Search

The pyrophosphate mimic and broad spectrum antiviral phosphonoformic acid (PFA, foscarnet) was shown to freeze the pre-translocational state of the reverse transcriptase (RT) complex of the human immunodeficiency virus type 1 (HIV-1). However, PFA lacks a specificity domain, which is seen as a major reason for toxic side effects associated with the clinical use of this drug. Here, we studied the mechanism of inhibition of HIV-1 RT by the 4-chlorophenylhydrazone of mesoxalic acid (CPHM) and demonstrate that this compound also blocks RT translocation. Hot spots for inhibition with PFA or CPHM occur at template positions with a bias toward pre-translocation. Mutations at active site residue Asp-185 compromise binding of both compounds. Moreover, divalent metal ions are required for the formation of ternary complexes with either of the two compounds. However, CPHM contains both an anchor domain that likely interacts with the catalytic metal ions and a specificity domain. Thus, although the inhibitor binding sites may partly overlap, they are not identical. The K65R mutation in HIV-1 RT, which reduces affinity to PFA, increases affinity to CPHM. Details with respect to the binding sites of the two inhibitors are provided on the basis of mutagenesis studies, structure-activity relationship analyses with newly designed CPHM derivatives, and in silico docking experiments. Together, these findings validate the pre-translocated complex of HIV-1 RT as a specific target for the development of novel classes of RT inhibitors.

Objectives The increasing prevalence of mutations in HIV-1 reverse transcriptase (RT) that confer resistance to existing NRTIs and NNRTIs underscores the need to develop RT inhibitors with novel mode-of-inhibition and distinct resistance profiles. Methods Biochemical assays were employed to identify inhibitors of RT activity and characterize their mode of inhibition. The antiviral activity of the inhibitors was assessed by cell-based assays using laboratory HIV-1 isolates and MT4 cells. RT variants were purified via avidin affinity columns. Results Compound A displayed equal or greater potency against many common NNRTI-resistant RTs (K103N and Y181C RTs) relative to WT RT. Despite possessing certain NNRTI-like properties, such as being unable to inhibit an engineered variant of RT lacking an NNRTI-binding pocket, we found that compound A was dependent on Mg2+ for binding to RT. Optimization of compound A led to more potent analogues, which retained similar activities against WT and K103N mutant viruses with submicromolar potency in a cell-based assay. One of the analogues, compound G, was crystallized in complex with RT and the structure was determined at 2.6 A resolution. The structure indicated that compound G simultaneously interacts with the active site (Asp186), the highly conserved primer grip region (Leu234 and Trp229) and the NNRTI-binding pocket (Tyr188). Conclusions These findings reveal a novel class of RT bifunctional inhibitors that are not sensitive to the most common RT mutations, which can be further developed to address the deficiency of current RT inhibitors.

Lung fibrosis is characterized by excessive accumulation of extracellular matrix components leading to progressive airflow limitation. Distinct profibrotic pathways converge on the activation of transforming growth factor-beta (TGF-beta), a central growth factor implicated in most fibroproliferative diseases. Recently, enforced expression of bioactive human TGF-beta1 (hTGF-beta1) in lungs of transgenic mice was shown to recapitulate several key pathophysiologies observed in fibrotic disorders of the lung, including cellular inflammation, tissue fibrosis, and myofibroblast hyperplasia. Inducible expression of hTGF-beta1 in this system provided a unique opportunity to characterize TGF-beta-driven mechanisms that precede and/or follow the onset of inflammation and fibrosis. Using gene expression profiling in lungs, we demonstrate temporal activation of key genetic programs regulating cell movement and invasiveness, inflammation, organ remodeling, and fibrosis. Consistent with our gene expression data, multiple soluble mediators associated with inflammation and tissue remodeling were markedly elevated in the bronchoalveolar lavage fluid of mice expressing hTGF-beta1. We observe significant TGF-beta1-driven infiltration of F4/80+ mononuclear cells producing bioactive arginase, a marker of alternatively activated macrophages. Finally, we identified a common "fibrosis" gene signature when comparing our findings with published data derived from preclinical and clinical studies.

