covalent bond with lysine 210 of MAL. We confirm in mouse and human cells that o-vanillin inhibits TLR2 but not TLR4 signalling, independently of MAL, suggesting it may covalently modify TLR2 signalling complexes directly. Reactive aldehyde-containing small molecules such as o-vanillin may target multiple proteins in the cell.
Subject(s)
Benzaldehydes , Lysine , Toll-Like Receptor 2 , Humans , Animals , Mice , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Myeloid Differentiation Factor 88/metabolism , Toll-Like Receptors/metabolism , Membrane Glycoproteins/metabolism , Receptors, Interleukin-1/metabolismABSTRACT
Virus-glycan interactions play a crucial role in the infection process of many viruses. NMR spectroscopy has emerged as a powerful tool for studying these interactions at the molecular level. In this article, we review several published papers and reports that have highlighted the application of NMR spectroscopy in understanding the complex questions of how viruses engage with and bind to receptor glycans. The use of saturation transfer difference (STD) NMR spectroscopy has demonstrated itself as highly advantageous in investigating the interaction between glycans and intact virions or virus-like particles (VLPs). The broad NMR signal linewidth of virions and VLPs allows efficient saturation without affecting the glycan signals. The advantage of this approach is that the viral capsid environment in protein organization and function is not ignored and therefore provides a more biologically relevant model for exploring the interactions between the virus and the host cell glycans. We will review some examples of using NMR spectroscopy to study influenza cell tropism, rotaviruses, and noroviruses.
Subject(s)
Polysaccharides , Proteins , Ligands , Polysaccharides/chemistry , Polysaccharides/metabolism , Magnetic Resonance Spectroscopy/methods , Proteins/chemistry , Virion/metabolism , Protein BindingABSTRACT
Simple alkyl-sulfonylacetamides have potent antitubercular activity and significantly decrease mycolic acid levels in mycobacteria. Although these compounds were originally designed to inhibit the ketoacyl synthase domain of fatty acid synthase, structure-activity relationships and biochemical evidence do not fully support fatty acid synthase as the target. In 2004, an enzyme family involved in the activation and transfer of fatty acids as acyl-adenylates was identified in mycobacteria, separate from the universal acetyl-CoA carrier mechanism. These fatty acyl-AMP ligases (FAAL), encoded by the FadD family play important roles in the biosynthesis of mycolic acids along with fatty acid metabolism and are hypothesised here to be the molecular target of the sulfonylacetamides. Due to structural similarities with the ligase's natural substrate, it is believed these compounds are exerting action via competitive inhibition of these highly potent molecular targets. The primary aim of this investigation was to synthesize an extended library of sulfonylacetamide derivatives, building upon existing structural activity relations to validate the molecular mechanism with the aid of molecular modelling, while also attempting to explore novel structural isosteres for further drug design and development. Sulfonylacetamide derivatives were modified based on the putative molecular target resulting in derivatives with improved activities towards Mycobacteriumtuberculosis (H37Rv). The most active novel derivatives reported were 19, 22b, 22c and 46 displaying MIC90 levels of 1.4, 16.0, 13.0 and 5.9 µg/mL, respectively.
