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Biomedical subjects

Carlos F Barbas

Publications and source records attributed to Carlos F Barbas.

At least 37 records · Page 2Linked to original sources
PubMed2005

Development of zinc finger domains for recognition of the 5'-CNN-3' family DNA sequences and their use in the construction of artificial transcription factors.

Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. Although many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a three-base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18-base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 of the 16 5'-CNN-3' subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis, and de novo design. Furthermore, these domains were used to generate a highly specific six-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up- and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5'-CNN-3'-, 5'-GNN-3'-, and 5'-ANN-3'-containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.

Amino Acid Sequence↗
PubMed2005

Intrabodies targeting the Kaposi sarcoma-associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells.

Kaposi sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen-1 (LANA1) is essential for the maintenance and segregation of viral episomes in KSHV latently infected B cells. We report development of intracellular, rabbit-derived antibodies generated by phage display technology, which bind to N-terminal LANA1 epitopes and neutralize the chromosome-binding activity of LANA1. Although these cloned single-chain variable fragments (scFvs) show relatively low binding affinities for the LANA1 viral antigen in in vitro assays, they nonetheless outcompete KSHV-seropositive human sera for LANA1 epitope binding. In heterologous cells, intracellular intrabody expression inhibits LANA1-dependent plasmid maintenance of both an artificial plasmid containing KSHV LANA1 binding sequences and a bacterial artificial chromosome containing the entire KSHV genome. In KSHV naturally infected primary effusion lymphoma cells, intracellular intrabody expression causes a reduction or loss of the typical LANA1 punctate, nuclear pattern. This morphologically apparent LANA1 dispersion correlates to loss of viral episome by molecular analysis. These data suggest a novel approach to antiherpes viral therapy and confirm LANA1 is critical target for neutralization of KSHV viral latency.

Amino Acid Sequence↗
PubMed2005

Genetic reprogramming of tumor cells by zinc finger transcription factors.

Cancer arises by the accumulation of genetic alterations in DNA leading to aberrant gene transcription. Expression-profiling studies have correlated genomewide expression signatures with malignancy. However, functional analysis elucidating the contribution and synergy of genes in specific cancer cell phenotypes remains a formidable obstacle. Herein, we describe an alternative genetic approach for identification of genes involved in tumor progression by using a library of zinc finger artificial transcription factors (ATFs) and functional screening of tumor cells as a source of genetic plasticity and clonal selection. We isolated a six-zinc finger transcriptional activator (TF 20-VP, TF 20 containing the VP64 activator domain) that acts to reprogram a drug-sensitive, poorly invasive, and nonmetastatic cell line into a cell line with a drug-resistant, highly invasive, and metastatic phenotype. Differential expression profiles of cells expressing TF 20-VP followed by functional studies, both in vitro and in animal models, revealed that invasion and metastasis requires co-regulation of multiple target genes. Significantly, the E48 antigen, associated with poor metastasis-free survival in head and neck cancer, was identified as one specific target of TF 20-VP. We have shown phenotypic modulation of tumor cell behavior by E48 expression, including enhanced cell migration in vitro and tumor cell dissemination in vivo. This study demonstrates the use of ATFs to identify the group of genes that cooperate during tumor progression. By co-regulating multiple targets, ATFs can be used as master genetic switches to reprogram and modulate complex neoplastic phenotypes.

Animals↗
PubMed2005

Crystallization and preliminary X-ray crystallographic analysis of Aart, a designed six-finger zinc-finger peptide, bound to DNA.

Crystals of a designed six-finger zinc-finger protein, Aart, bound to a 22-base-pair duplex DNA containing a consensus binding site have been obtained. Crystals grew by hanging-drop vapor diffusion from solutions containing polyethylene glycol 4000 as the precipitating agent. The irregularly shaped crystals belong to space group P1, with unit-cell parameters a = 41.95, b = 71.76, c = 74.73 A, alpha = 100.87, beta = 96.22, gamma = 106.33 degrees . There are most likely to be two protein-DNA complexes in the asymmetric unit. A complete native data set has been collected from a high-energy synchrotron source to a resolution of 2.95 A at 100 K, with an Rmerge of 9.3%.