The basis for the differential repressive effects of antiestrogens on transactivation by estrogen receptor-α (ERα) remains incompletely understood. Here, we show that the full antiestrogen ICI182,780 and, to a lesser extent, the selective ER modulator raloxifene (Ral), induce accumulation of exogenous ERα in a poorly soluble fraction in transiently transfected HepG2 or stably transfected MDA-MB231 cells and of endogenous receptor in MCF7 cells. ERα remained nuclear in HepG2 cells treated with either compound. Replacement of selected hydrophobic residues of ERα ligand-binding domain helix 12 (H12) enhanced receptor solubility in the presence of ICI182,780 or Ral. These mutations also increased transcriptional activity with Ral or ICI182,780 on reporter genes or on the endogenous estrogen target gene TFF1 in a manner requiring the integrity of the N-terminal AF-1 domain. The antiestrogen-specific effects of single mutations suggest that they affect receptor function by mechanisms other than a simple decrease in hydrophobicity of H12, possibly due to relief from local steric hindrance between these residues and the antiestrogen side chains. Fluorescence anisotropy experiments indicated an enhanced regional stabilization of mutant ligand-binding domains in the presence of antiestrogens. H12 mutations also prevent the increase in bioluminescence resonance energy transfer between ERα monomers induced by Ral or ICI182,780 and increase intranuclear receptor mobility in correlation with transcriptional activity in the presence of these antiestrogens. Our data indicate that ICI182,780 and Ral locally alter the ERα ligand binding structure via specific hydrophobic residues of H12 and decrease its transcriptional activity through tighter association with an insoluble nuclear structure.

Apolipoprotein E (apoE) plays a major role in lipoprotein metabolism by mediating the binding of apoE-containing lipoproteins to receptors. The role of hepatic apoE in the catabolism of apoE-free lipoproteins such as low density lipoprotein (LDL) and high density lipoprotein-3 (HDL3) is however, unclear. We analyzed the importance of hepatic apoE by comparing human LDL and HDL3 metabolism in primary cultures of hepatic cells from control C57BL/6J and apoE knockout (KO) mice. Binding analysis showed that the maximal binding capacity (Bmax) of LDL, but not of HDL3, is increased by twofold in the absence of apoE synthesis/secretion. Compared to control hepatic cells, LDL and HDL3 holoparticle uptake by apoE KO hepatic cells, as monitored by protein degradation, is reduced by 54 and 77%, respectively. Cleavage of heparan sulfate proteoglycans (HSPG) by treatment with heparinase I reduces LDL association by 21% in control hepatic cells. Thus, HSPG alone or a hepatic apoE-HSPG complex is partially involved in LDL association with mouse hepatic cells. In apoE KO, but not in normal hepatic cells, the same treatment increases LDL uptake/degradation by 2.4-fold suggesting that in normal hepatic cells, hepatic apoE increases LDL degradation by masking apoB-100 binding sites on proteoglycans. Cholesteryl ester (CE) association and CE selective uptake (CE/protein association ratio) from LDL and HDL3 by mouse hepatic cells were not affected by the absence of apoE expression. We also show that 69 and 72% of LDL-CE hydrolysis in control and apoE KO hepatic cells, respectively, is sensitive to chloroquine revealing the importance of a pathway linked to lysosomes. In contrast, HDL3-CE hydrolysis is only mediated by a nonlysosomal pathway in both control and apoE KO hepatic cells. Overall, our results indicate that hepatic apoE increases the holoparticle uptake pathway of LDL and HDL3 by mouse hepatic cells, that HSPG devoid of apoE favors LDL binding/association but impairs LDL uptake/degradation and that apoE plays no significant role in CE selective uptake from either human LDL or HDL3 lipoproteins.