Subject(s)
Mycobacterium tuberculosis , Acetamides/pharmacology , Antitubercular Agents/pharmacology , Mycolic Acids/metabolism , Fatty Acids/metabolism , Fatty Acid SynthasesABSTRACT
Escherichia coli signal peptidase I (LepB) has been shown to inefficiently cleave secreted proteins with aromatic amino acids at the second position after the signal peptidase cleavage site (P2'). The Bacillus subtilis exported protein TasA contains a phenylalanine at P2', which in B. subtilis is cleaved by a dedicated archaeal-organism-like signal peptidase, SipW. We have previously shown that when the TasA signal peptide is fused to maltose binding protein (MBP) up to the P2' position, the TasA-MBP fusion protein is cleaved very inefficiently by LepB. However, the precise reason why the TasA signal peptide hinders cleavage by LepB is not known. In this study, a set of 11 peptides were designed to mimic the inefficiently cleaved secreted proteins, wild-type TasA and TasA-MBP fusions, to determine whether the peptides interact with and inhibit the function of LepB. The binding affinity and inhibitory potential of the peptides against LepB were assessed by surface plasmon resonance (SPR) and a LepB enzyme activity assay. Molecular modeling of the interaction between TasA signal peptide and LepB indicated that the tryptophan residue at P2 (two amino acids before the cleavage site) inhibited the active site serine-90 residue on LepB from accessing the cleavage site. Replacing the P2 tryptophan with alanine (W26A) allowed for more efficient processing of the signal peptide when the TasA-MBP fusion was expressed in E. coli. The importance of this residue to inhibit signal peptide cleavage and the potential to design LepB inhibitors based on the TasA signal peptide are discussed. IMPORTANCE Signal peptidase I is an important drug target, and understanding its substrate is critically important to develop new bacterium-specific drugs. To that end, we have a unique signal peptide that we have shown is refractory to processing by LepB, the essential signal peptidase I in E. coli, but previously has been shown to be processed by a more human-like signal peptidase found in some bacteria. In this study, we demonstrate how the signal peptide can bind but is unable to be processed by LepB, using a variety of methods. This can inform the field on how to better design drugs that can target LepB and understand the differences between bacterial and human-like signal peptidases.
Subject(s)
Escherichia coli Proteins , Escherichia coli , Humans , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Substrate Specificity , Tryptophan/metabolism , Amino Acid Sequence , Protein Sorting SignalsABSTRACT
There is a requirement for an objective method to determine a safe level of low-level military occupational blast, having recognised it can lead to neurological damage. The purpose of the current study was to evaluate the effect of artillery firing training on the neurochemistry of frontline soldiers using two-dimensional (2D) COrrelated SpectroscopY (2D COSY) in a 3-T clinical MR scanner. Ten men considered to be of sound health were evaluated before and after a week-long live firing exercise in two ways. Prior to the live fire exercise, all participants were screened by a clinical psychologist using a combination of clinical interviews and psychometric tests, and were then scanned with 3-T MRI. The protocols included T1- and T2-weighted images for diagnostic reporting and anatomical localisation and 2D COSY to record any neurochemical effects from the firing. No changes to the structural MRI were recorded. Nine substantive and statistically significant changes in the neurochemistry were recorded as a consequence of firing training. Glutamine and glutamate, glutathione, and two of the seven fucose-α (1-2)-glycans were significantly increased. N-acetyl aspartate, myo-inositol + creatine, and glycerol were also increased. Significant decreases were recorded for the glutathione cysteine moiety and tentatively assigned glycan with a 1-6 linkage (F2: 4.00, F1: 1.31 ppm). These molecules are part of three neurochemical pathways at the terminus of the neurons providing evidence of early markers of disruption to neurotransmission. Using this technology, the extent of deregulation can now be monitored for each frontline defender on a personalised basis. The capacity to monitor early a disruption in neurotransmitters, using the 2D COSY protocol, can observe the effect of firing and may be used to prevent or limit these events.
ABSTRACT
The opportunistic fungus Aspergillus fumigatus causes a set of diseases ranging from allergy to lethal invasive mycosis. Within the human airways, A. fumigatus is embedded in a biofilm that forms not only a barrier against the host immune defense system, but also creates a physical barrier protecting the fungi from chemicals such as antifungal drugs. Novel therapeutic strategies aim at combining drugs that inhibit biofilm synthesis or disrupt existing biofilm with classical antimicrobials. One of the major constituents of A. fumigatus biofilm is the polysaccharide galactosaminogalactan (GAG) composed of α1,4-linked N-acetylgalactosamine, galactosamine, and galactose residues. GAG is synthesized on the cytosolic face of the plasma membrane and is extruded in the extracellular space, where it is partially deacetylated. The deacetylase Agd3 that mediates this last step is essential for the biofilm formation and full virulence of the fungus. In this work, a previously described enzyme-linked lectin assay, based on the adhesion of deacetylated GAG to negatively charged plates and quantification with biotinylated soybean agglutinin was adapted to screen microbial natural compounds, as well as compounds identified in in silico screening of drug libraries. Actinomycin X2, actinomycin D, rifaximin, and imatinib were shown to inhibit Agd3 activity in vitro. At a concentration of 100 µM, actinomycin D and imatinib showed a clear reduction in the biofilm biomass without affecting the fungal growth. Finally, imatinib reduced the virulence of A. fumigatus in a Galleria mellonella infection model in an Agd3-dependent manner.
