November 21, 2012

Peptide-based affinity adsorbents with high binding capacity for the purification of monoclonal antibodies

Filed under: Antibodies — Tags: — aapptec @ 8:30 pm

Peptide-based affinity adsorbents with high binding capacity for the purification of monoclonal antibodies
William Stanley Kish, Amith Dattathrai Naik, Stefano Menegatti, and Ruben G. Carbonell
Ind. Eng. Chem. Res., Just Accepted Manuscript
DOI: 10.1021/ie302345w
Publication Date (Web): November 14, 2012
Copyright © 2012 American Chemical Society

High binding capacity and selectivity are key features for the successful application of affinity adsorbents for antibody purification. This study presents the development of affinity resins based on hexapeptide ligand HWRGWV for recovering monoclonal antibodies from cell culture fluids. Methods are presented for the immobilization of the peptide ligand and its variants on polymethacrylate and agarose based chromatographic supports using two main coupling strategies. The first one involves the formation of a peptide bond between the amino groups on the substrate and the peptide C-terminus activated with the uronium coupling agent HATU. The second approach involves resin activation with iodoacetic acid, followed by coupling of a cysteine-terminated variant of the ligand to form a thioether bond. The reaction conditions of peptide coupling were optimized to maximize the binding capacity of the resulting adsorbents. The peptide resins were characterized by measuring their static IgG binding capacities. The measured static binding capacity ranged from 35 to 48 mg/mL. The dynamic binding capacities (DBC) of four selected adsorbents were also determined, and they ranged from of 35 to 42 mg/mL with a 5-minute residence time. All the resins exhibited high selectivity towards the Fc fragment of IgG. The affinity resins were used to purify two MAbs, a chimeric IgG1 and a humanized IgG4, from commercial CHO cell culture fluids. The resulting yields and purities for both MAbs were found to be in the range of 87 – 93% and > 94 % respectively, which compare well with the purity and yield values obtained using commercially available Protein A media. Finally, the peptide resin with the highest IgG binding capacity, HWRGWVC-WorkBeads, was tested for 20 DBC cycles which included cleaning in place with 0.1 M NaOH after every cycle. The resin showed a high degree of reusability and alkaline stability, as it maintained 90% of its initial capacity.

November 19, 2012

Antibacterial and leishmanicidal activities of temporin-SHd

Filed under: Peptide Antibiotics — Tags: — aapptec @ 5:30 pm

Antibacterial and leishmanicidal activities of temporin-SHd, a 17-residue long membrane-damaging peptide.
Abbassi F, Raja Z, Oury B, Gazanion E, Piesse C, Sereno D, Nicolas P, Foulon T, Ladram A., Biochimie. 2012 Oct 29. doi: 10.1016/j.biochi.2012.10.015. [Epub ahead of print]

Temporins are a family of short antimicrobial peptides (8-17 residues) that mostly show potent activity against Gram-positive bacteria. Herein, we demonstrate that temporin-SHd, a 17-residue peptide with a net charge of +2 (FLPAALAGIGGILGKLF(amide)), expressed a broad spectrum of antimicrobial activity. This peptide displayed potent antibacterial activities against Gram-negative and Gram-positive bacteria, including multi-drug resistant Staphylococcus aureus strains, as well as antiparasitic activity against promastigote and the intracellular stage (amastigote) of Leishmania infantum, at concentration not toxic for the macrophages. Temporin-SHd that is structured in a non-amphipathic α-helix in anionic membrane-mimetic environments, strongly and selectively perturbs anionic bilayer membranes by interacting with the polar head groups and acyl region of the phospholipids, with formation of regions of two coexisting phases: one phase rich in peptide and the other lipid-rich. The disruption of lipid packing within the bilayer may lead to the formation of transient pores and membrane permeation/disruption once a threshold peptide accumulation is reached. To our knowledge, Temporin-SHd represents the first known 17-residue long temporin expressing such broad spectrum of antimicrobial activity including members of the trypanosomatidae family. Additionally, since only a few shorter members (13 residues) of the temporin family are known to display antileishmanial activity (temporins-TA, -TB and -SHa), SHd is an interesting tool to analyze the antiparasitic mechanism of action of temporins.
Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Retro-Inverso Version of Catestatin Shows Prolonged Efficacy in Mouse Model of Hypertension

