ABSTRACT
Current treatments for tumors expressing epidermal growth factor receptor (EGFR) include anti-EGFR monoclonal antibodies, often used in conjunction with the standard chemotherapy, radiation therapy, or other EGFR inhibitors. While monoclonal antibody treatment is efficacious in many patients, drawbacks include its high cost of treatment and side effects associated with multiple drug infusions. As an alternative to monoclonal antibody treatments, we have focused on peptide-based vaccination to trigger natural anti-tumor antibodies. Here, we demonstrate that peptides based on a region of the EGFR extracellular domain IV break immune tolerance to EGFR and elicit anti-tumor immunity. Mice immunized with isoforms of EGFR peptide p580-598 generated anti-EGFR antibody and T-cell responses. Iso-aspartyl (iso-Asp)-modified EGFR p580 immune sera inhibit in vitro growth of EGFR overexpressing human A431 tumor cells, as well as promote antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies induced by Asp and iso-Asp p580 bound homologous regions of the EGFR family members HER2 and HER3. EGFR p580 immune sera also inhibited the growth of the human tumor cell line MDA-MB-453 that expresses HER2 but not EGFR. Asp and iso-Asp EGFR p580 induced antibodies were also able to inhibit the in vivo growth of EGFR-expressing tumors. These data demonstrate that EGFR peptides from a region of the EGFR extracellular domain IV promote anti-tumor immunity, tumor cell killing, and antibodies that are cross reactive with ErbB family members.
Subject(s)
Antibodies, Monoclonal/pharmacology , Breast Neoplasms/immunology , ErbB Receptors/immunology , Peptide Fragments/immunology , Receptor, ErbB-2/immunology , Receptor, ErbB-3/immunology , A549 Cells , Animals , Antibody-Dependent Cell Cytotoxicity , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , ErbB Receptors/antagonists & inhibitors , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptide Fragments/administration & dosage , Phosphorylation , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-3/antagonists & inhibitors , Signal Transduction , Tumor Cells, Cultured , VaccinationABSTRACT
Clostridium difficile, a Gram-positive, spore-forming anaerobic bacterium, is the leading cause of infectious diarrhea among hospitalized patients. C. difficile is frequently associated with antibiotic treatment, and causes diseases ranging from antibiotic-associated diarrhea to life-threatening pseudomembranous colitis. The severity of C. difficile infections is exacerbated by the emergence of hypervirulent and multidrug-resistant strains, which are difficult to treat and are often associated with increased mortality rates. Alanine racemase (Alr) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the reversible racemization of L- and D-alanine. Since D-alanine is an essential component of the bacterial cell-wall peptidoglycan, and there are no known Alr homologs in humans, this enzyme is being tested as an antibiotic target. Cycloserine is an antibiotic that inhibits Alr. In this study, the catalytic properties and crystal structures of recombinant Alr from the virulent and multidrug-resistant C. difficile strain 630 are presented. Three crystal structures of C. difficile Alr (CdAlr), corresponding to the complex with PLP, the complex with cycloserine and a K271T mutant form of the enzyme with bound PLP, are presented. The structures are prototypical Alr homodimers with two active sites in which the cofactor PLP and cycloserine are localized. Kinetic analyses reveal that the K271T mutant CdAlr has the highest catalytic constants reported to date for any Alr. Additional studies are needed to identify the basis for the high catalytic activity. The structural and activity data presented are first steps towards using CdAlr for the development of structure-based therapeutics for C. difficile infections.
