RSSPeptide and glycopeptide dendrimer apple trees as enzyme models and for biomedical applications.
Source
Department of Chemistry and Biochemistry, University of Berne/Freiestrasse 3, 3012, Berne, Switzerland. jean-louis.reymond@ioc.unibe.ch.
Abstract
Solid phase peptide synthesis (SPPS) provides peptides with a dendritic topology when diamino acids are introduced in the sequences. Peptide dendrimers with one to three amino acids between branches can be prepared with up to 38 amino acids (MW ∼ 5,000 Da). Larger peptide dendrimers (MW ∼ 30,000) were obtained by a multivalent chloroacetyl cysteine (ClAc) ligation. Structural studies of peptide dendrimers by CD, FT-IR, NMR and molecular dynamics reveal molten globule states containing up to 50% of α-helix. Esterase and aldolase peptide dendrimers displaying dendritic effects and enzyme kinetics (k(cat)/k(uncat)∼ 10(5)) were designed or discovered by screening large combinatorial libraries. Strong ligands for Pseudomonas aeruginosa lectins LecA and LecB able to inhibit biofilm formation were obtained with glycopeptide dendrimers. Efficient ligands for cobalamin, cytotoxic colchicine conjugates and antimicrobialpeptide dendrimers were also developed showing the versatility of dendritic peptides. Complementing the multivalency, the amino acid composition of the dendrimers strongly influenced the catalytic or biological activity obtained demonstrating the importance of the "apple tree" configuration for protein-like function in peptide dendrimers.
- PMID:
- 22274649
- [PubMed - as supplied by publisher]
Prime boost vaccination approaches with different conjugates of a new HIV-1 gp41 epitope encompassing the membrane proximal external region induce neutralizing antibodies in mice.
Source
Georg-Speyer-Haus, Institute for Biomedical Research, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt, Germany.
Abstract
Peptide mimics of epitopes for pathogen-specific antibodies present in patient sera can be selected based on the phage display technology. Such mimotopes potentially represent vaccine candidates in case they are able to induce neutralizing antibodies upon vaccination. Here we analyze the immunogenicity of different conjugates of epitope EC26-2A4 localizing to the membrane proximal external region (MPER) of the HIV-1 transmembrane protein gp41. The EC26-2A4 epitope, which overlaps with that of the broadly neutralizing monoclonal antibody (mAb) 2F5, was coupled to sequential oligopeptide carriers (SOC) or to palmitoyl acid for better immunogenicity. Upon prime-boost immunizations of mice, the peptide conjugates induced EC26-2A4 specific antibodies in all settings and mice sera neutralized HIV-1SF162.LS in standardized neutralization assays. Thus, the EC26-2A4 MPER epitope represents a promising vaccine candidate for further analysis in larger animals with respect to the breadth of the neutralizing antibodies induced.
Copyright © 2012 Elsevier Ltd. All rights reserved.
Atrial natriuretic peptide-Fc, ANP-Fc, fusion proteins: semi-synthesis, in vitro activity and pharmacokinetics in rats.
Abstract
Atrial natriuretic peptide (ANP) may be a useful molecule for the treatment of cardiovascular diseases due to its potent natriuretic effects. In an effort to prolong the short in vivo half-life of ANP, fusions of the peptide to the Fc domain of IgG were generated using a semi-synthetic methodology. Synthetic ANP peptides were synthesized with thioesters at either the N- or C-termini of the peptide and subsequently linked to the N-terminus of recombinantly-expressed Fc using native chemical ligation. The linker length between the ANP and Fc moieties was varied between 2, 11 or 16 amino acids. In addition, either one ("monomeric") or two ("dimeric") ANP peptides were linked to Fc to study whether this modification had an effect on in vitro activity and/or in vivo half-life. The various constructs were studied for in vitro activity using a cell-based cGMP assay. The ANP-Fc fusion constructs were between 16 and ~375-fold weaker than unconjugated ANP in this assay, and a trend was observed where the most potent conjugates were those with longer linkers and in the dimeric configuration. The pharmacokinetics of several constructs were assessed in rats, and the half-life of the ANP-Fc's were found to be approximately two orders of magnitude longer than that of the unconjugated peptide. There was no significant difference in terminal half-life between the monomeric and dimeric constructs (2.8-5.5 h), but a trend was observed where the Cmax of the monomeric constructs was approximately 3-fold higher than the dimeric constructs, although the origin of this effect is not understood. These novel ANP-Fc fusion constructs hold promise for future therapeutic application in the treatment of cardiovascular diseases.