Amino Acid Sequence↗
PubMed2005

Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesis in vivo.

The endothelial cell receptor-tyrosine kinases, VEGF receptor 2 (VEGF-R2) and Tie-2, and their ligands, vascular endothelial growth factor (VEGF) and angiopoietins 1 and 2, respectively, play key roles in tumor angiogenesis. Several studies suggest that the VEGF receptor pathway and the Tie-2 pathway are independent and essential mediators of angiogenesis, leading to the hypothesis that simultaneous interference with both pathways should result in additive effects on tumor growth. In this study, a human melanoma xenograft model (M21) was used to analyze the effects of simultaneous intradiabody depletion of vascular endothelial growth receptor-R2 and Tie-2 on tumor angiogenesis and tumor xenograft growth. The intradiabodies were expressed from recombinant adenovirus delivered through subtumoral injection. Blockade of both VEGF-R2 and Tie-2 pathways simultaneously or the VEGF receptor pathway alone resulted in a significant inhibition of tumor growth and tumor angiogenesis (92.2% and 74.4%, respectively). In addition, immunohistochemical staining of intradiabody-treated tumors demonstrated a decreased number of tumor-associated blood vessels versus control treatment. Previous studies with intrabodies had demonstrated that the Tie-2 receptor pathway was essential for tumor growth. The simultaneous blockade of the VEGF and Tie-2 pathways resulted in effective inhibition of tumor growth and demonstrated the potential of simultaneous targeting of multiple pathways as a therapeutic strategy.

Animals↗
PubMed2005

Development of small designer aldolase enzymes: catalytic activity, folding, and substrate specificity.

Small (24-35 amino acid residues) peptides that catalyze carbon-carbon bond transformations including aldol, retro-aldol, and Michael reactions in aqueous buffer via an enamine mechanism have been developed. Peptide phage libraries were created by appending six randomized amino acid residues to the C-terminus or to the N-terminus of an 18-mer alpha-helix peptide containing lysine residues. Reaction-based selection with 1,3-diketones was performed to trap the amino groups of reactive lysine residues that were necessary for the catalysis via an enamine mechanism by formation of stable enaminones. The selected 24-mer peptides catalyzed the reactions with improved activities. The improved activities were correlated with improved folded states of the peptides. The catalyst was then improved with respect to substrate specificity by appending a phage display-derived substrate-binding module. The resulting 35-mer peptide functioned with a significant proportion of the catalytic proficiency of larger protein catalysts. These results indicate that small designer enzymes with good rate acceleration and excellent substrate specificity can be created by combination of design and reaction-based selection from libraries.

Aldehydes↗
PubMed2005

Maspin alters the carcinoma proteome.

Maspin, a member of the serine protease inhibitor (serpin) family, is a tumor suppressor in breast and prostate cancer. To address molecular mechanisms underlying maspin's activity, we restored its expression in invasive carcinoma cells and analyzed the resulting changes by shotgun proteomics. Using a mass spectrometry-based multidimensional proteomic method, we observed changes to the expression of approximately 27% of the detectable proteome. In particular, we noted changes to the expression of proteins that regulate cytoskeletal architecture, cell death, and protein turnover. In each case, changes in protein expression were accompanied by measurable changes in tumor cell phenotype. Thus, maspin-expressing cells exhibit a more prominent actin cytoskeleton, a reduced invasive capacity, an increased rate of spontaneous apoptosis, and an altered proteasome function. These observations reveal for the first time the far reaching effects of maspin on multiple protein networks and a new hypothesis of maspin function based on the regulation of proteasome function.

Apoptosis↗
PubMed2005

Direct amino acid-catalyzed asymmetric desymmetrization of meso-compounds: tandem aminoxylation/O-N bond heterolysis reactions.