Subject(s)
Alanine Racemase/chemistry , Clostridioides difficile/enzymology , Drug Resistance, Multiple, Bacterial , Amino Acid Sequence , Chromatography, Gel , Clostridioides difficile/drug effects , Crystallography, X-Ray , Dimerization , Molecular Sequence Data , Protein Conformation , Recombinant Proteins/chemistry , Sequence Homology, Amino AcidABSTRACT
Semaphorin 7A (Sema7A) is a membrane-associated/secreted protein that plays an essential role in connecting the vertebrate neuronal and immune systems. However, the role of Sema7A has not been elucidated in viral pathogenesis. In this study, we show that abrogation of Sema7A protects mice from lethal West Nile virus (WNV) infection. Mice lacking Sema7A showed increased survival, reduced viral burden, and less blood-brain barrier permeability upon WNV infection. Increased Sema7A levels were evident in murine tissues, as well as in murine cortical neurons and primary human macrophages upon WNV infection. Treatment with Sema7A Ab blocked WNV infection in both of these cell types. Furthermore, Sema7A positively regulates the production of TGF-ß1 and Smad6 to facilitate WNV pathogenesis in mice. Collectively, these data elucidate the role of Sema7A in shared signaling pathways used by the immune and nervous systems during viral pathogenesis that may lead to the development of Sema7A-blocking therapies for WNV and possibly other flaviviral infections.
Subject(s)
Antigens, CD/physiology , Semaphorins/physiology , Signal Transduction/immunology , Smad6 Protein/physiology , Transforming Growth Factor beta1/physiology , West Nile virus/immunology , West Nile virus/pathogenicity , Animals , Cell Line , Cells, Cultured , Cerebral Cortex/immunology , Cerebral Cortex/metabolism , Cerebral Cortex/virology , Disease Models, Animal , Female , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Knockout , Virus Replication/immunologyABSTRACT
West Nile virus (WNV), and related flaviviruses such as tick-borne encephalitis, Japanese encephalitis, yellow fever and dengue viruses, constitute a significant global human health problem. However, our understanding of the molecular interaction of such flaviviruses with mammalian host cells is limited. WNV encodes only 10 proteins, implying that it may use many cellular proteins for infection. WNV enters the cytoplasm through pH-dependent endocytosis, undergoes cycles of translation and replication, assembles progeny virions in association with endoplasmic reticulum, and exits along the secretory pathway. RNA interference (RNAi) presents a powerful forward genetics approach to dissect virus-host cell interactions. Here we report the identification of 305 host proteins that affect WNV infection, using a human-genome-wide RNAi screen. Functional clustering of the genes revealed a complex dependence of this virus on host cell physiology, requiring a wide variety of molecules and cellular pathways for successful infection. We further demonstrate a requirement for the ubiquitin ligase CBLL1 in WNV internalization, a post-entry role for the endoplasmic-reticulum-associated degradation pathway in viral infection, and the monocarboxylic acid transporter MCT4 as a viral replication resistance factor. By extending this study to dengue virus, we show that flaviviruses have both overlapping and unique interaction strategies with host cells. This study provides a comprehensive molecular portrait of WNV-human cell interactions that forms a model for understanding single plus-stranded RNA virus infection, and reveals potential antiviral targets.
