-
Biological & Pharmaceutical Bulletin 2016Bisphosphonates (BPs) are typical anti-bone-resorptive drugs, with nitrogen-containing BPs (N-BPs) being stronger than non-nitrogen-containing BPs (non-N-BPs). However,...
Bisphosphonates (BPs) are typical anti-bone-resorptive drugs, with nitrogen-containing BPs (N-BPs) being stronger than non-nitrogen-containing BPs (non-N-BPs). However, N-BPs have inflammatory/necrotic effects, while the non-N-BPs clodronate and etidronate lack such side effects. Pharmacological studies have suggested that clodronate and etidronate can (i) prevent the side effects of N-BPs in mice via inhibition of the phosphate transporter families SLC20 and/or SLC34, through which N-BPs enter soft-tissue cells, and (ii) also inhibit the phosphate transporter family SLC17. Vesicular transporters for the pain transmitters glutamate and ATP belong to the SLC17 family. Here, we examined the hypothesis that clodronate and etidronate may enter neurons through SLC20/34, then inhibit SLC17-mediated transport of glutamate and/or ATP, resulting in their decrease, and thereby produce analgesic effects. We analyzed in mice the effects of various agents [namely, intrathecally injected clodronate, etidronate, phosphonoformic acid (PFA; an inhibitor of SLC20/34), and agonists of glutamate and ATP receptors] on the nociceptive responses to intraplantar injection of capsaicin. Clodronate and etidronate produced analgesic effects, and these effects were abolished by PFA. The analgesic effects were reduced by N-methyl-D-aspartate (agonist of the NMDA receptor, a glutamate receptor) and α,β-methylene ATP (agonist of the P2X-receptor, an ATP receptor). SLC20A1, SLC20A2, and SLC34A1 were detected within the mouse lumbar spinal cord. Although we need direct evidence, these results support the above hypothesis. Clodronate and etidronate may be representatives of a new type of analgesic drug. Such drugs, with both anti-bone-resorptive and unique analgesic effects without the adverse effects associated with N-BPs, might be useful for osteoporosis.
Topics: Acetic Acid; Adenosine Triphosphate; Analgesics; Animals; Bone Density Conservation Agents; Capsaicin; Clodronic Acid; Etidronic Acid; Excitatory Amino Acid Agonists; Foscarnet; Glutamic Acid; Lumbar Vertebrae; Mice; Mice, Inbred BALB C; N-Methylaspartate; Pain; Sodium-Phosphate Cotransporter Proteins; Spinal Cord; Substance P
PubMed: 27150146
DOI: 10.1248/bpb.b15-00882 -
Biomedizinische Technik. Biomedical... Oct 2015A deeper knowledge on the effects of the degradation of magnetic nanoparticles on their magnetic properties is required to develop tools for the identification and...
BACKGROUND
A deeper knowledge on the effects of the degradation of magnetic nanoparticles on their magnetic properties is required to develop tools for the identification and quantification of magnetic nanoparticles in biological media by magnetic means.
METHODS
Citric acid and phosphonoacetic acid-coated magnetic nanoparticles have been degraded in a medium that mimics lysosomal conditions. Magnetic measurements and transmission electron microscopy have been used to follow up the degradation process.
RESULTS
Particle size is reduced significantly in 24 h at pH 4.5 and body temperature. These transformations affect the magnetic properties of the compounds. A reduction of the interparticle interactions is observed just 4 h after the beginning of the degradation process. A strong paramagnetic contribution coming from the degradation products appears with time.
CONCLUSIONS
A model for the in vivo degradation of magnetic nanoparticles has been followed to gain insight on the changes of the magnetic properties of iron oxides during their degradation. The degradation kinetics is affected by the particle coating, in our case being the phosphonoacetic acid-coated particles degraded faster than the citric acid-coated ones.