[reaction: see text] A practical organocatalytic process for the synthesis of optically active, highly substituted alpha-hydroxy-ketones was achieved through asymmetric desymmetrization (ADS) of prochiral ketones. The ADS and O-N bond reduction reaction of prochiral ketone with nitrosobenzene in the presence of a catalytic amount of chiral amine or amino acid produced the tandem ADS/O-N bond reduced products as single diastereomers with good yields and excellent enantiomeric excesses.

Amines↗
PubMed2005

Mimicking dihydroxy acetone phosphate-utilizing aldolases through organocatalysis: a facile route to carbohydrates and aminosugars.

[reaction: see text] A practical and environmentally friendly organocatalytic strategy designed to mimic the DHAP aldolases has been developed and shown to be effective in the preparation of carbohydrates and aminosugars. (S)-Proline and (S)-2-pyrrolidine-tetrazole catalyzed the aldol reaction between dihydroxy acetone variants such as 1,3-dioxan-5-one and 2,2-dimethyl-1,3-dioxan-5-one with aldehydes to give the corresponding polyols in good yields with very high ees.

Aldehyde-Lyases↗
PubMed2005

Total synthesis of LFA-1 antagonist BIRT-377 via organocatalytic asymmetric construction of a quaternary stereocenter.

A catalytic route for enantioselective total synthesis of cell adhesion inhibitor BIRT-377 is described. The quaternary stereocenter was constructed through l-proline-derived, tetrazole-catalyzed direct asymmetric alpha-amination of 3-(4-bromophenyl)-2-methylpropanal with dibenzyl azodicarboxylate. In the course of these studies, a one-pot trifluoro acetylation/selective benzyloxycarbonyl deprotection method was developed. [structure: see text]

Aldehydes↗
PubMed2005

Targeting tumor angiogenesis with adenovirus-delivered anti-Tie-2 intrabody.

Inhibition of tumor angiogenesis is a promising approach for cancer therapy. As an endothelial cell-specific receptor kinase expressed almost exclusively on the surface of vascular endothelium, Tie-2 has an important role in tumor angiogenesis. To explore the therapeutic potential of blocking Tie-2 receptor-interaction pathway, an adenoviral vector was used to deliver a recombinant single-chain antibody fragment rabbit intrabody (pAd-2S03) capable of inhibition of both mouse and human Tie-2 surface expression. pAd-2S03 was given to mice with well-established primary tumors, either a human Kaposi's sarcoma (SLK) or a human colon carcinoma (SW1222). The intrabody significantly inhibited growth of both tumors (75% and 63%, respectively) when compared with pAd-GFP control-treated tumors (P < 0.01). Histopathologic analysis of cryosections taken from mice treated with pAd-2S03 revealed a marked decrease in vessel density, which was reduced by >87% in both tumor models when compared with control-treated tumors (P < 0.01). In contrast, human Tie-2-monospecific pAd-1S05 intrabody did not affect the growth of tumors, indicating that the antitumor effect of pAd-2S03 was due to the inhibition of tumor angiogenesis in these murine models. Our results show that the Tie-2 receptor pathway is essential for both SLK sarcoma and SW1222 colon carcinoma xenograft growth. The present study shows the potential utility of antiangiogenic agents that target the endothelium-specific receptor Tie-2 for down-regulation or genetic deletion.

Adenoviridae↗
PubMed2005

Custom zinc-finger nucleases for use in human cells.

Genome engineering through homologous recombination (HR) is a powerful instrument for studying biological pathways or creating treatment options for genetic disorders. In mammalian cells HR is rare but the creation of targeted DNA double-strand breaks stimulates HR significantly. Here, we present a method to generate, evaluate, and optimize rationally designed endonucleases that promote HR. The DNA-binding domains were synthesized by assembling predefined zinc-finger modules selected by phage display. Attachment of a transcriptional activation domain allowed assessment of DNA binding in reporter assays, while fusion with an endonuclease domain created custom nucleases that were tested for their ability to stimulate HR in episomal and chromosomal gene repair assays. We demonstrate that specificity, expression kinetics, and protein design are crucial parameters for efficient gene repair and that our two-step assay allows one to go quickly from design to testing to successful employment of the custom nucleases in human cells.