Subject(s)
Aspergillus fumigatus , Polysaccharides , Humans , Dactinomycin , Imatinib Mesylate , Polysaccharides/metabolism , Aspergillus fumigatus/metabolism , BiofilmsABSTRACT
Siglec-1, also known as Sialoadhesin (Sn) and CD169 is highly conserved among vertebrates and with 17 immunoglobulin-like domains is Siglec-1 the largest member of the Siglec family. Expression of Siglec-1 is found primarily on dendritic cells (DCs), macrophages and interferon induced monocyte. The structure of Siglec-1 is unique among siglecs and its function as a receptor is also different compared to other receptors in this class as it contains the most extracellular domains out of all the siglecs. However, the ability of Siglec-1 to internalize antigens and to pass them on to lymphocytes by allowing dendritic cells and macrophages to act as antigen presenting cells, is the main reason that has granted Siglec-1's key role in multiple human disease states including atherosclerosis, coronary artery disease, autoimmune diseases, cell-cell signaling, immunology, and more importantly bacterial and viral infections. Enveloped viruses for example have been shown to manipulate Siglec-1 to increase their virulence by binding to sialic acids present on the virus glycoproteins allowing them to spread or evade immune response. Siglec-1 mediates dissemination of HIV-1 in activated tissues enhancing viral spread via infection of DC/T-cell synapses. Overall, the ability of Siglec-1 to bind a variety of target cells within the immune system such as erythrocytes, B-cells, CD8+ granulocytes and NK cells, highlights that Siglec-1 is a unique player in these essential processes.
Subject(s)
Communicable Diseases , Sialic Acid Binding Ig-like Lectin 1 , Animals , Humans , Sialic Acid Binding Ig-like Lectin 1/metabolism , Sialic Acid Binding Immunoglobulin-like Lectins/metabolism , Sialic Acids , ImmunoglobulinsABSTRACT
Interactions between sialic acid (Sia) and sialic acid-binding immunoglobulin-like lectins (siglecs) regulate the immune system, with aberrations contributing to pathologies such as autoimmunity, infectious disease and cancer. Over the last decade, several multivalent Sia ligands have been synthesized to modulate the Sia-binding affinity of proteins/lectins. Here, we report a novel class of multivalent siglec probes through the decoration of α(2,6)-sialyllactose ligands on inherently fluorescent carbon dots (CD). We show that the preference of α(2,3)-linked Sia for siglec-1 can be altered by increasing the multivalence of Sia ligands present on the CD, and that a locally high glycan concentration can have a direct effect on linkage specificity. Additionally, micromolar (IC50 â¼ 70 µM) interaction of α(2,6)-sialyllactose-CD (6-CD) with siglec-2 (CD22) revealed it was capable of generating a significant cytotoxic effect on Burkitt's Lymphoma (BL) Daudi B cells. This phenonomen was attributed to 6-CD's ability to form trans interactions with CD22 on masked BL Daudi cells as a direct result of clustering of the Sia moiety on the CD surface. Overall, our glycoengineered carbon dots represent a novel high affinity molecular probe with multiple applications in sialoglycoscience and medicine.