Fibrin Specific Peptides Derived by Phage Display: Characterization ofPeptides and Conjugates for Imaging.
Abstract
Peptides that bind to fibrin but not to fibrinogen or serum albumin were selected from phage display libraries as targeting moieties for thrombus molecular imaging probes. Three classes of cyclic peptides (cyclized via disulfide bond between two Cys) were identified with consensus sequences XArXCPY(G/D)LCArIX (Ar=aromatic, Tn6), X2CXYYGTCLX (Tn7), and NHGCYNSYGVPYCDYS (Tn10). These peptides bound to fibrin at ~2 sites with Kd=4.1 µM, 4.0 µM, and 8.7 µM, respectively, whereas binding to fibrinogen was at least 100 - fold weaker. The peptides also bind to the fibrin degradation product DD(E) with similar affinity to that measured for fibrin. The Tn7 and Tn10 peptides bind to the same site on fibrin while the Tn6 peptides bind to a unique site. Alanine scanning identified the N- and C-terminal ends of the Tn6 and Tn7 peptides as most tolerant to modification. Peptide conjugates with either fluorescein or diethylenetriaminepentaaceto gadolinium(III) (GdDTPA) at the N-terminus were prepared for potential imaging applications and these retained fibrin binding affinity and specificity in plasma. Relaxivity and binding studies on the GdDTPA derivatives revealed that an N-terminal glycyl linker had modest effect on fibrin affinity but resulted in lower fibrin-bound relaxivity.
NGR-based strategies for targeting delivery of chemotherapeutics to tumor vasculature.
Source
Department of Medicinal Chemistry, School of Pharmaceutical Sciences, ShanDong University, 44, West Wenhua Road, 250012,Jinan, ShanDong, P.R. China.
Abstract
In the last decades, NGR-containing peptides have been proved useful for ligand-directed targeted delivery of various chemotherapeutic drugs to tumor vasculature. Aminopeptidase N (APN; CD13) has been demonstrated to be a key binding site for NGR peptides on tumor vasculature. For drug targeting, chemical means have been applied to couple NGR-peptides to small molecule drugs, such as cytokines, antiangiogenic compounds, viral particles, contrast agents, DNA complexes, and other biologic response modifiers. Some products have shown impressive results in preclinical animal models, such as NGR-TNF which was currently tested in Phase III trials. In this article we will review the process of NGR-to-isoDGR transition and provide suggestions for the design of the diverse NGR peptide-chemotherapeuticsconjugates.
- PMID:
- 22263803
- [PubMed - as supplied by publisher]
Metallopolymer-Peptide Hybrid Materials: Synthesis and Self-Assembly of Functional, Polyferrocenylsilane-Tetrapeptide Conjugates.
Source
School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 1TS (UK), Fax: (+44) 117-925-1295, Fax: (+44) 117-929-0509.
Abstract
Conjugates of poly(ferrocenyldimethylsilane) (PFDMS) with Ac-(GA)(2) -OH, Ac-A(4) -OH, Ac-G(4) -OH and Ac-V(4) -OH have been prepared by reaction of the tetrapeptide units with the amino-terminated metallopolymer. The number average degree of polymerisation (DP(n) ) of the PFDMS was approximately 20 and comparable materials with shorter (DP(n) ≈10) and/or amorphous chains have been prepared by the same procedure. Poly(ferrocenylethylmethylsilane) (PFEMS) was employed for the latter purpose. All conjugates were characterised by GPC, MALDI-TOF MS, NMR and IR spectroscopy. With the exception of Ac-V(4) -PFDMS(20) , all materials exhibited some anti-parallel β-sheet structure in the solid state. The self-assembly of the conjugates was studied in toluene by DLS. The vast majority of the materials, irrespective of peptide sequence or chain crystallinity, afforded fibres consisting of a peptidic core surrounded by a PFS corona. These fibres were found in the form of cross-linked networks by TEM and AFM. The accessibility of the chemically reducing PFS corona has been demonstrated by the localised formation of silver nanoparticles on the surface of the fibres.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Bombesin analogues for gastrin-releasing peptide receptor imaging.