Filed under: Potential Peptide Drug — Tags: , — aapptec @ 4:52 pm

Novel Peptide Isomer Strategy for Stable Inhibition of Catecholamine Release: Application to Hypertension.
Biswas N, Gayen J, Mahata M, Su Y, Mahata SK, O’Connor DT., Hypertension. 2012, 60(6), 1552-1559. doi: 10.1161/HYPERTENSIONAHA.112.202127 Epub 2012 Nov 5.

Although hypertension remains the most potent and widespread cardiovascular risk factor, its pharmacological treatment has achieved only limited success. The chromogranin A-derived fragment catestatin inhibits catecholamine release by acting as an endogenous nicotinic cholinergic antagonist and can rescue hypertension in the setting of chromogranin A-targeted ablation. Here, we undertook novel peptide chemistry to synthesize isomers of catestatin: normal/wild-type as well as a retro-inverso (R-I) version, with not only inversion of chirality (L→D amino acids) but also reversal of sequence (carboxyl→amino). The R-I peptide was entirely resistant to proteolytic digestion and displayed enhanced potency as well as preserved specificity of action toward nicotinic cholinergic events: catecholamine secretion, agonist desensitization, secretory protein transcription, and cationic signal transduction. Structural modeling suggested similar side-chain orientations of the wild-type and R-I isomers, whereas circular dichroism spectroscopy documented inversion of chirality. In vivo, the R-I peptide rescued hypertension in 2 mouse models of the human trait: monogenic chromogranin A-targeted ablation, with prolonged efficacy of the R-I version and a polygenic model, with magnified efficacy of the R-I version. These results may have general implications for generation of metabolically stable mimics of biologically active peptides for cardiovascular pathways. The findings also point the way toward a potential new class of drug therapeutics for an important risk trait and, more generally, open the door to broader applications of the R-I strategy in other pathways involved in cardiovascular biology, with the potential for synthesis of diagnostic and therapeutic probes for both physiology and disease.

Antimicrobial Peptides Effective Against Multidrug-Resistant Stain of Mycobacterium tuberculosis

Filed under: Peptide Antibiotics,Potential Peptide Drug — Tags: — aapptec @ 4:42 pm

Activity of LL-37, CRAMP and antimicrobial peptide-derived compounds E2, E6 and CP26 against Mycobacterium tuberculosis.
Rivas-Santiago B, Rivas Santiago CE, Castañeda-Delgado JE, León-Contreras JC, Hancock RE, Hernandez-Pando R., Int J Antimicrob Agents. 2012 Nov 7. doi: 10. 1016/j.ijantimicag.2012.09.015. [Epub ahead of print]

Tuberculosis (TB) is a major worldwide health problem in part due to the lack of development of new treatments and the emergence of new strains such as multidrug-resistant (MDR) and extensively drug-resistant strains that are threatening and impairing the control of this disease. In this study, the efficacy of natural and synthetic cationic antimicrobial (host defence) peptides that have been shown often to possess broad-spectrum antimicrobial activity was tested. The natural antimicrobial peptides human LL-37 and mouse CRAMP as well as synthetic peptides E2, E6 and CP26 were tested for their activity against Mycobacterium tuberculosis both in in vitro and in vivo models. The peptides had moderate antimicrobial activities, with minimum inhibitory concentrations ranging from 2μg/mL to 10μg/mL. In a virulent model of M. tuberculosis lung infection, intratracheal therapeutic application of these peptides three times a week at doses of ca. 1mg/kg led to significant 3-10-fold reductions in lung bacilli after 28-30 days of treatment. The treatments worked both against the drug-sensitive H37Rv strain and a MDR strain. These results indicate that antimicrobial peptides might constitute a novel therapy against TB.
Copyright © 2012. Published by Elsevier B.V.