ABSTRACT
Campylobacter jejuni responds to extracellular stimuli via transducer-like chemoreceptors (Tlps). Here, we describe receptor-ligand interactions of a unique paralogue family of dCache_1 (double Calcium channels and chemotaxis) chemoreceptors: Tlp2, Tlp3, and Tlp4. Phylogenetic analysis revealed that Tlp2, Tlp3, and Tlp4 receptors may have arisen through domain duplications, followed by a divergent evolutionary drift, with Tlp3 emerging more recently, and unexpectedly, responded to glycans, as well as multiple organic and amino acids with overlapping specificities. All three Tlps interacted with five monosaccharides and complex glycans, including Lewis's antigens, P antigens, and fucosyl GM1 ganglioside, indicating a potential role in host-pathogen interactions. Analysis of chemotactic motility of single, double, and triple mutants indicated that these chemoreceptors are likely to work together to balance responses to attractants and repellents to modulate chemotaxis in C. jejuni. Molecular docking experiments, in combination with saturation transfer difference nuclear magnetic resonance spectroscopy and competition surface plasmon resonance analysis, illustrated that the ligand-binding domain of Tlp3 possess one major binding pocket with two overlapping, but distinct binding sites able to interact with multiple ligands. A diverse sensory repertoire could provide C. jejuni with the ability to modulate responses to attractant and repellent signals and allow for adaptation in host-pathogen interactions. IMPORTANCE Campylobacter jejuni responds to extracellular stimuli via transducer-like chemoreceptors (Tlps). This remarkable sensory perception mechanism allows bacteria to sense environmental changes and avoid unfavorable conditions or to maneuver toward nutrient sources and host cells. Here, we describe receptor-ligand interactions of a unique paralogue family of chemoreceptors, Tlp2, Tlp3, and Tlp4, that may have arisen through domain duplications, followed by a divergent evolutionary drift, with Tlp3 emerging more recently. Unlike previous reports of ligands interacting with sensory proteins, Tlp2, Tlp3, and Tlp4 responded to many types of chemical compounds, including simple and complex sugars such as those present on human blood group antigens and gangliosides, indicating a potential role in host-pathogen interactions. Diverse sensory repertoire could provide C. jejuni with the ability to modulate responses to attractant and repellent signals and allow for adaptation in host-pathogen interactions.
Subject(s)
Bacterial Proteins , Campylobacter jejuni , Humans , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Campylobacter jejuni/genetics , Ligands , Molecular Docking Simulation , Phylogeny , ChemotaxisABSTRACT
Extra-intestinal pathogenic Escherichia coli (ExPEC) belong to a critical priority group of antibiotic resistant pathogens. ExPEC establish gut reservoirs that seed infection of the urinary tract and bloodstream, but the mechanisms of gut colonisation remain to be properly understood. Ucl fimbriae are attachment organelles that facilitate ExPEC adherence. Here, we investigated cellular receptors for Ucl fimbriae and Ucl expression to define molecular mechanisms of Ucl-mediated ExPEC colonisation of the gut. We demonstrate differential expression of Ucl fimbriae in ExPEC sequence types associated with disseminated infection. Genome editing of strains from two common sequence types, F11 (ST127) and UTI89 (ST95), identified a single nucleotide polymorphism in the ucl promoter that changes fimbriae expression via activation by the global stress-response regulator OxyR, leading to altered gut colonisation. Structure-function analysis of the Ucl fimbriae tip-adhesin (UclD) identified high-affinity glycan receptor targets, with highest affinity for sialyllacto-N-fucopentose VI, a structure likely to be expressed on the gut epithelium. Comparison of the UclD adhesin to the homologous UcaD tip-adhesin from Proteus mirabilis revealed that although they possess a similar tertiary structure, apart from lacto-N-fucopentose VI that bound to both adhesins at low-micromolar affinity, they recognize different fucose- and glucose-containing oligosaccharides. Competitive surface plasmon resonance analysis together with co-structural investigation of UcaD in complex with monosaccharides revealed a broad-specificity glycan binding pocket shared between UcaD and UclD that could accommodate these interactions. Overall, our study describes a mechanism of adaptation that augments establishment of an ExPEC gut reservoir to seed disseminated infections, providing a pathway for the development of targeted anti-adhesion therapeutics.
Subject(s)
Escherichia coli Infections , Extraintestinal Pathogenic Escherichia coli , Adhesins, Bacterial/metabolism , Adhesins, Escherichia coli/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Infections/metabolism , Extraintestinal Pathogenic Escherichia coli/genetics , Extraintestinal Pathogenic Escherichia coli/metabolism , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/metabolism , Humans , Intestinal Diseases , Polysaccharides/metabolismABSTRACT
Here, we present ultrastructural analyses showing that incoming HIV are captured near the lymphocyte surface in a virion-glycan-dependent manner. Biophysical analyses show that removal of either virion- or cell-associated N-glycans impairs virus-cell binding, and a similar glycan-dependent relationship is observed between purified HIV envelope (Env) and primary T cells. Trimming of N-glycans from either HIV or Env does not inhibit protein-protein interactions. Glycan arrays reveal HIV preferentially binds to N-acetylglucosamine and mannose. Interfering with these glycan-based interactions reduces HIV infectivity. These glycan interactions are distinct from previously reported glycan-lectin and non-specific electrostatic charge-based interactions. Specific glycan-glycan-mediated attachment occurs prior to virus entry and enhances efficiency of infection. Binding and fluorescent imaging data support glycan-glycan interactions as being responsible, at least in part, for initiating contact between HIV and the host cell, prior to viral Env-cellular CD4 engagement.