Topics: Biomimetic Materials; Body Fluids; Citric Acid; Coated Materials, Biocompatible; Electric Impedance; Kinetics; Lysosomes; Magnetite Nanoparticles; Materials Testing; Particle Size; Phosphonoacetic Acid; Temperature
PubMed: 26035106
DOI: 10.1515/bmt-2015-0043 -
PLoS Pathogens Jan 2018Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to...
Detection of viral nucleic acids plays a critical role in the induction of intracellular host immune defences. However, the temporal recruitment of immune regulators to infecting viral genomes remains poorly defined due to the technical difficulties associated with low genome copy-number detection. Here we utilize 5-Ethynyl-2'-deoxyuridine (EdU) labelling of herpes simplex virus 1 (HSV-1) DNA in combination with click chemistry to examine the sequential recruitment of host immune regulators to infecting viral genomes under low multiplicity of infection conditions. Following viral genome entry into the nucleus, PML-nuclear bodies (PML-NBs) rapidly entrapped viral DNA (vDNA) leading to a block in viral replication in the absence of the viral PML-NB antagonist ICP0. This pre-existing intrinsic host defence to infection occurred independently of the vDNA pathogen sensor IFI16 (Interferon Gamma Inducible Protein 16) and the induction of interferon stimulated gene (ISG) expression, demonstrating that vDNA entry into the nucleus alone is not sufficient to induce a robust innate immune response. Saturation of this pre-existing intrinsic host defence during HSV-1 ICP0-null mutant infection led to the stable recruitment of PML and IFI16 into vDNA complexes associated with ICP4, and led to the induction of ISG expression. This induced innate immune response occurred in a PML-, IFI16-, and Janus-Associated Kinase (JAK)-dependent manner and was restricted by phosphonoacetic acid, demonstrating that vDNA polymerase activity is required for the robust induction of ISG expression during HSV-1 infection. Our data identifies dual roles for PML in the sequential regulation of intrinsic and innate immunity to HSV-1 infection that are dependent on viral genome delivery to the nucleus and the onset of vDNA replication, respectively. These intracellular host defences are counteracted by ICP0, which targets PML for degradation from the outset of nuclear infection to promote vDNA release from PML-NBs and the onset of HSV-1 lytic replication.
Topics: Cell Line; Cell Line, Transformed; Cells, Cultured; Click Chemistry; Gene Deletion; Gene Expression Regulation, Viral; Herpes Simplex; Herpesvirus 1, Human; Host-Pathogen Interactions; Humans; Immunity, Innate; Inclusion Bodies, Viral; Kinetics; Lysogeny; Mutation; Nuclear Proteins; Phosphoproteins; Promyelocytic Leukemia Protein; RNA Interference; Reverse Transcriptase Inhibitors; Ubiquitin-Protein Ligases; Viral Proteins; Virus Internalization; Virus Replication
PubMed: 29309427
DOI: 10.1371/journal.ppat.1006769 -
American Journal of Transplantation :... Jan 2019
Topics: Antiviral Agents; Control Groups; Cord Blood Stem Cell Transplantation; Encephalitis; Foscarnet; Ganciclovir; Herpesvirus 6, Human; Humans; Prospective Studies; Syndrome
PubMed: 30102463
DOI: 10.1111/ajt.15069 -
Journal of Virology Oct 2018Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186)...