Subject(s)
RNA Interference , West Nile Fever/genetics , West Nile Fever/virology , West Nile virus/physiology , Computational Biology , Dengue Virus/physiology , Endoplasmic Reticulum/metabolism , Gene Expression Profiling , Genome, Human , HIV , HeLa Cells , Humans , Immunity/genetics , Monocarboxylic Acid Transporters/deficiency , Monocarboxylic Acid Transporters/genetics , Monocarboxylic Acid Transporters/metabolism , Muscle Proteins/deficiency , Muscle Proteins/genetics , Muscle Proteins/metabolism , Protein Binding , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/genetics , Vesiculovirus , Virus ReplicationABSTRACT
Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/metabolism , Azo Compounds , Fibroblasts/metabolism , Histocompatibility Antigens Class II/metabolism , Intramolecular Oxidoreductases , Macrophage Migration-Inhibitory Factors , Trypan Blue/chemistry , Trypan Blue/pharmacology , Allosteric Regulation/drug effects , Antigens, Differentiation, B-Lymphocyte/chemistry , Azo Compounds/chemistry , Azo Compounds/pharmacology , Cells, Cultured , Fibroblasts/cytology , Histocompatibility Antigens Class II/chemistry , Humans , Intramolecular Oxidoreductases/antagonists & inhibitors , Intramolecular Oxidoreductases/chemistry , Intramolecular Oxidoreductases/metabolism , Macrophage Migration-Inhibitory Factors/antagonists & inhibitors , Macrophage Migration-Inhibitory Factors/chemistry , Macrophage Migration-Inhibitory Factors/metabolism , Protein Binding/drug effects , Protein Structure, QuaternaryABSTRACT
Human glioma pathogenesis-related protein 1 (GLIPR1) is a membrane protein that is highly upregulated in brain cancers but is barely detectable in normal brain tissue. GLIPR1 is composed of a signal peptide that directs its secretion, a conserved cysteine-rich CAP (cysteine-rich secretory proteins, antigen 5 and pathogenesis-related 1 proteins) domain and a transmembrane domain. GLIPR1 is currently being investigated as a candidate for prostate cancer gene therapy and for glioblastoma targeted therapy. Crystal structures of a truncated soluble domain of the human GLIPR1 protein (sGLIPR1) solved by molecular replacement using a truncated polyalanine search model of the CAP domain of stecrisp, a snake-venom cysteine-rich secretory protein (CRISP), are presented. The correct molecular-replacement solution could only be obtained by removing all loops from the search model. The native structure was refined to 1.85â Å resolution and that of a Zn2+ complex was refined to 2.2â Å resolution. The latter structure revealed that the putative binding cavity coordinates Zn2+ similarly to snake-venom CRISPs, which are involved in Zn2+-dependent mechanisms of inflammatory modulation. Both sGLIPR1 structures have extensive flexible loop/turn regions and unique charge distributions that were not observed in any of the previously reported CAP protein structures. A model is also proposed for the structure of full-length membrane-bound GLIPR1.
Subject(s)
Neoplasm Proteins/chemistry , Nerve Tissue Proteins/chemistry , Amino Acid Sequence , Binding Sites , Conserved Sequence , Crystallography, X-Ray , Humans , Membrane Proteins , Models, Molecular , Molecular Sequence Data , Neoplasm Proteins/metabolism , Nerve Tissue Proteins/metabolism , Protein Conformation , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Snake Venoms/chemistry , Structural Homology, Protein , Zinc/metabolismABSTRACT
West Nile virus is an emerging pathogen that can cause fatal neurological disease. A recombinant human mAb, mAb11, has been described as a candidate for the prevention and treatment of West Nile disease. Using a yeast surface display epitope mapping assay and neutralization escape mutant, we show that mAb11 recognizes the fusion loop, at the distal end of domain II of the West Nile virus envelope protein. Ab mAb11 cross-reacts with all four dengue viruses and provides protection against dengue (serotypes 2 and 4) viruses. In contrast to the parental West Nile virus, a neutralization escape variant failed to cause lethal encephalitis (at higher infectious doses) or induce the inflammatory responses associated with blood-brain barrier permeability in mice, suggesting an important role for the fusion loop in viral pathogenesis. Our data demonstrate that an intact West Nile virus fusion loop is critical for virulence, and that human mAb11 targeting this region is efficacious against West Nile virus infection. These experiments define the molecular determinant on the envelope protein recognized by mAb11 and demonstrate the importance of this region in causing West Nile encephalitis.