Subject(s)
HIV Antibodies/pharmacology , HIV Infections/drug therapy , Polysaccharides/metabolism , Virus Internalization/drug effects , Antibodies, Neutralizing/metabolism , Cell Membrane/metabolism , Glycosylation/drug effects , HIV Antibodies/metabolism , HIV Infections/metabolism , HIV-1/drug effects , HIV-1/immunology , Humans , Virion/metabolism , env Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
The Helicobacter pylori chemoreceptor TlpA plays a role in dampening host inflammation during chronic stomach colonization. TlpA has a periplasmic dCache_1 domain, a structure that is capable of sensing many ligands; however, the only characterized TlpA signals are arginine, bicarbonate, and acid. To increase our understanding of TlpA's sensing profile, we screened for diverse TlpA ligands using ligand binding arrays. TlpA bound seven ligands with affinities in the low- to middle-micromolar ranges. Three of these ligands, arginine, fumarate, and cysteine, were TlpA-dependent chemoattractants, while the others elicited no response. Molecular docking experiments, site-directed point mutants, and competition surface plasmon resonance binding assays suggested that TlpA binds ligands via both the membrane-distal and -proximal dCache_1 binding pockets. Surprisingly, one of the nonactive ligands, glucosamine, acted as a chemotaxis antagonist, preventing the chemotaxis response to chemoattractant ligands, and acted to block the binding of ligands irrespective of whether they bound the membrane-distal or -proximal dCache_1 subdomains. In total, these results suggest that TlpA senses multiple attractant ligands as well as antagonist ones, an emerging theme in chemotaxis systems. IMPORTANCE Numerous chemotactic bacterial pathogens depend on the ability to sense a diverse array of signals through chemoreceptors to achieve successful colonization and virulence within their host. The signals sensed by chemoreceptors, however, are not always fully understood. This is the case for TlpA, a dCache_1 chemoreceptor of H. pylori that enables the bacterium to induce less inflammation during chronic infections. H. pylori causes a significant global disease burden, which is driven by the development of gastric inflammation. Accordingly, it is essential to understand the processes by which H. pylori modulates host inflammation. This work uncovers the signals that TlpA can sense and highlights the underappreciated ability to regulate chemotactic responses by antagonistic chemoreceptor ligands, which is an emerging theme among other chemotactic systems.
Subject(s)
Bacterial Proteins/metabolism , Chemoreceptor Cells/metabolism , Helicobacter pylori/genetics , Helicobacter pylori/metabolism , Bacterial Proteins/genetics , Chemotaxis , Glucosamine/metabolism , Ligands , Molecular Docking Simulation , Point MutationABSTRACT
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a recently emerged virus that causes coronavirus infectious disease 2019 (COVID-19). SARS-CoV-2 spike protein, like SARS-CoV-1, uses the angiotensin converting enzyme 2 (ACE2) as a cellular receptor to initiate infection. Compounds that interfere with the SARS-CoV-2 spike protein receptor binding domain protein (RBD)-ACE2 receptor interaction may function as entry inhibitors. Here, we used a dual strategy of molecular docking and surface plasmon resonance (SPR) screening of compound libraries to identify those that bind to human ACE2 or the SARS-CoV-2 spike protein receptor binding domain (RBD). Molecular modeling screening interrogated 57,641 compounds and focused on the region of ACE2 that is engaged by RBD of the SARS-CoV-2 spike glycoprotein and vice versa. SPR screening used immobilized human ACE2 and SARS-CoV-2 Spike protein to evaluate the binding of these proteins to a library of 3,141 compounds. These combined screens identified compounds from these libraries that bind at KD (equilibrium dissociation constant) <3 µM affinity to their respective targets, 17 for ACE2 and 6 for SARS-CoV-2 RBD. Twelve ACE2 binders and six of the RBD binders compete with the RBD-ACE2 interaction in an SPR-based competition assay. These compounds included registered drugs and dyes used in biomedical applications. A Vero-E6 cell-based SARS-CoV-2 infection assay was used to evaluate infection blockade by candidate entry inhibitors. Three compounds demonstrated dose-dependent antiviral in vitro potency-Evans blue, sodium lifitegrast, and lumacaftor. This study has identified potential drugs for repurposing as SARS-CoV-2 entry inhibitors or as chemical scaffolds for drug development.