Source
Department of Radiology, University of Missouri School of Medicine, Columbia, Missouri 65211, USA.
Abstract
OBJECTIVES:
The present study describes the design and development of a series of new bombesin (BBN) antagonistpeptide ligands of the form [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)], where Cu-64=a positron emitting radiometal; NO2A=1,4,7-triazacyclononane-1,4-diacetic acid; X=6-amino hexanoic acid, 8-amino octanoic acid or 9-Aminononanoic acid; and BBN(6-13)NHEt=Gln-Trp-Ala-Val-Gly-His-Leu-NHEt, an antagonist analogue of bombesin peptide for specific targeting of the gastrin-releasing peptide receptor (GRPR).
METHODS:
[NO2A-X-D-Phe(6)-BBN(6-13)NHEt] conjugates were manually conjugated with NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), and the resulting conjugates were labeled with (64)Cu to yield [(64)Cu-(NO2A-X-D-Phe(6)-BBN(6-13)NHEt)]. The metallated and nonmetallated conjugates were purified via reversed-phase high-performance liquid chromatography and characterized by electrospray ionization-mass spectrometry.
RESULTS:
Competitive displacement binding assays displayed nanomolar binding affinities toward human GRPR for all of the newly formed peptide analogues. Biodistribution studies showed very high uptake and retention of tumor-associated radioactivity in PC-3 (a prostate tumor model known to express the GRPR) tumor-bearing rodent models. The radiolabeled conjugates also exhibited rapid urinary excretion and very high tumor to background ratios. Micro-positron emission tomography (PET) molecular imaging investigations showed clear visualization of tumors in female PC-3 tumor-bearing mice 15 h postinjection.
CONCLUSION:
The biodistribution and molecular imaging study suggests that these conjugates can be considered as potential PET tracer candidates for the diagnosis of GRPR-positive tumors in human patients.
Published by Elsevier Inc.
Evaluation of the long-term effects of gastric inhibitory polypeptide-ovalbuminconjugates on insulin resistance, metabolic dysfunction, energy balance and cognition in high-fat-fed mice.
Source
School of Biomedical Sciences Research Institute, SAAD Centre for Pharmacy and Diabetes, University of Ulster, Coleraine BT52 1SA, UK.
Abstract
The effects of active immunisation with gastric inhibitory polypeptide (GIP) or (proline3)GIP-ovalbumin conjugates on insulin resistance, metabolic dysfunction, energy expenditure and cognition were examined in high-fat-fed mice. Normal mice were injected (subcutaneously) once every 14 d for 98 d with GIP-ovalbumin conjugates, with transfer to a high-fat diet on day 21. Active immunisation resulted in GIP antibody generation and significantly (P < 0·01 to P < 0·001) reduced circulating non-fasting plasma insulin concentrations compared to high-fat control mice from day 70 onwards. The glycaemic responses to intraperitoneal glucose or nutrient ingestion were significantly improved in all treated mice, with corresponding stimulated plasma insulin levels depressed compared to high-fat controls. These changes were associated with substantially (P < 0·001) improved glucose-lowering responses to exogenous insulin and decreases of muscle and fat TAG, pancreatic insulin, circulating total and LDL-cholesterol levels (P < 0·01 to P < 0·001). Treatment with GIP-ovalbumin conjugates was not associated with alterations in energy expenditure, indirect calorimetry or aspects of cognitive function. The observed changes were almost identical in GIP and (Pro3)GIP immunised mice and were independent of any effects on food intake or body weight. Further tests established that coupling of GIP peptides to ovalbumin abolished any intrinsic insulin-releasing or glucose-lowering activity. These results suggest that induction of GIP-neutralising antibodies with GIP-ovalbumin conjugates is an effective means of countering the metabolic abnormalities induced by high-fat feeding and does not adversely have an impact on a marker of cognition function or energy expenditure.