Novel Peptide Targets Non-Small Cell Lung Cancer

Filed under: Anti-cancer,Potential Peptide Drug — Tags: — aapptec @ 4:36 pm

A novel peptide (Thx) homing to non-small cell lung cancer identified by ex vivo phage display.
Koivistoinen A, Ilonen II, Punakivi K, Räsänen JV, Helin H, Sihvo EI, Bergman M, Salo JA., Clin Transl Oncol. 2012 Nov 10. doi: 10.1007/s12094-012-0959-z [Epub ahead of print]

AIM:
To identify linear peptide homing to non-small cell lung cancer (NSCLC) tumor cells using ex vivo phage display method.
MATERIALS AND METHODS:
Twenty-six clinical patient samples were used to identify linear homing peptide, which was exposed to NSCLC cell cultures and control cell lines to determine cell binding affinity and cell localization. Also, ex vivo biodistribution was analyzed using tumor-bearing mice.
RESULTS:
The panning yielded peptide enrichment with a core motif (A)/(S)RXPXXX. Based on this, an amino acid sequence, ARRPKLD, was selected for characterization and named Thx-peptide. The in vitro binding properties of Thx-peptide demonstrated selectivity toward NSCLC. Internalization assays showed that Thx-Alexa and fluorescein conjugates were located in a subset of perinuclearly located lysosomes of tumor cells. Thx-peptide appeared with fluorescein-labeled peptide and peptide-DTPA-chelator complex in adenocarcinoma xenografts in mice.
CONCLUSION:
Thx shows promise for targeted imaging and drug delivery.

Drug conjugated with EGF receptor-binding peptide targets drug resistant colon cancer cells

Filed under: Anti-cancer,Potential Peptide Drug — Tags: — aapptec @ 4:32 pm

Targeted delivery of doxorubicin through conjugation with EGF receptor-binding peptide overcomes drug resistance in human colon cancer cells.
Ai S, Jia T, Ai W, Duan J, Liu Y, Chen J, Liu X, Yang F, Tian Y, Huang Z., Br J Pharmacol. 2012 Nov 12. doi: 10.1111/bph.12055. [Epub ahead of print]

BACKGROUND AND PURPOSE:
Induction of multidrug resistance by doxorubicin (DOX), together with non-specific toxicities, has restrained DOX-based chemotherapy. We have recently conjugated DOX with EGFR-binding peptide EBP, and demonstrated the enhanced anticancer efficacy and reduced systemic toxicity of DOX-EBP conjugate by targeting EGFR-overexpressing tumours. Here we investigated whether DOX-EBP was capable of overcoming drug resistance and the underlying molecular mechanisms.
EXPERIMENTAL APPROACH:
DOX-resistant SW480/DOX cells were derived from non-resistant SW480 cells by stepwise exposure to increased DOX, and P-glycoprotein overexpression induced by DOX was confirmed by Western blotting. Cytocidal capacity and intracellular distribution of drugs were evaluated by MTT assay and fluorescence microscopy, respectively. EGFR-mediated endocytosis was tested by EGFR and endocytosis inhibition assays. Drug accumulation in tumour cells and murine xenografts was determined by HPLC.
KEY RESULTS:
The cytotoxicity and accumulation of DOX-EBP in SW480/DOX cells were almost the same as in SW480 cells, but those of free DOX were reduced. DOX-EBP accumulation was inhibited by both EGFR and endocytosis inhibitors, suggesting an EGFR-mediated endocytotic uptake. Tumour accumulation of DOX-EBP was significantly higher than free DOX in mice, and DOX-EBP remained at similar levels in DOX-resistant and non-resistant tumour tissues. Importantly, DOX-EBP, but not free DOX, was effective to inhibit solid tumour growth and increase survival rate of both sensitive and resistant models.
CONCLUSION AND IMPLICATIONS:
DOX-EBP is capable of overcoming DOX resistance of tumour cells and increasing in vivo antitumour efficacy and therefore may serve as a potent therapeutic agent for the treatment of resistant cancers.
© 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Identification of putative receptors for cancer-binding peptides