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, has caused more than 60 million cases worldwide with almost 1.5 million deaths as of November 2020. Repurposing existing drugs is the most rapid path to clinical intervention for emerging diseases. Using an in silico screen of 57,641 compounds and a biophysical screen of 3,141 compounds, we identified 22 compounds that bound to either the angiotensin converting enzyme 2 (ACE2) and/or the SARS-CoV-2 spike protein receptor binding domain (SARS-CoV-2 spike protein RBD). Nine of these drugs were identified by both screening methods. Three of the identified compounds, Evans blue, sodium lifitegrast, and lumacaftor, were found to inhibit viral replication in a Vero-E6 cell-based SARS-CoV-2 infection assay and may have utility as repurposed therapeutics. All 22 identified compounds provide scaffolds for the development of new chemical entities for the treatment of COVID-19.
Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antiviral Agents/pharmacology , COVID-19 Drug Treatment , Spike Glycoprotein, Coronavirus/metabolism , Virus Attachment/drug effects , Virus Replication/drug effects , Aminopyridines/pharmacology , Animals , Benzodioxoles/pharmacology , Cell Line , Chlorocebus aethiops , Drug Evaluation, Preclinical , Drug Repositioning , Evans Blue/pharmacology , Humans , Molecular Docking Simulation , Phenylalanine/analogs & derivatives , Phenylalanine/pharmacology , Protein Binding/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sulfones/pharmacology , Surface Plasmon Resonance , Vero CellsABSTRACT
1 Hâ NMR spectroscopic studies on the 1:1 adduct of the pentasaccharide Fondaparinux (FPX) and the substitution-inert polynuclear platinum complex TriplatinNC show significant modulation of geometry around the glycosidic linkages of the FPX constituent monosaccharides. FPX is a valid model for the highly sulfated cell signalling molecule heparan sulfate (HS). The conformational ratio of the 1 C4 :2 S0 forms of the FPX residue IdoA(2S) is altered from ca. 35:65 (free FPX) to ca. 75:25 in the adduct; the first demonstration of a small molecule affecting conformational changes on a HS oligosaccharide. Functional consequences of such binding are suggested to be inhibition of HS cleavage in MDA-MB-231 triple-negative breast cancer (TNBC) cells. We further describe inhibition of metastasis by TriplatinNC in the TNBC 4T1 syngeneic tumour model. Our work provides insight into a novel approach for design of platinum drugs (and coordination compounds in general) with intrinsic anti-metastatic potential.
Subject(s)
Antineoplastic Agents/chemistry , Glycosaminoglycans/chemistry , Iduronic Acid/chemistry , Organoplatinum Compounds/chemistry , Platinum/chemistry , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Density Functional Theory , Heparitin Sulfate/chemistry , Humans , Magnetic Resonance Spectroscopy , Molecular Conformation , Organoplatinum Compounds/chemical synthesis , Organoplatinum Compounds/pharmacologyABSTRACT
Liposomal delivery systems have significantly enhanced the efficacy and safety of chemotherapeutic agents compared to free (non-liposomal) formulations. Liposomes are vesicles made up of lipophilic bilayer and a hydrophilic core which provides perfect opportunity for their application as transport vehicle for various therapeutic and diagnostic agents. Doxorubicin is the most exploited chemotherapeutic agent for evaluation of different liposomal applications, as its physicochemical properties permit high drug entrapment and easy remote loading in pre-formulated liposomes. Pegylated liposomal doxorubicin clinically approved and, on the market, Doxil®, exemplifies the benefits offered upon the surface modification of liposome with polyethylene glycol. This unique formulation prolonged the drug residence time in the circulation and increased accumulation of doxorubicin in tumor tissue via passive targeting (enhanced permeability and retention effect). However, there is ample scope for further improvement in the efficiency of targeting tumors by coupling biological active ligands onto the liposome surface to generate intelligent drug delivery systems. Small biomolecules such as peptides, fraction of antibodies and carbohydrates have the potential to target receptors present on the surface of the malignant cells. Hence, active targeting of malignant cells using functionalised nanocarrier (liposomes encapsulated with doxorubicin) have been attempted which is reviewed in this article.