Base Sequence↗
PubMed2005

Zinc finger transcription factors designed for bispecific coregulation of ErbB2 and ErbB3 receptors: insights into ErbB receptor biology.

Signaling through the ErbB family of tyrosine kinase receptors in normal and cancer-derived cell lines contributes to cell growth and differentiation. In this work, we altered the levels of ErbB2 and ErbB3 receptors, individually and in combination, by using 6-finger and 12-finger synthetic zinc finger protein artificial transcription factors (ATFs) in an epidermoid squamous cell carcinoma line, A431. We successfully designed 12-finger ATFs capable of coregulating ErbB3 and ICAM-1 or ErbB2 and ErbB3. With ATFs, the effects of changes in ErbB2 and ErbB3 receptor levels were evaluated by using cell proliferation, cell migration, and cell signaling assays. Cell proliferation was increased when ErbB2 and ErbB3 were both overexpressed. Cell migration on collagen was decreased when ErbB2 was down-regulated, yet migration on laminin was significantly increased with ErbB3 overexpression. ErbB2 and ErbB3 overexpression also stimulated the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Our ATF approach has elucidated differences in ErbB receptor-mediated proliferation, migration, and intracellular signaling that cannot be explained merely by the presence or absence of particular ErbB receptors and emphasizes the dynamic nature of the ErbB signaling system. The transcription factor approach developed here provides a gene-economical route to the regulation of multiple genes and may be important for complex gene therapies.

Amino Acid Sequence↗
PubMed2005

Neutralization of Burkholderia pseudomallei protease by Fabs generated through phage display.

The isolation of therapeutic and functional protease inhibitors in vitro via combinatorial chemistry and phage display technology has been described previously. Here we report the construction of a combinatorial mouse-human chimeric antibody fragment (Fab) antibody library targeted against the protease of the tropical pathogen, Burkholderia pseudomallei. The resulting library was biopanned against the protease, and selected clones were analyzed for their ability to function as protease inhibitors. Three families of Fabs were identified by restriction fingerprinting, all of which demonstrated high specificity towards the protease of B. pseudomallei. Purified Fabs also demonstrated the capacity to inhibit B. pseudomallei protease activity in vitro, and this inhibitory property was exclusive to the pathogenic protease. Thus these recombinant antibodies are candidates for immunotherapy and tools to aid in further elucidation of the mechanism of action of the B. pseudomallei protease.

Amino Acid Sequence↗
PubMed2004

Exploring strategies for the design of artificial transcription factors: targeting sites proximal to known regulatory regions for the induction of gamma-globin expression and the treatment of sickle cell disease.

Artificial transcription factors can be engineered to interact with specific DNA sequences to modulate endogenous gene expression within cells. A significant hurdle to implementation of this approach is the selection of the appropriate DNA sequence for targeting. We reasoned that a good target site should be located in chromatin, where it is accessible to DNA-binding proteins, and it should be in the close vicinity of known transcriptional regulators of the gene. Here we have explored the efficacy of these criteria to guide our selection of potential regulators of gamma-globin expression. Several zinc finger-based transcriptional activators were designed to target the sites proximal to the -117-position of the gamma-globin promoter. This region is proximal to the binding sites of known and potential natural transcription factors. Design and study of three transcription factors identified the potent transcriptional activator, gg1-VP64-HA. This transcription factor was able to interact directly with the gamma-globin promoter and up-regulate expression of reporter gene constructs as well as the endogenous gene in a selective manner. Transfection of a gg1-VP64-HA expression vector or retroviral delivery of this transcription factor into the erythroleukemia cell line K562 resulted in an increase of fetal hemoglobin. The gamma-globin content of cells expressing gg1-vp64-HA showed up to 16-fold higher levels of fetal hemoglobin than the native K562 cell line. These transcriptional activators constitute a novel class of regulators of the globin locus that may be suitable for treatment of diseases arising from mutations in this locus such as sickle cell disease and thalassemic diseases.

Anemia, Sickle Cell↗