Subject(s)
Antibodies, Monoclonal/metabolism , Binding Sites, Antibody , Peptides/immunology , Viral Envelope Proteins/immunology , Viral Fusion Proteins/immunology , West Nile Fever/immunology , West Nile virus/pathogenicity , Animals , Antibodies, Monoclonal/therapeutic use , Cell Line , Cross Reactions , Dengue Virus/immunology , Dengue Virus/pathogenicity , Female , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Peptides/metabolism , Protein Structure, Tertiary , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Viral Envelope Proteins/metabolism , Viral Fusion Proteins/metabolism , West Nile Fever/therapy , West Nile Fever/virology , West Nile virus/immunologyABSTRACT
The Lyme disease agent, Borrelia burgdorferi, is maintained in a tick-mouse cycle. Here we show that B. burgdorferi usurps a tick salivary protein, Salp15 (ref. 3), to facilitate the infection of mice. The level of salp15 expression was selectively enhanced by the presence of B. burgdorferi in Ixodes scapularis, first indicating that spirochaetes might use Salp15 during transmission. Salp15 was then shown to adhere to the spirochaete, both in vitro and in vivo, and specifically interacted with B. burgdorferi outer surface protein C. The binding of Salp15 protected B. burgdorferi from antibody-mediated killing in vitro and provided spirochaetes with a marked advantage when they were inoculated into naive mice or animals previously infected with B. burgdorferi. Moreover, RNA interference-mediated repression of salp15 in I. scapularis drastically reduced the capacity of tick-borne spirochaetes to infect mice. These results show the capacity of a pathogen to use a secreted arthropod protein to help it colonize the mammalian host.
Subject(s)
Borrelia burgdorferi/physiology , Borrelia burgdorferi/pathogenicity , Ixodes/metabolism , Ixodes/microbiology , Lyme Disease/microbiology , Lyme Disease/transmission , Salivary Proteins and Peptides/metabolism , Animals , Antigens, Bacterial/metabolism , Bacterial Outer Membrane Proteins/metabolism , Ixodes/genetics , Mice , Mice, Inbred C3H , Protein Binding , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Salivary Glands/metabolism , Salivary Glands/microbiology , Salivary Proteins and Peptides/geneticsABSTRACT
Epidermal Growth Factor Receptor (EGFR) is overexpressed on a number of human cancers, and often is indicative of a poor outcome. Treatment of EGFR/HER2 overexpressing cancers includes monoclonal antibody therapy (cetuximab/trastuzumab) either alone or in conjunction with other standard cancer therapies. While monoclonal antibody therapy has been proven to be efficacious in the treatment of EGFR/HER2 overexpressing tumors, drawbacks include the lack of long-lasting immunity and acquired resistance to monoclonal therapy. An alternative approach is to induce a polyclonal anti-EGFR/HER2 tumor antigen response by vaccine therapy. In this phase I/II open-label study, we examined anti-tumor immunity in companion dogs with spontaneous EGFR expressing tumors. Canine cancers represent an outbred population in which the initiation, progression of disease, mutations and growth factors closely resemble that of human cancers. Dogs with EGFR expressing tumors were immunized with a short peptide of the EGFR extracellular domain with sequence homology to HER2. Serial serum analyses demonstrated high titers of EGFR/HER2 binding antibodies with biological activity similar to that of cetuximab and trastuzumab. Canine antibodies bound both canine and human EGFR on tumor cell lines and tumor tissue. CD8 T cells and IgG deposition were evident in tumors from immunized dogs. The antibodies inhibited EGFR intracellular signaling and inhibited tumor growth in vitro. Additionally, we illustrate objective responses in reducing tumors at metastatic sites in host animals. The data support the approach of amplifying anti-tumor immunity that may be relevant in combination with other immune modifying therapies such as checkpoint inhibitors.
ABSTRACT
Glioma pathogenesis-related protein 1 (GLIPR1) is a member of the CAP superfamily that includes proteins from a wide range of eukaryotic organisms. The biological functions of most CAP proteins, including GLIPR1, are unclear. GLIPR1 is up-regulated in aggressive glioblastomas and contributes to the invasiveness of cultured glioblastoma cells. In contrast, decreased GLIPR1 expression is associated with advanced prostate cancer. Forced GLIPR1 overexpression is pro-apoptotic in prostate cancer cells and is being tested in clinical trials as an experimental prostate-cancer therapy. Human GLIPR1 was expressed as a truncated soluble protein (sGLIPR1), purified and crystallized. Useful X-ray data have been collected to beyond 1.9â Å resolution from a crystal that belonged to the orthorhombic space group P2(1)2(1)2 with average unit-cell parameters a = 85.1, b = 79.5, c = 38.9â Å and either a monomer or dimer in the asymmetric unit.