B Lymphocytes Treated In Vitro with Antigen Coupled to Cholera Toxin B Subunit Induce Antigen-Specific Foxp3+ Regulatory T Cells and Protect against Experimental Autoimmune Encephalomyelitis.
Source
University of Gothenburg Vaccine Research Institute, Sahlgrenska Academy at the University of Gothenburg, SE-405 30, Göteborg, Sweden.
Abstract
The ability of activated B cells to protect against various experimental autoimmune or allergic diseases makes them attractive for use in cell-based therapies. We describe an efficient way to generate B cells with strong suppressive functions by incubating naive B cells with a relevant Ag conjugated to cholera toxin B subunit (CTB). This allows most B cells, irrespective of BCR, to take up and present Ag and induces their expression of latency-associated polypeptide (LAP)/TGF-β and after adoptive transfer also their production of IL-10. With OVA as model Ag, when naive T cells were cocultured in vitro with B cells pretreated with OVA conjugated to CTB (OVA/CTB) Ag-specific CD4(+) Foxp3 regulatory T (Treg) cells increased >50-fold. These cells effectively suppressed CD25(-)CD4(+) effector T (Teff) cells in secondary cultures. Adoptive transfer of OVA/CTB-treated B cells to mice subsequently immunized with OVA in CFA induced increase in Foxp3 Treg cells together with suppression and depletion of Teff cells. Likewise, adoptive transfer of B cells pulsed with myelin oligodendrocyte glycoprotein peptide(35-55) (MOGp) conjugated to CTB increased the number of Treg cells, suppressed MOGp-specific T cell proliferation and IL-17 and IFN-γ production, and prevented the development of experimental autoimmune encephalomyelitis. Similar effects were seen when B cells were given "therapeutically" to mice with early-stage experimental autoimmune encephalomyelitis. Our results suggest that B cells pulsed in vitro with relevant Ag/CTB conjugates may be used in cell therapy to induce Ag-specific suppression of autoimmune disease.
Evaluation of a HER2-targeting affibody molecule combining an N-terminal HEHEHE-tag with a GGGC chelator for (99m)Tc-labelling at the C terminus.
Source
Division of Molecular Biotechnology, School of Biotechnology, AlbaNova University Center, Royal Institute of Technology, Stockholm, Sweden.
Abstract
Affibody molecules are a class of small (ca.7 kDa) robust scaffold proteins with high potential as tracers for radionuclide molecular imaging in vivo. Incorporation of a cysteine-containing peptide-based chelator at the C terminus provides an opportunity for stable labelling with the radionuclide (99m)Tc. The use of a GGGC chelator at the C terminus has provided the lowest renal radioactivity retention of the previously investigated peptide-based chelators. Previously, it has also been demonstrated that replacement of the His(6)-tag with the negatively charged histidine-glutamate-histidine-glutamate-histidine-glutamate (HEHEHE)-tag permits purification of affibody molecules by immobilized metal ion affinity chromatography (IMAC) and provides low hepatic accumulation of radioactivity of conjugates site-specifically labelled at the C terminus using several different nuclides. We hypothesized that the combination of a HEHEHE-tag at the N terminus and a GGGC chelator at the C terminus of an affibody molecule would be a favourable format permitting IMAC purification and providing low uptake in excretory organs. To investigate this hypothesis, a (HE)(3)-Z(HER2:342)-GGGC affibody molecule was generated. It could be efficiently purified by IMAC and stably labelled with (99m)Tc. (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC preserved specific binding to HER2-expressing cells. In NMRI mice, hepatic uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was lower than the uptake of the control affibody molecules, (99m)Tc-Z(HER2:2395)-VDC and (99m)Tc-Z(HER2:342)-GGGC. At 1 and 4 h after injection, the renal uptake of (99m)Tc-(HE)(3)-Z(HER2:342)-GGGC was 2-3-fold lower than uptake of (99m)Tc-Z(HER2:2395)-VDC, but it was substantially higher than uptake of (99m)Tc-Z(HER2:342)-GGGC. Further investigation indicated that a fraction of (99m)Tc was chelated by the HEHEHE-tag which caused a higher accumulation of radioactivity in the kidneys. Thus, a combination of a HEHEHE-tag and the GGGC chelator in targeting scaffold proteins was found to be undesirable in the case of (99m)Tc labelling due to a partial loss of site-specificity of nuclide chelation.
