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1.
Biotechnol Prog ; 29(2): 415-24, 2013.
Article in English | MEDLINE | ID: mdl-23172735

ABSTRACT

Traditional metabolic engineering approaches, including homologous recombination, zinc-finger nucleases, and short hairpin RNA, have previously been used to generate biologics with specific characteristics that improve efficacy, potency, and safety. An alternative approach is to exogenously add soluble small interfering RNA (siRNA) duplexes, formulated with a cationic lipid, directly to cells grown in shake flasks or bioreactors. This approach has the following potential advantages: no cell line development required, ability to tailor mRNA silencing by adjusting siRNA concentration, simultaneous silencing of multiple target genes, and potential temporal control of down regulation of target gene expression. In this study, we demonstrate proof of concept of the siRNA feeding approach as a metabolic engineering tool in the context of increasing monoclonal antibody (MAb) afucosylation. First, potent siRNA duplexes targeting fut8 and gmds were dosed into shake flasks with cells that express an anti-CD20 MAb. Dose response studies demonstrated the ability to titrate the silencing effect. Furthermore, siRNA addition resulted in no deleterious effects on cell growth, final protein titer, or specific productivity. In bioreactors, antibodies produced by cells following siRNA treatment exhibited improved functional characteristics compared to antibodies from untreated cells, including increased levels of afucosylation (63%), a 17-fold improvement in FCgRIIIa binding, and an increase in specific cell lysis by up to 30%, as determined in an Antibody-Dependent Cellular Cytoxicity (ADCC) assay. In addition, standard purification procedures effectively cleared the exogenously added siRNA and transfection agent. Moreover, no differences were observed when other key product quality structural attributes were compared to untreated controls. These results establish that exogenous addition of siRNA represents a potentially novel metabolic engineering tool to improve biopharmaceutical function and quality that can complement existing metabolic engineering methods.


Subject(s)
Antibodies, Monoclonal/metabolism , Metabolic Engineering/methods , RNA, Small Interfering/genetics , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibody-Dependent Cell Cytotoxicity , Cell Line , Fucosyltransferases/genetics , Fucosyltransferases/metabolism , Gene Silencing , Humans , Metabolic Engineering/instrumentation , Protein Processing, Post-Translational , RNA, Small Interfering/metabolism
2.
J Wildl Dis ; 48(2): 530-3, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22493139

ABSTRACT

We compared prion infection rates among mule deer (Odocoileus hemionus) receiving pentosan polysulfate, tannic acid, tetracycline HCl, or no treatment 14 days before to 14 days after (dpi) oral inoculation with tonsil tissue homogenate. All deer were infected, but the rapid disease course (230-603 dpi) suggested our challenge was overwhelming.


Subject(s)
Deer , Pentosan Sulfuric Polyester/therapeutic use , Prions/pathogenicity , Tannins/therapeutic use , Tetracycline/therapeutic use , Wasting Disease, Chronic/drug therapy , Animals , Prions/administration & dosage , Survival Analysis , Time Factors , Wasting Disease, Chronic/mortality
3.
Bioorg Med Chem Lett ; 21(24): 7277-80, 2011 Dec 15.
Article in English | MEDLINE | ID: mdl-22061640

ABSTRACT

Starting from literature examples of nonsteroidal anti-inflammatory drugs (NSAIDs)-type carboxylic acid γ-secretase modulators (GSMs) and using a scaffold design approach, we identified 4-aminomethylphenylacetic acid 4 with a desirable γ-secretase modulation profile. Scaffold optimization led to the discovery of a novel chemical series, represented by 6b, having improved brain penetration. Further SAR studies provided analog 6q that exhibited a good pharmacological profile. Oral administration of 6q significantly reduced brain Aß42 levels in mice and rats.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Enzyme Inhibitors/chemistry , Phenylacetates/chemistry , Piperidines/chemistry , Administration, Oral , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/metabolism , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemical synthesis , Anti-Inflammatory Agents, Non-Steroidal/pharmacokinetics , Brain/metabolism , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacokinetics , Mice , Peptide Fragments/metabolism , Phenylacetates/chemical synthesis , Phenylacetates/pharmacokinetics , Piperidines/chemical synthesis , Piperidines/pharmacokinetics , Rats
4.
Antimicrob Agents Chemother ; 51(11): 3887-94, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17709470