Subject(s)
Neoplasm Proteins/chemistry , Nerve Tissue Proteins/chemistry , Crystallization , Crystallography, X-Ray , Gene Expression , Humans , Membrane Proteins , Neoplasm Proteins/genetics , Neoplasm Proteins/isolation & purification , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , SolubilityABSTRACT
Human Macrophage Migration Inhibitory Factor (MIF) is a trimeric cytokine implicated in a number of inflammatory and autoimmune diseases and cancer. We previously reported that the dye p425 (Chicago Sky Blue), which bound MIF at the interface of two MIF trimers covering the tautomerase and allosteric pockets, revealed a unique strategy to block MIF's pro-inflammatory activities. Structural liabilities, including the large size, precluded p425 as a medicinal chemistry lead for drug development. We report here a rational design strategy linking only the fragment of p425 that binds over the tautomerase pocket to the core of ibudilast, a known MIF allosteric site-specific inhibitor. The chimeric compound, termed L2-4048, was shown by X-ray crystallography to bind at the allosteric and tautomerase sites as anticipated. L2-4048 retained target binding and blocked MIF's tautomerase CD74 receptor binding, and pro-inflammatory activities. Our studies lay the foundation for the design and synthesis of smaller and more drug-like compounds that retain the MIF inhibitory properties of this chimera.
ABSTRACT
A polyvalent ELISA and plaque reduction neutralization tests (PRNTs) were used to measure serum antibodies to West Nile virus (WNV) in horses naturally exposed to or vaccinated against this flavivirus in Connecticut and New York State, USA. Relying on a PRNT as a 'gold standard', the main objective was to validate a modified ELISA containing a recombinant WNV envelope protein antigen. It was also important to assess specificity by testing sera from horses that had other, undiagnosed illnesses. Sera for the latter study were obtained from 43 privately owned horses during 1995-1996. Analyses by an ELISA and a PRNT confirmed the presence of WNV antibodies in 21 (91%) of 23 sera from naturally exposed horses and in 85% of the 20 vaccinated subjects; overall results for both study groups were highly concordant (91% agreement). Humoral responses of naturally exposed and immunized horses were similar. Both serological tests were useful in confirming past infections with WNV, but there was no evidence that horses with undiagnosed illnesses were exposed to WNV prior to a 1999 outbreak in Connecticut, USA.
Subject(s)
Antibodies, Viral/blood , Horse Diseases/immunology , Viral Vaccines/immunology , West Nile Fever/veterinary , West Nile virus/immunology , Animals , Antibodies, Viral/biosynthesis , Enzyme-Linked Immunosorbent Assay/standards , Enzyme-Linked Immunosorbent Assay/veterinary , Horse Diseases/blood , Horses , Neutralization Tests/standards , Neutralization Tests/veterinary , West Nile Fever/blood , West Nile Fever/immunologyABSTRACT
One hundred and ninety-one sera from horses that recently were exposed to West Nile virus (WNV) by either vaccination or natural infection or that were not vaccinated and remained free of infection were used to evaluate fluorescent microsphere immunoassays (MIAs) incorporating recombinant WNV envelope protein (rE) and recombinant nonstructural proteins (rNS1, rNS3, and rNS5) for detection of equine antibodies to WNV. The rE MIA had a diagnostic sensitivity and specificity, respectively, of 99.3% and 97.4% for detection of WNV antibodies in the serum of horses that were recently vaccinated or naturally infected with WNV, as compared to the plaque reduction neutralization test (PRNT). The positive rE MIA results were assumed to be WNV-specific because of the close agreement between this assay and the PRNT and the fact that unvaccinated control horses included in this study were confirmed to be free of exposure to the related St Louis encephalitis virus. The NS protein-based MIA were all less sensitive than either the rE MIA or PRNT (sensitivity 0-48.0), although the rNSI MIA distinguished horses vaccinated with the recombinant WNV vaccine from those that were immunized with the inactivated WNV vaccine (P < 0.0001) or naturally infected with WNV (P < 0.0001). The rE MIA would appear to provide a rapid, convenient, inexpensive, and accurate test for the screening of equine sera for the presence of antibodies to WNV.