Nanomolar cellular antisense activity of peptide nucleic acid (PNA) cholic acid ("umbrella") and cholesterol conjugates delivered by cationic lipids.
Abstract
Limited cellular uptake and low bioavailability of peptide nucleic acids (PNAs) have restricted widespread use of PNAs as antisense/antigene agents for cells in culture and not least for in vivo applications. We now report the synthesis and cellular antisense activity in cultured HeLa pLuc705 cells of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 μM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs, conjugated to cholesterol through an ester hemisuccinate linker or to cholic acid exhibited low nanomolar activity (EC50 ~ 25 nM). Excellent sequence specificity was retained as mismatch PNA conjugates did not show any significant antisense activity. Furthermore, we show that increasing the transfection volume improved transfection efficiency suggesting that accumulation (condensation) of the PNA/lipid complex on the cellular surface is part of the uptake mechanism. These results provide a novel, simple method for very efficient cellular delivery of PNA oligomers, especially using PNA-cholic acid conjugates which, in contrast to PNA-cholesterol conjugates exhibit sufficient water solubility. The results also question the generality of using cholic acid "umbrella" derivatives as a delivery modality for antisense oligomers.
Analytical measurement of PEGylated molecules.
Abstract
Attachment of polyethylene glycol (PEG) to proteins, peptides, liposomes, drugs, and nanoparticles can improve pharmaceutical pharmacokinetic properties and enhance in vivo biological efficacy. Since the first PEGylated product was approved by the Food and Drug Administration in 1990, increasing numbers of PEGylated compounds have entered clinical use. Successful clinical development of PEGylated pharmaceuticals requires accurate methods for the qualitative and quantitative analysis of intact PEG conjugates in biological fluids. In this article, we review assay methods that can be utilized for the detection and measurement of PEGylated pharmaceuticals in complex biological samples for determination of biodistribution and pharmacokinetic properties. In particular, we describe relevant colorimetric, chromatographic, radiolabeled, biological and enzyme-linked immunosorbent assays for the pharmacokinetic study of PEGylated molecules.
Therapeutic efficacy and toxicity of (225)Ac-labelled vs. (213)Bi-labelled tumour-homing peptides in a preclinical mouse model of peritoneal carcinomatosis.
Source
Department of Nuclear Medicine, Technische Universität München, Ismaninger Strasse 22, 81675, Munich, Germany.
Abstract
PURPOSE:
Targeted delivery of alpha-particle-emitting radionuclides is a promising novel option in cancer therapy. We generated stable conjugates of the vascular tumour-homing peptide F3 both with (225)Ac and (213)Bi that specifically bind to nucleolin on the surface of proliferating tumour cells. The aim of our study was to determine the therapeutic efficacy of (225)Ac-DOTA-F3 in comparison with that of (213)Bi-DTPA-F3.
METHODS:
ID(50) values of (213)Bi-DTPA-F3 and (225)Ac-DOTA-F3 were determined via clonogenic assays. The therapeutic efficacy of both constructs was assayed by repeated treatment of mice bearing intraperitoneal MDA-MB-435 xenograft tumours. Therapy was monitored by bioluminescence imaging. Nephrotoxic effects were analysed by histology.