ABSTRACT

Cyclic tetrapyrroles are among the most potent compounds with activity against transmissible spongiform encephalopathies (TSEs; or prion diseases). Here the effects of differential sulfonation and metal binding to cyclic tetrapyrroles were investigated. Their potencies in inhibiting disease-associated protease-resistant prion protein were compared in several types of TSE-infected cell cultures. In addition, prophylactic antiscrapie activities were determined in scrapie-infected mice. The activity of phthalocyanine was relatively insensitive to the number of peripheral sulfonate groups but varied with the type of metal bound at the center of the molecule. The tendency of the various phthalocyanine sulfonates to oligomerize (i.e., stack) correlated with anti-TSE activity. Notably, aluminum(III) phthalocyanine tetrasulfonate was both the poorest anti-TSE compound and the least prone to oligomerization in aqueous media. Similar comparisons of iron- and manganese-bound porphyrin sulfonates confirmed that stacking ability correlates with anti-TSE activity among cyclic tetrapyrroles.


Subject(s)
Metals/chemistry , Prion Diseases/prevention & control , Sulfonic Acids/chemistry , Tetrapyrroles/pharmacology , Animals , Blotting, Western , Cells, Cultured , Cricetinae , Dimerization , Gene Expression/drug effects , Indoles/chemistry , Indoles/pharmacology , Isoindoles , Metals/metabolism , Mice , Mice, Transgenic , Molecular Structure , PrPSc Proteins/genetics , PrPSc Proteins/metabolism , Prion Diseases/genetics , Prion Diseases/metabolism , Sulfonic Acids/metabolism , Tetrapyrroles/chemistry , Tetrapyrroles/metabolism
6.
Methods Enzymol ; 412: 223-34, 2006.
Article in English | MEDLINE | ID: mdl-17046661

ABSTRACT

The transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurodegenerative diseases of mammals. Protease-resistant prion protein (PrP-res) is only associated with TSEs and thus has been a target for therapeutic intervention. The most effective compounds known against scrapie in vivo are inhibitors of PrP-res in infected cells. Mouse neuroblastoma (N2a) cells have been chronically infected with several strains of mouse scrapie including RML and 22L. Also, rabbit epithelial cells that produce sheep prion protein in the presence of doxycycline (Rov9) have been infected with sheep scrapie. Here a high-throughput 96-well plate PrP-res inhibition assay is described for each of these scrapie-infected cell lines. With this dot-blot assay, thousands of compounds can easily be screened for inhibition of PrP-res formation. This assay is designed to find new PrP-res inhibitors, which may make good candidates for in vivo anti-scrapie testing. However, an in vitro assay can only suggest that a given compound might have in vivo anti-scrapie activity, which is typically measured as increased survival times. Methods for in vivo testing of compounds for anti-scrapie activity in transgenic mice, a much more lengthy and expensive process, are also discussed.


Subject(s)
PrPSc Proteins/antagonists & inhibitors , Scrapie/drug therapy , Scrapie/metabolism , Animals , Biological Assay , Cell Line , Cell Line, Tumor , Drug Design , Epithelial Cells/cytology , In Vitro Techniques , Mice , Mice, Transgenic , Neuroblastoma , PrPSc Proteins/metabolism , Rabbits , Sheep , Species Specificity
7.
Antimicrob Agents Chemother ; 50(10): 3447-9, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17005828

ABSTRACT

Combination treatment with pentosan polysulfate and Fe(III)meso-tetra(4-sulfonatophenyl)porphine in mice beginning 14 or 28 days after scrapie inoculation significantly increased survival times. This increase may be synergistic, implying that the compounds act cooperatively in vivo. Combination therapy may therefore be more effective for treatment of transmissible spongiform encephalopathies and other protein-misfolding diseases.