Subject(s)
Antibodies, Viral/blood , Horse Diseases/diagnosis , Horse Diseases/virology , Immunoassay/veterinary , Microspheres , West Nile Fever/veterinary , West Nile virus/immunology , Animals , Horse Diseases/blood , Horse Diseases/immunology , Horses/blood , Horses/immunology , Horses/virology , Viral Vaccines/immunology , West Nile Fever/diagnosis , West Nile Fever/prevention & controlABSTRACT
BACKGROUND: The presence of a Type III secretion system in clinical isolates of Pseudomonas aeruginosa is associated with severe disease and poor outcomes in infections caused by this pathogen. We describe an indirect enzyme-linked immunosorbent assay that rapidly and quantitatively detects two exotoxins, ExoU and ExoT, and two structural components, PopD and PcrV, of the P. aeruginosa Type III secretion system after in-vitro growth in a calcium-free minimal medium. METHODS: We used this assay to characterize the Type III secretion phenotype of 74 clinical isolates of P. aeruginosa. Findings were compared with results of standard immunoblotting and correlated with Type III secretion-dependent virulence of isolates toward cultured epithelial cells. RESULTS: Results of the ELISA assay were concordant with immunoblot detection of the secreted antigens for 73 of 74 isolates. The Type III secretion phenotype assessed by this immunoassay predicted bacterial virulence toward epithelial cells in vitro for all but five of the clinical isolates. CONCLUSION: The availability of an ELISA assay for rapid detection of Type III secreted virulence factors will facilitate large clinical studies to examine whether the Type III secretion phenotype of a P. aeruginosa isolate predicts the course of clinical disease in a patient and should be taken into account in determining optimal treatment strategies for infected patients.
Subject(s)
Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/pathogenicity , Antibodies, Monoclonal , Antigens, Bacterial/immunology , Antigens, Bacterial/isolation & purification , Bacterial Proteins/immunology , Bacterial Proteins/isolation & purification , Bacterial Toxins/immunology , Bacterial Toxins/isolation & purification , Enzyme-Linked Immunosorbent Assay/methods , Humans , Phenotype , Pore Forming Cytotoxic Proteins , Pseudomonas aeruginosa/genetics , VirulenceABSTRACT
Ixodes scapularis, the black-legged tick, vectors several human pathogens including Borrelia burgdorferi, the agent of Lyme disease in North America. Pathogen transmission to the vertebrate host occurs when infected ticks feed on the mammalian host to obtain a blood meal. Efforts to understand how the tick confronts host hemostatic mechanisms and imbibes a fluid blood meal have largely focused on the anticoagulation strategies of tick saliva. The blood meal that enters the tick gut remains in a fluid state for several days during the process of feeding, and the role of the tick gut in maintaining the blood-meal fluid is not understood. We now demonstrate that the tick gut produces a potent inhibitor of thrombin, a key enzyme in the mammalian coagulation cascade. Chromatographic fractionation of engorged tick gut proteins identified one predominant thrombin inhibitory activity associated with an approximately 18 kDa protein, henceforth referred to as Ixophilin. The ixophilin gene was preferentially transcribed in the guts of feeding nymphs. Expression began after 24 hours of feeding, coincident with the flow of host blood into the tick gut. Immunity against Ixophilin delayed tick feeding, and decreased feeding efficiency significantly. Surprisingly, immunity against Ixophilin resulted in increased Borrelia burgdorferi transmission to the host, possibly due to delayed feeding and increased transmission opportunity. These observations illuminate the potential drawbacks of targeting individual tick proteins in a functional suite. They also underscore the need to identify the "anticoagulome" of the tick gut, and to prioritize a critical subset of anticoagulants that could be targeted to efficiently thwart tick feeding, and block pathogen transmission to the vertebrate host.