RESULTS:
ID(50) values of (213)Bi-DTPA-F3 and (225)Ac-DOTA-F3 were 53 kBq/ml and 67 Bq/ml, respectively. The median survival of control mice treated with phosphate-buffered saline was 60 days after intraperitoneal inoculation of 1 × 10(7) MDA-MB-435 cells. Therapy with 6 × 1.85 kBq of (225)Ac-DOTA-F3 or 6 × 1.85 MBq of (213)Bi-DTPA-F3 prolonged median survival to 95 days and 97 days, respectively. While F3 labelled with short-lived (213)Bi (t (1/2) 46 min) reduced the tumour mass at early time-points up to 30 days after treatment, the antitumour effect of (225)Ac-DOTA-F3 (t (1/2) 10 days) increased at later time-points. The difference in the fraction of necrotic cells after treatment with (225)Ac-DOTA-F3 (43%) and with (213)Bi-DTPA-F3 (36%) was not significant. Though histological analysis of kidney samples revealed acute tubular necrosis and tubular oedema in 10-30% of animals after treatment with (225)Ac-DOTA-F3 or (213)Bi-DTPA-F3, protein casts were negligible (2%), indicating only minor damage to the kidney.
CONCLUSION:
Therapy with both (225)Ac-DOTA-F3 and (213)Bi-DTPA-F3 increased survival of mice with peritoneal carcinomatosis. Mild renal toxicity of both constructs favours future therapeutic application.
Potential of the gastric motility drug lorglumide in prostate cancer imaging.
Source
Department of Neuroradiology, University of Tübingen, Germany; Peptide Synthesis Laboratory, Interfaculty Institute of Biochemistry, University of Tübingen, Germany.
Abstract
The use of tissue-specific receptor ligands is a promising approach for cancer diagnostics and therapy. Lorglumide, a highly effective competitive ligand for the cholecystokinine-A receptor (CCKRA) was conjugated to a fluorescent dye and a magnetic resonance imaging (MRI) contrast agent to obtain a bifunctional marker for tissue with high CCKRA expression. An intermediate conjugate containing only lorglumide and a fluorescent dye was also produced. By performing CCKRA mRNA expression analysis on carcinoma cell lines we found that CCKRA is highly expressed in PC3 prostate carcinoma cells compared to U373 glioma and U2OS osteosarcoma cells. Uptake, specificity and detection sensitivity of both lorglumide conjugates was evaluated by confocal laser scanning microscopy, fluorescence activated cell sorting (FACS) and magnetic resonance relaxometry. While the conjugate containing only lorglumide and rhodamine isothiocyanate as fluorescent dye showed clearly higher uptake than the bifunctional conjugate in FACS analysis, both conjugates clearly showed preferential staining of the PC3 prostate carcinoma cells. Magnetic resonance relaxometry experiments with the bifunctional conjugate containing the MRI contrast agent gadolinium-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid confirmed the higher PC3-affinity of the lorglumide ligand. Confocal laser scanning microscopy images of PC3/U2OS mixed cell cultures incubated with the bifunctional conjugate also clearly showed PC3 preference and cytoplasmic dot-like staining concurring with uptake by receptor binding and subsequent receptor internalization. Considering these results, CCKRA ligands like lorglumide could play a role in the future design of prostate-cancer-specific markers.
Copyright © 2011 Elsevier B.V. All rights reserved.
MAP-mediated nuclear delivery of a cargo protein.
Source
Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California , Los Angeles, CA , USA.
Abstract
Radiolabeled cytochrome c (Cyt c), either as a free protein or as cell penetrating peptide (CPP)-conjugates, was tested for cellular uptake and nuclear transport in Human embryonic kidney293 (HEK293) cells and HeLa cells. Conjugation of Cyt c with either the amphipathic peptide model amphipathic peptide (MAP) or the cationic peptide oligoarginine via a disulfide linkage significantly increased the total internalization and nuclear localization of Cyt c in both cell lines, though to a greater extent following conjugation with MAP. The nuclear localization was also evaluated qualitatively by confocal laser scanning microscopy (CLSM). CLSM images depicted high amounts of colocalization of fluorescently labeled Cyt c-MAP and the nucleus in HEK293 cells. In addition, prevention of disulfide reduction at the cell surface, or comparison of reducible disulfide or non-reducible thioether MAP-conjugates, showed that maintenance of an intact conjugate using a stable linkage enhanced MAP-mediated nuclear delivery. Furthermore, nuclear transport of the MAP-cargo conjugate appears to be dependent on vesicle fusion events following internalization via endocytosis. The findings presented in this report demonstrate the MAP-mediated transport of a small protein such as Cyt c into the nuclear compartment, which can be used to improve current CPP-cargo delivery of macromolecules with nuclear biological functions.