Subject(s)
Pentosan Sulfuric Polyester , Porphyrins , Scrapie/drug therapy , Animals , Cricetinae , Drug Synergism , Drug Therapy, Combination , Mice , Pentosan Sulfuric Polyester/administration & dosage , Pentosan Sulfuric Polyester/therapeutic use , Porphyrins/administration & dosage , Porphyrins/therapeutic use , Scrapie/mortality , Survival Rate , Treatment Outcome
8.
Protein Sci ; 15(3): 609-19, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16452616

ABSTRACT

A central feature of transmissible spongiform encephalopathies (TSE or prion diseases) involves the conversion of a normal, protease-sensitive glycoprotein termed prion protein (PrP-sen) into a pro-tease-resistant form, termed PrP-res. The N terminus of PrP-sen has five copies of a repeating eight amino acid sequence (octapeptide repeat). The presence of one to nine extra copies of this motif is associated with a heritable form of Creutzfeld-Jakob disease (CJD) in humans. An increasing number of octapeptide repeats correlates with earlier CJD onset, suggesting that the rate at which PrP-sen misfolds into PrP-res may be influenced by these mutations. In order to determine if octapeptide repeat insertions influence the rate at which PrP-res is formed, we used a hamster PrP amyloid-forming peptide (residues 23-144) into which two to 10 extra octapeptide repeats were inserted. The spontaneous formation of protease-resistant PrP amyloid from these peptides was more rapid in response to an increased number of octapeptide repeats. Furthermore, experiments using full-length glycosylated hamster PrP-sen demonstrated that PrP-res formation also occurred more rapidly from PrP-sen molecules expressing 10 extra copies of the octapeptide repeat. The rate increase for PrP-res formation did not appear to be due to any influence of the octapeptide repeat region on PrP structure, but rather to more rapid binding between PrP molecules. Our data from both models support the hypothesis that extra octapeptide repeats in PrP increase the rate at which protease resistant PrP is formed which in turn may affect the rate of disease onset in familial forms of CJD.


Subject(s)
Amyloid/chemistry , Peptide Hydrolases/metabolism , Prions/chemistry , Amyloid/metabolism , Amyloid/ultrastructure , Animals , Cricetinae , Kinetics , Mutagenesis, Insertional , Prions/genetics , Prions/metabolism , Protein Binding , Repetitive Sequences, Amino Acid
9.
Antimicrob Agents Chemother ; 50(3): 1034-44, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16495266

ABSTRACT

Although transmissible spongiform encephalopathies (TSEs) are incurable, a key therapeutic approach is prevention of conversion of the normal, protease-sensitive form of prion protein (PrP-sen) to the disease-specific protease-resistant form of prion protein (PrP-res). Here degenerate phosphorothioate oligonucleotides (PS-ONs) are introduced as low-nM PrP-res conversion inhibitors with strong antiscrapie activities in vivo. Comparisons of various PS-ON analogs indicated that hydrophobicity and size were important, while base composition was only minimally influential. PS-ONs bound avidly to PrP-sen but could be displaced by sulfated glycan PrP-res inhibitors, indicating the presence of overlapping binding sites. Labeled PS-ONs also bound to PrP-sen on live cells and were internalized. This binding likely accounts for the antiscrapie activity. Prophylactic PS-ON treatments more than tripled scrapie survival periods in mice. Survival times also increased when PS-ONs were mixed with scrapie brain inoculum. With these antiscrapie activities and their much lower anticoagulant activities than that of pentosan polysulfate, degenerate PS-ONs are attractive new compounds for the treatment of TSEs.


Subject(s)
Oligonucleotides/pharmacology , Phosphates/chemistry , PrPSc Proteins/antagonists & inhibitors , Scrapie/metabolism , Scrapie/prevention & control , Animals , Base Composition , Cell Line , Cricetinae , Hydrophobic and Hydrophilic Interactions , Inhibitory Concentration 50 , Injections, Intraperitoneal , Injections, Subcutaneous , Mice , Mice, Transgenic , Molecular Weight , Nucleic Acid Conformation , Oligonucleotides/administration & dosage , Oligonucleotides/chemistry , PrPSc Proteins/genetics , Survival
10.
Antimicrob Agents Chemother ; 50(2): 759-61, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16436739

ABSTRACT

Prion diseases, including scrapie, are incurable neurodegenerative disorders. Some compounds can delay disease after a peripheral scrapie inoculation, but few are effective against advanced disease. Here, we tested multiple related porphyrins, but only Fe(III)meso-tetra(4-sulfonatophenyl)porphine injected into mouse brains after intracerebral scrapie inoculation substantially increased survival times.