Subject(s)
Arthropod Proteins/pharmacology , Gastrointestinal Tract/chemistry , Ixodes/chemistry , Thrombin/antagonists & inhibitors , Amino Acid Sequence , Animals , Arthropod Proteins/chemistry , Arthropod Proteins/genetics , Arthropod Proteins/immunology , Arthropod Proteins/isolation & purification , Female , Gene Expression , Humans , Ixodes/genetics , Mice , Molecular Sequence Data , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Sequence AlignmentABSTRACT
The genus Mycobacterium includes non-pathogenic species such as M. smegmatis, and pathogenic species such as M. tuberculosis, the causative agent of tuberculosis (TB). Treatment of TB requires a lengthy regimen of several antibiotics, whose effectiveness has been compromised by the emergence of resistant strains. New antibiotics that can shorten the treatment course and those that have not been compromised by bacterial resistance are needed. In this study, we report that thiadiazolidinones, a relatively little-studied heterocyclic class, inhibit the activity of mycobacterial alanine racemase, an essential enzyme that converts l-alanine to d-alanine for peptidoglycan synthesis. Twelve members of the thiadiazolidinone family were evaluated for inhibition of M. tuberculosis and M. smegmatis alanine racemase activity and bacterial growth. Thiadiazolidinones inhibited M. tuberculosis and M. smegmatis alanine racemases to different extents with 50% inhibitory concentrations (IC50) ranging from <0.03 to 28µM and 23 to >150µM, respectively. The compounds also inhibited the growth of these bacteria, including multidrug resistant strains of M. tuberculosis. The minimal inhibitory concentrations (MIC) for drug-susceptible M. tuberculosis and M. smegmatis ranged from 6.25µg/ml to 100µg/ml, and from 1.56 to 6.25µg/ml for drug-resistant M. tuberculosis. The in vitro activities of thiadiazolidinones suggest that this family of compounds might represent starting points for medicinal chemistry efforts aimed at developing novel antimycobacterial agents.
Subject(s)
Alanine Racemase/antagonists & inhibitors , Mycobacterium smegmatis/drug effects , Mycobacterium tuberculosis/drug effects , Thiadiazoles/pharmacology , Alanine Racemase/chemistry , Alanine Racemase/metabolism , Amino Acid Sequence , Catalysis , Molecular Sequence Data , Mycobacterium smegmatis/enzymology , Mycobacterium tuberculosis/enzymology , Sequence Homology, Amino Acid , Spectrometry, Mass, Electrospray Ionization , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
Methicillin-resistant Staphylococcus aureus (MRSA) is a human pathogen and a major cause of hospital-acquired infections. New antibacterial agents that have not been compromised by bacterial resistance are needed to treat MRSA-related infections. We chose the S. aureus cell wall synthesis enzyme, alanine racemase (Alr) as the target for a high-throughput screening effort to obtain novel enzyme inhibitors, which inhibit bacterial growth. Among the 'hits' identified was a thiadiazolidinone with chemical properties attractive for lead development. This study evaluated the mode of action, antimicrobial activities, and mammalian cell cytotoxicity of the thiadiazolidinone family in order to assess its potential for development as a therapeutic agent against MRSA. The thiadiazolidones inhibited Alr activity with 50% inhibitory concentrations (IC50) ranging from 0.36 to 6.4 µM, and they appear to inhibit the enzyme irreversibly. The series inhibited the growth of S. aureus, including MRSA strains, with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 µg/ml. The antimicrobial activity showed selectivity against Gram-positive bacteria and fungi, but not Gram-negative bacteria. The series inhibited human HeLa cell proliferation. Lead development centering on the thiadiazolidinone series would require additional medicinal chemistry efforts to enhance the antibacterial activity and minimize mammalian cell toxicity.