Optimization of Peptide-Based ELISA for Serological Diagnostics: A Retrospective Study of Human Monkeypox Infection.
Source
1 Najít Technologies, Inc. , Portland, Oregon.
Abstract
Abstract Although smallpox has been eradicated, other diseases caused by virulent orthopoxviruses such as monkeypox virus (MPV) remain endemic in remote areas of western and central sub-Saharan Africa, and represent a potential biothreat due to international travel and/or inadvertent exposure. Unfortunately, extensive antigenic cross-reactivity among orthopoxviruses presents a challenge to serological diagnosis. We previously reported a 20mer peptide-based ELISA that identified recent MPV infection with >90% sensitivity and >90% specificity. However, the sensitivity of this approach was not determined with samples obtained at later time points after antibody titers had declined from their peak levels. To improve assay sensitivity for detecting MPV-specific antibodies at later time points, we compared diagnostic 20mer peptides to 30mer peptides. In addition, optimal 30mer peptides were tested in combination or after conjugating selected peptides to a carrier protein (bovine serum albumin) to further improve assay performance. An optimized combination of four unconjugated 30mer peptides provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 45% sensitivity for detecting MPV infection at >2 years post-infection, and 99% specificity. However, an optimized combination of two peptide conjugates provided 100% sensitivity for detecting MPV infection at 2-6 months post-infection, 90% sensitivity for detecting MPV infection at >2 years post-infection, and 97% specificity.Peptide-based ELISA tests provide a relatively simple approach for serological detection of MPV infection. Moreover, the systematic approach used here to optimize diagnostic peptide reagents is applicable to developing improved diagnostics to a broad range of other viruses, and may be particularly useful for distinguishing between closely-related viruses within the same genus or family.
Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging.
Source
1. DKFZ, Central Peptide Synthesis Unit, INF 580, D-69120 Heidelberg, Germany.
Abstract
Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB) gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized.
64Cu Labeled AmBaSar-RGD2 for micro-PET Imaging of Integrin αvβ3 Expression.
Source
Molecular Imaging Center, Department of Radiology, Keck School of Medicine, University of SouthernCalifornia, 1510 San Pablo Street, Room #350, Los Angeles, California 90033, USA; pconti@usc.edu.
Abstract
Integrin αvβ3 plays a critical role in tumor-induced angiogenesis and metastasis. Previously, a 64Cu-AmBaSar- RGD monomer with high in vivo stability compared with 64Cu-DOTA-RGD was developed for integrin αvβ3 PET imaging. It has been established that dimeric RGD peptides have higher receptor-binding affinity and superior in vivo kinetics compared with monomeric RGD peptides due to the polyvalency effect. In this context, we synthesized and evaluated 64Cu-labeled AmBaSar dimeric RGD conjugates (64Cu-AmBaSar-RGD2) for PET imaging of integrin αvβ3 expression. The dimeric RGD peptide was conjugated with a cage-like chelator AmBaSar and labeled with 64Cu. Cell binding, microPET imaging, receptor blocking, and biodistribution studies of 64Cu-AmBaSar-RGD2 were conducted in the U87MG human glioblastoma xenograft model. AmBaSar-RGD2 conjugate was obtained in reasonable yield (45.0 ± 2.5%, n= 4) and the identity was confirmed by HPLC and MS (found 1779.8, calculated m/z for [M+H]+ M: C81H125N27O19 1779.9). 64Cu-AmBaSar-RGD2 was obtained with high radiochemical yield (92.0 ± 1.3%) and purity (≥ 98.0%) under mild conditions (pH 5.0~5.5, 23~37 °C) in 30 min. The specific activity of 64Cu-AmBaSar-RGD2 was estimated to be 15-22 GBq/µmol at the end of synthesis. Based on microPET imaging and biodistribution studies, 64Cu-AmBaSar-RGD2 has demonstrated higher tumor uptake at selected time points than 64Cu-AmBaSar-RGD. At 20 h p.i., the tumor uptake reached 0.65 ± 0.05 %ID/g for 64Cu-AmBaSar-RGD and 1.76 ± 0.38 %ID/g for 64Cu-AmBaSar-RGD2, respectively. The integrin αvβ3 targeting specificity was confirmed by blocking experiments. Therefore, the new tracer 64Cu-AmBaSar- RGD2 exhibited better tumor-targeting efficacy and more favorable in vivo pharmacokinetics than the 64Cu labeled RGD monomer due to the polyvalency effect.