Epstein-Barr virus (EBV) ZEBRA protein activates the EBV lytic cycle. Cellular AP-1 proteins with alanine-to-serine [AP-1(A/S)] substitutions homologous to ZEBRA(S186) assume some functions of EBV ZEBRA. These AP-1(A/S) mutants bind methylated EBV DNA and activate expression of some EBV genes. Here, we compare expression of 67 viral genes induced by ZEBRA versus expression induced by AP-1(A/S) proteins. AP-1(A/S) activated 24 genes to high levels and 15 genes to intermediate levels; activation of 28 genes by AP-1(A/S) was severely impaired. We show that AP-1(A/S) proteins are defective at stimulating viral lytic DNA replication. The impairment of expression of many late genes compared to that of ZEBRA is likely due to the inability of AP-1(A/S) proteins to promote viral DNA replication. However, even in the absence of detectable viral DNA replication, AP-1(A/S) proteins stimulated expression of a subgroup of late genes that encode viral structural proteins and immune modulators. In response to ZEBRA, expression of this subgroup of late genes was inhibited by phosphonoacetic acid (PAA), which is a potent viral replication inhibitor. However, when the lytic cycle was activated by AP-1(A/S), PAA did not reduce expression of this subgroup of late genes. We also provide genetic evidence, using the BMRF1 knockout bacmid, that these genes are true late genes in response to ZEBRA. AP-1(A/S) binds to the promoter region of at least one of these late genes, BDLF3, encoding an immune modulator. Mutant c-Jun and c-Fos proteins selectively activate expression of EBV lytic genes, including a subgroup of viral late genes, in the absence of viral DNA replication. These findings indicate that newly synthesized viral DNA is not invariably required for viral late gene expression. While viral DNA replication may be obligatory for late gene expression driven by viral transcription factors, it does not limit the ability of cellular transcription factors to activate expression of some viral late genes. Our results show that expression of all late genes may not be strictly dependent on viral lytic DNA replication. The c-Fos A151S mutation has been identified in a human cancer. c-Fos A151S in combination with wild-type c-Jun activates the EBV lytic cycle. Our data provide proof of principle that mutant cellular transcription factors could cause aberrant regulation of viral lytic cycle gene expression and play important roles in EBV-associated diseases.
Topics: Amino Acid Substitution; Antigens, Viral; Antiviral Agents; Binding Sites; Cell Line, Tumor; DNA Methylation; DNA, Viral; Gene Expression Regulation; HEK293 Cells; Herpesvirus 4, Human; Host-Pathogen Interactions; Humans; Lymphocytes; Membrane Glycoproteins; Mutation; Phosphonoacetic Acid; Promoter Regions, Genetic; Protein Binding; Signal Transduction; Trans-Activators; Transcription Factor AP-1; Viral Proteins; Virus Replication
PubMed: 30021895
DOI: 10.1128/JVI.01062-18 -
Journal of Virology Apr 2015DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. In spite of similarities in structure and function, the RB69...
UNLABELLED
DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. In spite of similarities in structure and function, the RB69 enzyme is relatively resistant to foscarnet, requiring the mutation V478W in helix N to promote the closed conformation of the enzyme to make it susceptible to the antiviral. Here, we generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility and viral replicative capacity. The mutation W781V in HSV-1 induced resistance to foscarnet, acyclovir, and ganciclovir (3-, 14-, and 3-fold increases in the 50% effective concentrations [EC50s], respectively). The recombinant HCMV mutant harboring the W780V mutation was slightly resistant to foscarnet (a 1.9-fold increase in the EC50) and susceptible to ganciclovir. Recombinant HSV-1 and HCMV mutants had altered viral replication kinetics. The apparent inhibition constant values of foscarnet against mutant UL30 and UL54 DNA polymerases were 45- and 4.9-fold higher, respectively, than those against their wild-type counterparts. Structural evaluation of the tryptophan position in the UL54 DNA polymerase suggests that the bulkier phenylalanine (fingers domain) and isoleucine (N-terminal domain) could induce a tendency toward the closed conformation greater than that for UL30 and explains the modest effect of the W780V mutation on foscarnet susceptibility. Our results further suggest a role of the tryptophan in helix N in conferring HCMV and especially HSV-1 susceptibility to foscarnet and the possible contribution of other residues localized at the interface between the fingers and N-terminal domains.