Filed under: Anti-cancer,Potential Peptide Drug — Tags: — aapptec @ 4:24 pm

High-throughput identification of putative receptors for cancer-binding peptides using biopanning and microarray analysis.
Ferraro DJ, Bhave SR, Kotipatruni RP, Hunn JC, Wildman SA, Hong C, Dadey DY, Muhoro LK, Jaboin JJ, Thotala D, Hallahan DE., Integr Biol (Camb). 2012 Nov 13. doi: 10.1039/C2IB20187A [Epub ahead of print]

Phage-display peptide biopanning has been successfully used to identify cancer-targeting peptides in multiple models. For cancer-binding peptides, identification of the peptide receptor is necessary to demonstrate the mechanism of action and to further optimize specificity and target binding. The process of receptor identification can be slow and some peptides may turn out to bind ubiquitous proteins not suitable for further drug development. In this report, we describe a high-throughput method for screening a large number of peptides in parallel to identify peptide receptors, which we have termed “reverse biopanning.” Peptides can then be selected for further development based on their receptor. To demonstrate this method, we screened a library of 39 peptides previously identified in our laboratory to bind specifically to cancers after irradiation. The reverse biopanning process identified 2 peptides, RKFLMTTRYSRV and KTAKKNVFFCSV, as candidate ligands for the protein tax interacting protein 1 (TIP-1), a protein previously identified in our laboratory to be expressed in tumors and upregulated after exposure to ionizing radiation. We used computational modeling as the initial method for rapid validation of peptide–TIP-1 binding. Pseudo-binding energies were calculated to be −360.645 kcal mol−1, −487.239 kcal mol−1, and −595.328 kcal mol−1 for HVGGSSV, TTRYSRV, and NVFFCSV respectively, suggesting that the peptides would have at least similar, if not stronger, binding to TIP-1 compared to the known TIP-1 binding peptide HVGGSSV. We validated peptide binding in vitro using electrophoretic mobility shift assay, which showed strong binding of RKFLMTTRYSRV and the truncated form TTRYSRV. This method allows for the identification of many peptide receptors and subsequent selection of peptides for further drug development based on the peptide receptor.

Tumor-Penetrating Peptide Designed

Filed under: Anti-cancer,Cell Penetrating Peptides — Tags: — aapptec @ 4:17 pm

De Novo Design of a Tumor-Penetrating Peptide.
Alberici L, Roth L, Sugahara KN, Agemy L, Kotamraju VR, Teesalu T, Bordignon C, Traversari C, Rizzardi GP, Ruoslahti E., Cancer Res. 2012 Nov 14. doi: 10.1158/0008-5472.CAN-12-1668 [Epub ahead of print]

Poor penetration of anti-tumor drugs into the extravascular tumor tissue is often a major factor limiting the efficacy of cancer treatments. Our group has recently described a strategy to enhance tumor penetration of chemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequence motifs: RGD, which binds to αvβ3/5 integrins on tumor endothelia and tumor cells and a cryptic CendR motif (R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleaved to expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds to neuropilin-1, activating a tissue-penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass. Here we describe the design of a new tumor-penetrating peptide based on the current knowledge of homing sequences and internalizing receptors. The tumor-homing motif in the new peptide is the NGR sequence, which binds to endothelial CD13. The NGR sequence was placed in the context of a CendR motif (RNGR), and this sequence was embedded in the iRGD framework. The resulting peptide (CRNGRGPDC, iNGR) homed to tumor vessels and penetrated into tumor tissue more effectively than the standard NGR peptide. iNGR induced greater tumor penetration of coupled nanoparticles and co-administered compounds than NGR. Doxorubicin given together with iNGR was significantly more efficacious than the drug alone. These results show that a tumor-specific, tissue-penetrating peptide can be constructed from known sequence elements. This principle may be useful in designing tissue-penetrating peptides for other diseases.