Easy and Efficient 111Indium Labeling of Long-Term Stored DTPA Conjugated Protein.
Source
Department of Clinical Physiology&Nuclear Medicine KF 4011, Rigshospitalet, DK 2100 - Copenhagen, Denmark; bhesse@rh.dk.
Abstract
The labelling efficiency of long-term stored DTPA-conjugates has not been reported previously even though DTPA has been in extensive use as metal chelator in the development of radiopharmaceuticals and contrast agents. DTPA is often used as a bifunctional chelating agent conjugated to tumor targeting vehicles such as monoclonal antibodies and receptor directed peptides. The purpose of this study was to monitor the labelling efficiency of a DTPA-conjugate on long-term storage using 111In-chloride at two different temperatures and incubation times for the In-labelling. Method: Cyclic-diethylene-triamine-pentaacetic acid (cDTAP) was conjugated to a polyclonal immunoglobulin-G (IgG) in borate buffer, pH 8.2 at +4?C for 4 hours. Then the DTPA-conjugate was dialyzed against 50 mmol/l sodium citrate buffer saline, pH 6.0 and stored at -80° C in aliquots of 1 mg/0.5 ml. The DTPA-conjugate was labeled with 111In-chloride in citrate buffer, pH 6. The labelling reaction was incubated at room temperature (RT) for 30 min and at +4?C for 90 min. Determination of labelling efficiency was performed using ITLC and an instant chromatography scanner equipped with a NaI crystal. The labelling efficiency of the DTPA-conjugate was monitored every third month for 12 months. Results: The median labelling efficiencies varied between 92 and 96% during the whole period. The two combinations of incubation times and temperatures (30 min at RT and 90 min at +4°C) had no affect on labelling efficiency of the DTPA-conjugate, stored for 12 months. Conclusion: Our study shows that 111In-labelling can easily be performed within 30 min at RT for thermo-stable proteins like polyclonal, DTPA-conjugated IgG stored long-term at -80°C with a high 111In-labelling efficiency.
Treatment of prostate carcinoma with (Galectin-3)-targeted HPMA copolymer-(G3-C12)-5-Fluorouracil conjugates.
Source
Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, No. 17, Block 3, Southern Renmin Road, Chengdu 610041, PR China.
Abstract
Galectin-3 (Gal-3), over-expressed on a variety of human tumor cells, is a potential binding site for targeted metastatic prostate cancer therapy. The aim of this study was to develop a G3-C12-mediated drug delivery system based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers targeting to Gal-3-expressed human PC-3 prostate carcinoma cells. 5-Fluorouracil (5-Fu), an anti-tumor agent, was selected as a model drug. G3-C12, a binding peptide, which specifically binds to the carbohydrate-recognition domain (CRD) of Gal-3, was attached to HPMA copolymers as a targeting moiety. Compared with non-targeted conjugates (P-Fu), Gal-3-targeted HPMA copolymer-(G3-C12)-5-Fuconjugates (P-(G3-C12)-Fu) displayed a superior intracellular internalization followed by enhanced cytotoxicity and apoptosis-induction. Subsequently, the in vitro migration study on PC-3 cells indicated that P-(G3-C12)-Fu was able to efficiently inhibit the cell migration ability after wounding. On PC-3 tumor-bearing mice model, G3-C12-modified copolymers showed a higher tumor accumulation coupled with a faster clearance from blood circulation than non-modified ones. Finally, Gal-3-targeted conjugates significantly improved the anti-tumor activity of 5-Fu in nude mice bearing PC-3 tumor xenografts. Consequently, G3-C12 would be a promising targeting moiety for cell-specific prostate cancer therapy in future.
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