RESUMEN
A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau4RDΔK (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range.
Asunto(s)
Portadores de Fármacos/química , Péptidos/química , Composición de Medicamentos , Liberación de Fármacos , Humanos , Modelos Moleculares , Biblioteca de Péptidos , Preparaciones Farmacéuticas/administración & dosificación , Polietilenglicoles/química , Solubilidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Cell models of tauopathy were generated in order to study mechanisms of neurodegeneration involving abnormal changes of tau. They are based on neuroblastoma cell lines (N2a) that inducibly express different forms of the repeat domain of tau (tau(RD)), e.g. the 4-repeat domain of tau with the wild-type sequence, the repeat domain with the DeltaK280 mutation ("pro-aggregation mutant"), or the repeat domain with DeltaK280 and two proline point mutations ("anti-aggregation mutant"). The data indicate that the aggregation of tau(RD) is toxic, and that aggregation and toxicity can be prevented by low molecular weight compounds, notably compounds based on the N-phenylamine core. Thus the cell models are suitable for developing aggregation inhibitor drugs.
Asunto(s)
Compuestos de Anilina/metabolismo , Tauopatías/tratamiento farmacológico , Tauopatías/genética , Animales , Línea Celular Tumoral/patología , Evaluación Preclínica de Medicamentos , Humanos , Modelos Biológicos , Mutación , Neuroblastoma/patología , Estructura Terciaria de ProteínaRESUMEN
Tau is a highly soluble protein, yet it aggregates abnormally in Alzheimer's disease. Here, we address the question of proteolytic processing of tau and the nucleation of aggregates by tau fragments. We show in neuronal cell models that fragments of the repeat domain of tau containing mutations of FTDP17 (frontotemporal dementia with parkinsonism linked to chromosome 17), produced by endogenous proteases, can induce the aggregation of full-length tau. Fragments are generated by successive cleavages, first N-terminally between K257 and S258, then C-terminally around residues 353-364; conversely, when the N-terminal cleavage is inhibited, no fragmentation and aggregation takes place. The C-terminal truncation and the coaggregation of fragments with full-length tau depends on the propensity for beta-structure. The aggregation is modulated by phosphorylation but does not depend on it. Aggregation but not fragmentation as such is toxic to cells; conversely, toxicity can be prevented by inhibiting either aggregation or proteolysis. The results reveal a novel pathway of abnormal tau aggregation in neuronal cells.
Asunto(s)
Procesamiento Proteico-Postraduccional , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Hidrólisis , Ratones , Microscopía Fluorescente , Modelos Biológicos , Modelos Químicos , Neuroblastoma/patología , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Fosforilación , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Solubilidad , Trombina/química , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
Progressive multifocal leukoencephalopathy (PML) is a fatal, demyelinating disease caused by JC virus (JCV) in patients with severe immunosuppression. We studied the JCV-specific cellular and humoral immune response in 7 healthy donors (HD), 6 human immunodeficiency virus-1 (HIV-1)-infected patients without PML (HIV), 4 HIV-1-negative patients with PML (PML), and 8 HIV-1-positive patients with PML (HIV/PML). As antigens, recombinant virus-like particles of the major structural protein VP1 (VP1-VLP) of JCV, tetanus toxoid (TT), or the mitogen phytohemagglutinin (PHA) were used. Proliferation of peripheral blood mononuclear cells (PBMC) after stimulation with the VP1-VLP was significantly suppressed in PML and HIV/PML patients compared to HD. After antigen stimulation the production of interferon-gamma (IFN-gamma) was reduced in PML, in HIV/PML, and in HIV patients. The production of interleukin-10 (IL-10), however, was elevated in HIV/PML patients. Neither proliferation nor cytokine production correlated with the presence of JCV DNA in PBMC. The immunoglobulin G serum antibody titer to the VP1-VLP was slightly elevated in HIV, elevated in PML, and highly elevated in HIV/PML patients compared to HD. The development of PML appears to coincide with a general impairment of the Th1-type T-helper cell function of cell-mediated immunity.
Asunto(s)
Infecciones por VIH/inmunología , Virus JC/inmunología , Leucoencefalopatía Multifocal Progresiva/inmunología , Proteínas Estructurales Virales/inmunología , Citocinas/inmunología , VIH-1 , Humanos , Inmunoglobulina G/inmunología , Leucocitos Mononucleares/inmunología , Reacción en Cadena de la PolimerasaRESUMEN
Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease caused by the human polyomavirus JCV. The hypervariable noncoding transcriptional control region (TCR) largely regulates replication of JCV in glial cells. Two distinct types of the TCR can be distinguished. Type II is derived from the archetype sequence. All type I TCRs, including the prototypical Mad-1 isolate contain a 23 bp deletion at nucleotide position 36. In a prospective study, TCR-DNA could be amplified and sequenced in 16/29 (55%) suspect cases of PML from the cerebrospinal fluid (CSF) and in 14/28 (50%) urine samples. Sequencing of the CSF-TCR identified Mad-1 like sequences in 5/17 (29.5%) instances and a type II TCR in 12/17 (70.5%) of cases. Of 14 urine TCRs, 12 (86%) displayed the archetype sequence, while two showed complex rearrangements. In all type II TCR sequences, the tst-1/oct-6 binding sites present in regions C and E of Mad-1 were missing. In 11/12 type II TCR sequences the pentanucleotide repeat in region A showed a G to T substitution of one nucleotide at position 36 relative to the Mad-1 TCR. All type II TCRs contained an Sp1 binding site at the beginning of region B. Of the 12 TCR type II sequences, 10 (83%) were of the 'D-retaining' pattern. In eight of these (80%) additional juxtapositioned nuclear factor 1, glial factor 1 and/or AP-1 binding motifs were created by duplications and/or insertions in region D. These findings indicate that type II TCRs are frequently present in PML and suggest to use TCR type II constructs for in vitro and in vivo studies of the evaluation of the functional role of DNA binding motifs.
Asunto(s)
Regulación Viral de la Expresión Génica , Virus JC/genética , Leucoencefalopatía Multifocal Progresiva/virología , Activación Transcripcional/genética , Secuencia de Bases , ADN Viral/líquido cefalorraquídeo , ADN Viral/genética , ADN Viral/orina , Humanos , Virus JC/aislamiento & purificación , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , TATA Box/genéticaRESUMEN
OBJECTIVE: Before parturition are there useful and valid predictors of successful or unsuccessful vaginal birth after previous cesarean birth that could be used to enhance the obstetric care of a patient and her pregnancy? STUDY DESIGN: The clinical course and outcome of all patients who attempted vaginal birth after cesarean delivery at one level III center during 1989 were evaluated to identify factors prognostic of a successful or unsuccessful patient group; use of this information in stepwise logistic regression and cluster analysis was disappointing. RESULTS: No single criterion or optimal clusters of factors were found and no equation achieved greater than 75% predictability of outcome with acceptable sensitivity and specificity. CONCLUSIONS: Before parturition prediction of outcome of vaginal birth after cesarean delivery is tenuous regardless of past obstetric history or recent clinical parameters. Thus it seems appropriate to encourage a trial of labor in almost all patients with a prior low-segment uterine incision (transverse or vertical) unless there is a strong physician or patient-derived contraindication to such an undertaking.