IMPORTANCE
DNA polymerases of the Herpesviridae and bacteriophage RB69 belong to the α-like DNA polymerase family. However, the RB69 DNA polymerase is relatively resistant to the broad-spectrum antiviral agent foscarnet. The mutation V478W in helix N of the fingers domain caused the enzyme to adopt a closed conformation and to become susceptible to the antiviral. We generated recombinant herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) mutants harboring the revertant in UL30 (W781V) and UL54 (W780V) DNA polymerases, respectively, to further investigate the impact of this tryptophan on antiviral drug susceptibility. The W781V mutation in HSV-1 induced resistance to foscarnet, whereas the W780V mutation in HCMV slightly decreased drug susceptibility. This study suggests that the different profiles of susceptibility to foscarnet of the HSV-1 and HCMV mutants could be related to subtle conformational changes resulting from the interaction between residues specific to each enzyme that are located at the interface between the fingers and the N-terminal domains.
Topics: Acyclovir; Analysis of Variance; Animals; Antiviral Agents; Chlorocebus aethiops; Cytomegalovirus; DNA-Directed DNA Polymerase; Drug Resistance, Viral; Foscarnet; Ganciclovir; Gene Transfer Techniques; Herpesvirus 1, Human; Humans; Kinetics; Models, Molecular; Mutation, Missense; Protein Structure, Secondary; Vero Cells
PubMed: 25673718
DOI: 10.1128/JVI.03360-14 -
Scientific Reports Jun 2018Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior...
Multidrug-resistant bacterial strains are a rapidly emerging healthcare threat; therefore it is critical to develop new therapies to combat these organisms. Prior antibacterial strategies directly target pathogen growth or viability. Host-directed strategies to increase antimicrobial defenses may be an effective alternative to antibiotics and reduce development of resistant strains. In this study, we demonstrated the efficacy of a pyrimidine synthesis inhibitor, N-phosphonacetyl-L-aspartate (PALA), to enhance clearance of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Acinetobacter baumannii strains by primary human dermal fibroblasts in vitro. PALA did not have a direct bactericidal effect, but enhanced cellular secretion of the antimicrobial peptides human β-defensin 2 (HBD2) and HBD3 from fibroblasts. When tested in porcine and human skin explant models, a topical PALA formulation was efficacious to enhance MRSA, P. aeruginosa, and A. baumannii clearance. Topical PALA treatment of human skin explants also resulted in increased HBD2 and cathelicidin (LL-37) production. The antimicrobial actions of PALA required expression of nucleotide-binding, oligomerization domain 2 (NOD2), receptor-interacting serine/threonine-protein kinase 2 (RIP2), and carbamoyl phosphatase synthase II/aspartate transcarbamylase/dihydroorotase (CAD). Our results indicate that PALA may be a new option to combat multidrug-resistant bacterial infections of the skin through enhancement of an integral pathway of the cutaneous innate immune defense system.
Topics: Animals; Aspartic Acid; Bacteria; Dermis; Drug Resistance, Multiple, Bacterial; HEK293 Cells; Humans; Immunity, Innate; Nod2 Signaling Adaptor Protein; Phosphonoacetic Acid; Pyrimidines; Signal Transduction; Skin Diseases, Bacterial; Swine
PubMed: 29880914
DOI: 10.1038/s41598-018-27012-0 -
ACS Chemical Biology Aug 2016HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase...
HIV-1 reverse transcriptase (RT) catalytically incorporates individual nucleotides into a viral DNA strand complementing an RNA or DNA template strand; the polymerase active site of RT adopts multiple conformational and structural states while performing this task. The states associated are dNTP binding at the N site, catalytic incorporation of a nucleotide, release of a pyrophosphate, and translocation of the primer 3'-end to the P site. Structural characterization of each of these states may help in understanding the molecular mechanisms of drug activity and resistance and in developing new RT inhibitors. Using a 38-mer DNA template-primer aptamer as the substrate mimic, we crystallized an RT/dsDNA complex that is catalytically active, yet translocation-incompetent in crystals. The ability of RT to perform dNTP binding and incorporation in crystals permitted obtaining a series of structures: (I) RT/DNA (P-site), (II) RT/DNA/AZTTP ternary, (III) RT/AZT-terminated DNA (N-site), and (IV) RT/AZT-terminated DNA (N-site)/foscarnet complexes. The stable N-site complex permitted the binding of foscarnet as a pyrophosphate mimic. The Mg(2+) ions dissociated after catalytic addition of AZTMP in the pretranslocated structure III, whereas ions A and B had re-entered the active site to bind foscarnet in structure IV. The binding of foscarnet involves chelation with the Mg(2+) (B) ion and interactions with K65 and R72. The analysis of interactions of foscarnet and the recently discovered nucleotide-competing RT inhibitor (NcRTI) α-T-CNP in two different conformational states of the enzyme provides insights for developing new classes of polymerase active site RT inhibitors.