Disease associated with Clostridium difficile infection is caused by the actions of the homologous toxins TcdA and TcdB on colonic epithelial cells. Binding to target cells triggers toxin internalization into acidified vesicles, whereupon cryptic segments from within the 1,050-aa translocation domain unfurl and insert into the bounding membrane, creating a transmembrane passageway to the cytosol. Our current understanding of the mechanisms underlying pore formation and the subsequent translocation of the upstream cytotoxic domain to the cytosol is limited by the lack of information available regarding the identity and architecture of the transmembrane pore. Here, through systematic perturbation of conserved sites within predicted membrane-insertion elements of the translocation domain, we uncovered highly sensitive residues—clustered between amino acids 1,035 and 1,107—that when individually mutated, reduced cellular toxicity by as much as >1,000-fold. We demonstrate that defective variants are defined by impaired pore formation in planar lipid bilayers and biological membranes, resulting in an inability to intoxicate cells through either apoptotic or necrotic pathways. These findings along with the unexpected similarities uncovered between the pore-forming “hotspots” of TcdB and the well-characterized α-helical diphtheria toxin translocation domain provide insights into the structure and mechanism of formation of the translocation pore for this important class of pathogenic toxins.

Introduction: The hypertensive double-transgenic (dTG) rat strain, expressing human renin and angiotensinogen, develops severe hypertension and organ damage and 50% of individuals die by 7 weeks of age. Here, we characterise a variation of this model in which animals present stable hypertension.Materials and methods: The effect of renin—angiotensin system blockers on blood pressure was determined with adult dTG rats treated with enalapril from 3 to 12 weeks of age. Tissue expression levels of renin and angiotensinogen were determined in dTG rats and rhesus monkeys by quantitative PCR.Results: Upon withdrawal from enalapril, mean arterial pressure (MAP) rose to 160—180 mmHg, with 95% of the female dTG rats surviving for 6 to 12 months, In Sprague-Dawley (SD) rats and rhesus monkeys, renin mRNA was absent or weakly expressed in most tissues, except for the kidneys and adrenals. In dTG rats, human renin expression was high in many additional tissues. The expression of human angiotensinogen in dTG rats followed a similar tissue pattern to SD and rhesus monkey angiotensinogen. Oral dosing of aliskiren, enalapril or losartan provided a similar maximal reduction in MAP and duration of efficacy in telemetrised dTG rats.Conclusions: Enalapril-pretreated dTG rats are suitable for long-term MAP monitoring and sequential evaluation of human renin inhibitors.

The rapid emergence and the prevalence of resistance mutations in HIV-1 reverse transcriptase (RT) underscore the need to identify RT inhibitors with novel binding modes and mechanisms of inhibition. Recently, two structurally distinct inhibitors, phosphonoformic acid (foscarnet) and INDOPY-1 were shown to disrupt the translocational equilibrium of RT during polymerization through trapping of the enzyme in the pre- and the post-translocation states, respectively. Here, we show that foscarnet and INDOPY-1 additionally display a shared novel inhibitory preference with respect to substrate primer identity. In RT-catalyzed reactions using RNA-primed substrates, translocation inhibitors were markedly less potent at blocking DNA polymerization than in equivalent DNA-primed assays; i.e. the inverse pattern observed with marketed non-nucleoside inhibitors that bind the allosteric pocket of RT. This potency profile was shown to correspond with reduced binding on RNA·DNA primer/template substrates versus DNA·DNA substrates. Furthermore, using site-specific footprinting with chimeric RNA·DNA primers, we demonstrate that the negative impact of the RNA primer on translocation inhibitor potency is overcome after 18 deoxyribonucleotide incorporations, where RT transitions primarily into polymerization-competent binding mode. In addition to providing a simple means to identify similarly acting translocation inhibitors, these findings suggest a broader role for the primer-influenced binding mode on RT translocation equilibrium and inhibitor sensitivity.