Subject(s)
Porphyrins/therapeutic use , Prion Diseases/drug therapy , Animals , Blood-Brain Barrier , Mice , Mice, Transgenic , Porphyrins/pharmacokinetics
11.
J Virol ; 80(2): 596-604, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16378962

ABSTRACT

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP(CWD)) was used as an indicator of CWD infection. Although no PrP(CWD) was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP(CWD)-positive clone out of 51. This clone, designated MDB(CWD), has maintained stable PrP(CWD) production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP(CWD)-positive subclones out of 30, one of which was designated MDB(CWD2). The MDB(CWD2) cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP(CWD) accumulation in MDB(CWD) cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP(CWD) inhibitors and suggests that these compounds have potential to be active against CWD in vivo.


Subject(s)
Cell Line, Transformed/metabolism , Deer , Pentosan Sulfuric Polyester/pharmacology , Peptide Hydrolases/pharmacology , Porphyrins/pharmacology , Prions/antagonists & inhibitors , Wasting Disease, Chronic , Animals , Cells, Cultured , Drug Resistance , Prions/biosynthesis
12.
J Virol ; 80(2): 1044-6, 2006 Jan.
Article in English | MEDLINE | ID: mdl-16379006

ABSTRACT

In view of the effectiveness of antimalaria drugs inhibiting abnormal protease-resistant prion protein (PrP-res) formation in scrapie agent-infected cells, we tested other antimalarial compounds for similar activity. Mefloquine (MF), a quinoline antimalaria drug, was the most active compound tested against RML and 22L mouse scrapie agent-infected cells, with 50% inhibitory concentrations of approximately 0.5 and approximately 1.2 microM, respectively. However, MF administered to mice did not delay the onset of intraperitoneally inoculated scrapie agent, the result previously observed with quinacrine. While most anti-scrapie agent compounds inhibit PrP-res formation in vitro, many PrP-res inhibitors have no activity in vivo. This underscores the importance of testing promising candidates in vivo.


Subject(s)
Mefloquine/administration & dosage , Prions , Scrapie/prevention & control , Animals , Antimalarials/administration & dosage , Antimalarials/pharmacology , Drug Evaluation, Preclinical , Injections, Intraperitoneal , Mefloquine/pharmacology , Mice , Peptide Hydrolases/metabolism , Prions/antagonists & inhibitors , Prions/metabolism
13.
Neurosci Lett ; 388(2): 106-11, 2005 Nov 11.
Article in English | MEDLINE | ID: mdl-16039063

ABSTRACT

The transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases. A primary therapeutic target for TSE intervention has been a protease-resistant form of prion protein known as PrP(Sc) or PrP-res. In vitro testing of mouse scrapie-infected cell cultures has identified many PrP-res inhibitors that also have activity in vivo. Here we identify 32 new inhibitors of two strains of mouse scrapie PrP-res. Furthermore, to investigate the species-specificity of these and other PrP-res inhibitors, we have developed a high-throughput cell culture assay based on Rov9 cells chronically-infected with sheep scrapie. Of 32 inhibitors of murine PrP-res that were also tested in the Rov9 cells, only six showed inhibitory activity against sheep PrP-res. The three most potent inhibitors of both murine and ovine PrP-res formation (with 50% inhibition at < or =5 microM) were tannic acid, pentosan polysulfate and Fe(III) deuteroporphyrin 2,4-bisethyleneglycol. The latter two have anti-mouse scrapie activity in vivo. These results identify new inhibitors of murine and ovine PrP-res formation and reinforce the idea that compounds effective against PrP-res from one species or strain cannot be assumed to be active against others.