Subject(s)
Antineoplastic Agents , Doxorubicin , Antineoplastic Agents/therapeutic use , Doxorubicin/analogs & derivatives , Drug Delivery Systems , Liposomes , Polyethylene GlycolsABSTRACT
NTHi is a human-adapted pathogen that colonizes the human respiratory tract. Strains of NTHi express multiple adhesins; however, there is a unique, mutually exclusive relationship between the major adhesins Hia and HMW1 and HMW2 (HMW1/2). Approximately 25% of NTHi strains express Hia, a phase-variable autotransporter protein that has a critical role in colonization of the host nasopharynx. The remaining 75% of strains express HMW1/2. Previous work has shown that the HMW1 and HMW2 proteins mediate binding to 2-3- and 2-6-linked sialic acid glycans found in the human respiratory tract. Here, we show that the high-affinity binding domain of Hia, binding domain 1 (BD1), is responsible for binding to α2-6-sialyllactosamine (2-6 SLN) glycans. BD1 is highly specific for glycans that incorporate the form of sialic acid expressed by humans, N-acetylneuraminic acid (Neu5Ac). We further show that Hia has lower-affinity binding activity for 2-3-linked sialic acid and that this binding activity is mediated via a distinct domain. Thus, Hia with its dual binding activities functionally mimics the combined activities of the HMW1 and HMW2 adhesins. In addition, we show that Hia has a role in biofilm formation by strains of NTHi that express the adhesin. Knowledge of the binding affinity of this major NTHi adhesin and putative vaccine candidate will direct and inform development of future vaccines and therapeutic strategies for this important pathogen.IMPORTANCE Host-adapted bacterial pathogens like NTHi have evolved specific mechanisms to colonize their restricted host niche. Relatively few of the adhesins expressed by NTHi have been characterized as regards their binding affinity at the molecular level. In this work, we show that the major NTHi adhesin Hia preferentially binds to Neu5Ac-α2-6-sialyllactosamine, the form of sialic acid expressed in humans. The receptors targeted by Hia in the human airway mirror those targeted by influenza A virus and indicates the broad importance of sialic acid glycans as receptors for microbes that colonize the human airway.
Subject(s)
Adhesins, Bacterial/metabolism , Haemophilus Infections/metabolism , Haemophilus Infections/microbiology , Haemophilus influenzae/physiology , Receptors, Cell Surface/metabolism , Respiratory Mucosa/metabolism , Respiratory Mucosa/microbiology , Adhesins, Bacterial/chemistry , Amino Acid Sequence , Binding Sites , Biofilms , Host-Pathogen Interactions , Humans , Models, Molecular , Molecular Conformation , Molecular Structure , Protein BindingABSTRACT
Cholesterol-dependent cytolysins (CDCs) form pores in cholesterol-rich membranes, but cholesterol alone is insufficient to explain their cell and host tropism. Here, we show that all eight major CDCs have high-affinity lectin activity that identifies glycans as candidate cellular receptors. Streptolysin O, vaginolysin, and perfringolysin O bind multiple glycans, while pneumolysin, lectinolysin, and listeriolysin O recognize a single glycan class. Addition of exogenous carbohydrate receptors for each CDC inhibits toxin activity. We present a structure for suilysin domain 4 in complex with two distinct glycan receptors, P1 antigen and αGal/Galili. We report a wide range of binding affinities for cholesterol and for the cholesterol analog pregnenolone sulfate and show that CDCs bind glycans and cholesterol independently. Intermedilysin binds to the sialyl-TF O-glycan on its erythrocyte receptor, CD59. Removing sialyl-TF from CD59 reduces intermedilysin binding. Glycan-lectin interactions underpin the cellular tropism of CDCs and provide molecular targets to block their cytotoxic activity.