Subject(s)
Alanine Racemase/antagonists & inhibitors , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/pharmacology , Methicillin-Resistant Staphylococcus aureus/enzymology , Thiadiazoles/chemistry , Thiadiazoles/pharmacology , Alanine Racemase/metabolism , Anti-Bacterial Agents/administration & dosage , Drug Delivery Systems/methods , HeLa Cells , Humans , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Thiadiazoles/classificationABSTRACT
BACKGROUND: In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target. METHODOLOGY/PRINCIPAL FINDINGS: To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the 'hits' for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells. CONCLUSIONS/SIGNIFICANCE: This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.
Subject(s)
Alanine Racemase/antagonists & inhibitors , Anti-Infective Agents/pharmacology , Enzyme Inhibitors/classification , Enzyme Inhibitors/pharmacology , High-Throughput Screening Assays/methods , Mycobacterium tuberculosis/drug effects , Alanine Dehydrogenase/metabolism , Alanine Racemase/chemistry , Alanine Racemase/metabolism , Alanine Racemase/pharmacology , Anti-Infective Agents/analysis , Anti-Infective Agents/chemistry , Anti-Infective Agents/classification , Cell Death/drug effects , Enzyme Inhibitors/analysis , Enzyme Inhibitors/chemistry , HeLa Cells , Humans , Inhibitory Concentration 50 , Kinetics , Mass Spectrometry , Microbial Sensitivity Tests , Substrate Specificity/drug effectsABSTRACT
West Nile virus (WNV) is an emerging human pathogen for which specific antiviral therapy has not been developed. The therapeutic potential of RNA interference (RNAi) as a sequence-specific inhibitor of WNV has been well demonstrated. Although multiple siRNA targets have been identified within the genomic coding region, targets within the untranslated regions (UTR), which encode cis-acting regulatory elements, remain relatively unknown. In WNV and other flaviviruses, UTRs are located at the genomic termini. These regions form complex secondary structures, which pose difficulty when designing effective siRNA targets. In this study, we report the identification of siRNA targets in the WNV 3' UTR. These targets were selected by siRNA predictor algorithms, and synthesized as short hairpin RNA sequences from a plasmid-based expression system. Vero cells stably expressing these sequences had greatly diminished ability to support WNV replication but not the related dengue virus, demonstrating that the siRNAs were effective and suppressed WNV viral replication in a sequence-specific manner. The siRNAs developed in this study could function as potential antiviral therapeutics and as genetic tools to investigate the role of 3' UTR in WNV pathogenesis.
Subject(s)
3' Untranslated Regions , Antiviral Agents/pharmacology , RNA, Small Interfering/genetics , RNA, Viral/genetics , Virus Replication/drug effects , West Nile virus/genetics , Animals , Base Sequence , Chlorocebus aethiops , Dengue Virus/genetics , Dengue Virus/physiology , Humans , Molecular Sequence Data , RNA, Small Interfering/pharmacology , Vero Cells , Viral Plaque Assay , West Nile virus/physiologyABSTRACT
In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hydroxide was stable for greater than 8months at 4 degrees C. The recombinant vaccine candidate was evaluated for immunogenicity and protective efficacy in several animal models. In mouse and hamster WNV challenge models, the vaccine candidate induced viral protection that correlated with anti-rWNV-E immunogenicity and WNV neutralizing antibody titers. The rWNV-E vaccine candidate was used to boost horses previously immunized with the Fort Dodge inactivated WNV vaccine and also to induce WNV neutralizing titers in naïve foals that were at least 14weeks of age. Furthermore, the vaccine candidate was found safe when high doses were injected into rats, with no detectable treatment-related clinical adverse effects. These observations demonstrate that baculovirus-produced rWNV-E can be formulated with aluminum hydroxide to produce a stable and safe vaccine which induces humoral immunity that can protect against WNV infection.