Subject(s)
PrPSc Proteins/antagonists & inhibitors , Scrapie/drug therapy , Scrapie/metabolism , Tannins/pharmacology , Animals , Cell Line , Deuteroporphyrins/pharmacology , Enzyme Inhibitors/pharmacology , Epithelial Cells/cytology , Ferric Compounds/pharmacology , Mice , Pentosan Sulfuric Polyester/pharmacology , PrPSc Proteins/metabolism , Rabbits , Sheep , Species Specificity
14.
J Gen Virol ; 85(Pt 8): 2479-2483, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15269390

ABSTRACT

In vitro inhibitors of the accumulation of abnormal (protease-resistant) prion protein (PrP-res) can sometimes prolong the lives of scrapie-infected rodents. Here, transgenic mice were used to test the in vivo anti-scrapie activities of new PrP-res inhibitors, which, because they are approved drugs or edible natural products, might be considered for clinical trials in humans or livestock with transmissible spongiform encephalopathies (TSEs). These inhibitors were amodiaquine, thioridazine, thiothixene, trifluoperazine, tetrandrine, tannic acid and polyphenolic extracts of tea, grape seed and pine bark. Test compounds were administered for several weeks beginning 1-2 weeks prior to, or 2 weeks after, intracerebral or intraperitoneal 263K scrapie challenge. Tannic acid was also tested by direct preincubation with inoculum. None of the compounds significantly prolonged the scrapie incubation periods. These results highlight the need to assess TSE inhibitors active in cell culture against TSE infections in vivo prior to testing these compounds in humans and livestock.


Subject(s)
PrPSc Proteins/antagonists & inhibitors , Animals , Cells, Cultured , Hydrolyzable Tannins/pharmacology , Mice , Mice, Transgenic , Scrapie/drug therapy
15.
Clin Infect Dis ; 37(12): 1654-8, 2003 Dec 15.
Article in English | MEDLINE | ID: mdl-14689348

ABSTRACT

We measured plasma tafenoquine concentrations in Thai soldiers given a monthly regimen of tafenoquine to determine whether these concentrations adequately suppressed malarial infections on the Thai-Cambodian border. After receiving a treatment course of artesunate and doxycycline, 104 male soldiers were administered a loading dose of tafenoquine (400 mg daily for 3 days), followed by tafenoquine monthly (400 mg every 4 weeks) for 5 months. Consecutive monthly mean (+/- standard deviation) trough plasma tafenoquine concentrations were 223+/-41, 127+/-29, 157+/-51, 120+/-24, and 88+/-20 ng/mL. Only 1 soldier developed malaria during the study. At the time of malaria diagnosis, his plasma tafenoquine concentration was 40 ng/mL, which was approximately 3-fold lower than the trough concentrations of the other soldiers. Although low tafenoquine concentrations appear to be uncommon, additional investigations are needed to determine the relationship between plasma tafenoquine concentrations and suppression of malaria.


Subject(s)
Aminoquinolines/blood , Antimalarials/blood , Adult , Artemisinins/blood , Artesunate , Chemoprevention , Double-Blind Method , Doxycycline/blood , Humans , Male , Middle Aged , Military Personnel , Sesquiterpenes/blood , Thailand
17.
J Virol ; 77(19): 10288-94, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12970413

ABSTRACT

Transmissible spongiform encephalopathies (TSEs) are fatal, untreatable neurodegenerative diseases associated with the accumulation of a disease-specific form of prion protein (PrP) in the brain. One approach to TSE therapeutics is the inhibition of PrP accumulation. Indeed, many inhibitors of the accumulation of PrP associated with scrapie (PrP(Sc)) in scrapie-infected mouse neuroblastoma cells (ScN(2)a) also have antiscrapie activity in rodents. To expedite the search for potential TSE therapeutic agents, we have developed a high-throughput screening assay for PrP(Sc) inhibitors using ScN(2)a cells in a 96-well format. A library of 2000 drugs and natural products was screened in ScN(2)a cells infected with scrapie strain RML (Chandler) or 22L. Forty compounds were found to have concentrations causing 50% inhibition (IC(50)s) of PrP(Sc) accumulation of

Subject(s)
Biological Products/pharmacology , Flavonoids , PrPSc Proteins/antagonists & inhibitors , Animals , Antimalarials/pharmacology , Histamine Antagonists/pharmacology , Humans , Phenols/pharmacology , Polymers/pharmacology , Polyphenols
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