The authors have devised a continuous fluorescence-based assay to measure HIV reverse transcriptase (RT) polymerase activity for both high-throughput screening (HTS) and mechanistic characterization of inhibitors. The designed substrate is composed of a recessed DNA primer annealed to a DNA template that is labeled at the 5'-terminus with a donor fluorophore (AlexaFluor 488). RT-catalyzed incorporation of an acceptor-labeled deoxyuridine (dUTP-AlexaFluor 555) at the 3'-terminus of the fully extended DNA primer juxtaposes donor and acceptor fluorophores, resulting in robust fluorescence resonance energy transfer that can be monitored kinetically in real time. The assay is sensitive, permitting the use of low enzyme concentrations (

SummaryClostridium difficile causes life-threatening diarrhea through the actions of its homologous toxins TcdA and TcdB on human colonocytes. Therapeutic agents that block toxin-induced damage are urgently needed to prevent the harmful consequences of toxin action that are not addressed with current antibiotic-based treatments. Here, we developed an imaging-based phenotypic screen to identify small molecules that protected human cells from TcdB-induced cell rounding. A series of structurally diverse compounds with antitoxin activity were identified and found to act through one of a small subset of mechanisms, including direct binding and sequestration of TcdB, inhibition of endosomal maturation, and noncompetitive inhibition of the toxin glucosyltransferase activity. Distinct classes of inhibitors were used further to dissect the determinants of the toxin-mediated necrosis phenotype occurring at higher doses of toxin. These findings validate and inform novel targeting strategies for discovering small molecule agents to treat C. difficile infection.

Étant donné l’importance des compétences langagières dans le parcours scolaire d’un individu ainsi que dans son développement personnel, professionnel et social, l’objectif de ce texte est de présenter un portrait des compétences langagières des élèves au début de leur parcours dans les CFER et à la suite d’une première année de formation. Différentes épreuves diagnostiques ont été utilisées de manière à évaluer l’ensemble des composantes des compétences langagières. Plus spécifiquement, seront présentés dans cet article les résultats obtenus (1) pour le volet lecture, aux épreuves de compréhension écrite, d’identification de mots et de conscience phonologique; (2) pour le volet écriture, à l’épreuve de production de mots et (3) pour le volet oral, aux épreuves de compréhension d’énoncés et de structure du récit, et de vocabulaire fonctionnel. Les analyses permettront de démontrer l’évolution des compétences langagières chez les jeunes scolarisés dans les CFER et les relations existantes entre les résultats aux différentes épreuves., Considering the importance of language skills in terms of making one’s way through school, as well as for personal, professional, and social development, the objective of this article is to present a portrait of the language skills of students when they first enter the CFERs and after one year of training. A variety of diagnostic tests were used to evaluate the different components of their language skills. More specifically, the article presents results obtained 1) for the reading section, on written comprehension, word identification, and phonological awareness tests, 2) for the writing section, on word production tests, and 3) for the oral section, on tests on the comprehension of statements and story structure, and on functional vocabulary tests. The analyses of the results will allow the researchers to demonstrate the development of language skills among youngsters attending CFERs and the relationships among the results of the different tests., Dada la importancia de la competencia lingüística en el trayecto escolar, el desarrollo personal, profesional y social de un individuo, el presente artículo tiene como finalidad presentar un retrato de la competencia lingüística de alumnos que comienzan su trayectoria en los CFER, después de un año de formación. Diferentes exámenes diagnósticos fueron empleados en la evaluación del conjunto de componentes de la competencia lingüística. Más específicamente, en este artículo se presentan los resultados obtenidos 1) en lectura, en una prueba de comprensión escrita, identificación de palabras y consciencia fonológica; 2) en escritura, en una prueba de producción de palabras y 3) a nivel oral, en los exámenes de comprensión de enunciados, de estructura narrativa y de vocabulario funcional. Los análisis permitirán demostrar la evolución de la competencia lingüística entre los jóvenes escolarizados en los CFER y las relaciones que se pueden establecer entre los resultados en las diferentes pruebas.