Topics: Crystallography, X-Ray; Diphosphates; Drug Design; Foscarnet; HIV Reverse Transcriptase; Hydrolysis; Nucleotides; Protein Conformation; Reverse Transcriptase Inhibitors
PubMed: 27192549
DOI: 10.1021/acschembio.6b00187 -
Antimicrobial Agents and Chemotherapy Aug 2015A total of 302 clinical herpes simplex virus 1 (HSV-1) strains, collected over 4 decades from 1973 to 2014, were characterized retrospectively for drug resistance. All...
Sequence Analysis of Herpes Simplex Virus 1 Thymidine Kinase and DNA Polymerase Genes from over 300 Clinical Isolates from 1973 to 2014 Finds Novel Mutations That May Be Relevant for Development of Antiviral Resistance.
A total of 302 clinical herpes simplex virus 1 (HSV-1) strains, collected over 4 decades from 1973 to 2014, were characterized retrospectively for drug resistance. All HSV-1 isolates were analyzed genotypically for nonsynonymous mutations in the thymidine kinase (TK) and DNA polymerase (Pol) genes. The resistance phenotype against acyclovir (ACV) and/or foscarnet (FOS) was examined in the case of novel, unclear, or resistance-related mutations. Twenty-six novel natural polymorphisms could be detected in the TK gene and 69 in the DNA Pol gene. Furthermore, three novel resistance-associated mutations (two in the TK gene and one in the DNA Pol gene) were analyzed, and eight known but hitherto unclear amino acid substitutions (two encoded in TK and six in the DNA Pol gene) could be clarified. Between 1973 and 2014, the distribution of amino acid changes related to the natural gene polymorphisms of TK and DNA Pol remained largely stable. Resistance to ACV was confirmed phenotypically for 16 isolates, and resistance to ACV plus FOS was confirmed for 1 isolate. Acyclovir-resistant strains were observed from the year 1995 onwards, predominantly in immunosuppressed patients, especially those with stem cell transplantation, and the number of ACV-resistant strains increased during the last 2 decades. The data confirm the strong genetic variability among HIV-1 isolates, which is more pronounced in the DNA Pol gene than in the TK gene, and will facilitate considerably the rapid genotypic diagnosis of HSV-1 resistance.
Topics: Acyclovir; Adolescent; Adult; Aged; Aged, 80 and over; Antiviral Agents; Child; Child, Preschool; DNA-Directed DNA Polymerase; Drug Resistance, Viral; Female; Foscarnet; Genotype; Herpes Simplex; Herpesvirus 1, Human; Humans; Infant; Infant, Newborn; Male; Middle Aged; Mutation; Polymorphism, Genetic; Retrospective Studies; Sequence Analysis, DNA; Thymidine Kinase; Viral Proteins; Young Adult
PubMed: 26055375
DOI: 10.1128/AAC.00977-15 -
Biochemistry Jun 2015Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three...
Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO₂ from the carboxyphosphate intermediate to biotin.
Topics: Adenosine Diphosphate; Adenosine Triphosphate; Bacterial Proteins; Bicarbonates; Biocatalysis; Biotin; Carbon-Nitrogen Ligases; Catalytic Domain; Crystallography, X-Ray; Databases, Protein; Foscarnet; Haemophilus influenzae; Models, Molecular; Molecular Conformation; Organophosphorus Compounds; Phosphates; Protein Conformation; Protein Subunits
PubMed: 26020841
DOI: 10.1021/acs.biochem.5b00340