Smac Peptide enhances TRAIL- or paclitaxel-induced inhibition of cell growth in ovarian cancer cells

Filed under: Anti-cancer,Potential Peptide Drug — Tags: — aapptec @ 4:11 pm

Smac peptide potentiates TRAIL- or paclitaxel-mediated ovarian cancer cell death in vitro and in vivo.
Mao HL, Pang Y, Zhang X, Yang F, Zheng J, Wang Y, Liu P., Oncol Rep. 2012 Nov 9.  doi: 10.3892/or.2012.2132. [Epub ahead of print]

Second mitochondria-derived activator of caspases (Smac) is a recently identified protein that is released from mitochondria in response to apoptotic stimuli and promotes apoptosis by antagonizing the inhibitor of apoptosis proteins (IAPs). Our previous study showed that ectopic overexpression of Smac sensitizes drug-resistant tumor cells to TRAIL- or paclitaxel-induced apoptosis in vitro. The present study was designed to explore the effect of the synthesized Smac N7 peptide in a human ovarian cancer cell line and xenograft model. The results showed that the single-agent Smac N7 had a non-cytotoxic effect, but it effectively enhanced TRAIL- or paclitaxel-induced inhibition of cell proliferation in a dose-dependent manner, even in TRAIL-resistant A2780 cells. When Smac N7 was combined with TRAIL or paclitaxel in treating A2780 cell tumor xenografts, synergistic anticancer effects were achieved. Furthermore, the combination therapy caused less damage in normal tissues and more apoptosis in tumor xenografts compared with TRAIL or paclitaxel alone. Increased apoptosis was associated with the downregulation of XIAP, survivin and the increased activity of caspase-3, along with an increased amount of cleaved PARP. In conclusion, this Smac N7 peptide is a promising candidate for ovarian cancer combination therapy, and Smac may be the target for the development of a novel class of anticancer drugs.

November 14, 2012

Efficient oxidative folding and site-specific labeling of human hepcidin to study its interaction with receptor ferroportin.

Filed under: Uncategorized — Tags: — aapptec @ 10:25 pm

Efficient oxidative folding and site-specific labeling of human hepcidin to study its interaction with receptor ferroportin.
Luo X, Jiang Q, Song G, Liu YL, Xu ZG, Guo ZY.,  FEBS J.  2012, 279, 3166-75. doi: 10.1111/j.1742-4658.2012.08695.x

Hepcidin is a small disulfide-rich peptide hormone that plays a key role in the regulation of iron homeostasis by binding and mediating the degradation of the cell membrane iron efflux transporter, ferroportin. Since it is a small peptide, chemical synthesis is a suitable approach for the preparation of mature human hepcidin. However, oxidative folding of synthetic hepcidin is extremely difficult due to its high cysteine content and high aggregation propensity. To improve its oxidative folding efficiency, we propose a reversible S-modification approach. Introduction of eight negatively charged sulfonate moieties into synthetic hepcidin significantly decreased its aggregation propensity and, under optimized conditions, dramatically increased the refolding yield. The folded hepcidin displayed a typical disulfide-constrained β-sheet structure and could induce internalization of enhanced green fluorescent protein (EGFP) tagged ferroportin in transfected HEK293 cells. In order to study interactions between hepcidin and its receptor ferroportin, we propose a general approach for site-specific labeling of synthetic hepcidin analogues by incorporation of an L-propargylglycine during chemical synthesis. Following efficient oxidative refolding, a hepcidin analogue with Met20 replaced by L-propargylglycine was efficiently mono-labeled by a red fluorescent dye through click chemistry. The labeled hepcidin was internalized into the transfected cells together with the EGFP-tagged ferroportin, suggesting direct binding between hepcidin and ferroportin. The labeled hepcidin was also a suitable tool to visualize internalization of overexpressed or even endogenously expressed ferroportin without tags. We anticipate that the present refolding and labeling approaches could also be used for other synthetic peptides.
© 2012 The Authors Journal compilation © 2012 FEBS.

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