All posts in Raw-Journal Peptide Science

The ubiquitin–proteasome pathway (UPP) influences essential cellular functions including cell growth, differentiation, apoptosis, signal transduction, antigen processing and inflammatory responses. The main proteolytic component of the UPP is the 26S proteasome, which is responsible for the turnover of many cellular proteins and represents an attractive target for the treatment of pathologies such as cancer, as well as inflammatory, immune and neurodegenerative diseases. Natural and synthetic proteasome inhibitors having different chemical structures and potency have been discovered. We report herein the synthesis, proteasome inhibition and modelling studies of novel C-terminal isoxazoline vinyl ester pseudopeptides. Some new compounds that contain a C-terminal extended conjugation inhibit β1 and especially β5 proteasomal catalytic subunits with IC₅₀ values ranging from 10 to 100 µm. These results will permit further optimization based on these structural moieties to develop more active and selective molecules. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

We report the synthesis, proteasome inhibition and modelling studies of novel C-terminal isoxazoline vinyl ester pseudopeptides. Some new compounds inhibit proteasomal catalytic subunits with an IC₅₀ around the micromolar concentration.

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a β^2,2-amino acid with either two 2-naphthyl-methylene or two para-CF₃-benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC₅₀) 11–70 μM] compared to the linear tetrapeptide counterparts (IC₅₀ 18.7 to >413 μM). The most potent cyclic tetrapeptide c3 had a 16.5-fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9-fold) preference for Ramos cells (IC₅₀ 23 μM) compared to healthy human lung fibroblast cells (MRC-5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22–28 μM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide-based cancer drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Detection of structure–activity relationship of anticancer potency and selectivity for linear and cyclic tetrapeptides containing an achiral β^2,2-amino acid are reported. Pharmacokinetic investigation of the interaction with the major transporter protein human serum albumin shows binding to drug site II is exclusively through the hydrophobic parts of the peptides and is in the micromolar range. In vitro testing indicates in vivo implication for the potency of these peptides when human serum albumin is present.

Self-assembly of PAs composed of palmitic acid and several repeated heptad peptide sequences, C₁₅H₃₁CO-(IEEYTKK)_n-NH₂ (n = 1–4, represented by PA1–PA4), was investigated systematically. The secondary structures of the PAs were characterized by CD. PA3 and PA4 (n = 3 and 4, respectively) showed an α-helical structure, whereas PA1 and PA2 (n = 1 and 2, respectively) did not display an α-helical conformations under the tested conditions. The morphology of the self-assembled peptides in aqueous medium was studied by transmission electron microscopy. As the number of heptad repeats in the PAs increased, the nanostructure of the self-assembled peptides changed from nanofibers to nanovesicles. Changes of the secondary structures and the self-assembly morphologies of PA3 and PA4 in aqueous medium with various cations were also studied. The critical micelle concentrations were determined using a pyrene fluorescence probe. In conclusion, this method may be used to design new peptide nanomaterials. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

We designed a series of four peptide amphiphiles (PAs) with varied heptad repeated peptide headgroup lengths. As the number of heptad repeats in the PAs increased, the secondary structures of the PAs tended to display α-helical conformations, and the nanostructure of the self-assembled peptides changed from nanofibers to nanovesicles. This research provides a useful way of modulating the self-assembly of α-helical PAs.

To screen and identify the novel probe markers binding hepatocellular carcinoma specifically and sensitively, a phage-displayed 12-mer peptide library was used to make biopanning with the modified protocols on HepG2 cells. After four rounds of panning, the consensus sequences were obtained, and the PC28, a phage clone with most specific and sensitive binding to HepG2 cells, was identified as the best positive clone. The peptide probe HCSP4 (sequence SLDSTHTHAPWP) was synthesized based on the sequencing result of PC28. The specificity and sensitivity of HCSP4 were primarily analyzed using immunofluorescence, flow cytometry, and other methods. The results show that HCSP4 can bind to hepatocellular carcinoma cells with satisfactory specificity and sensitivity. It may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The specific binding to HepG2 cells indicates that HCSP4-fluorescein isothiocyanate (FITC) may be a promising lead candidate for molecular imaging and targeted drug delivery in the diagnosis and therapy of hepatocellular carcinoma. A, B: HCSP4-FITC binds to HepG2 cells specifically; C, D: the nuclei (C) and membrane (D) of HepG2 cells are displayed using DAPI and Dil staining methods; E, F: the merged photos show that HCSP4-FITC binds to the surfaces of HepG2 cells specifically.

CXCR4 is a G-protein-coupled receptor involved in a number of physiological processes in the hematopoietic and immune systems. CXCL12/CXCR4 axis plays a central role in diseases, such as HIV, cancer, WHIM syndrome, rheumatoid arthritis, pulmonary fibrosis, and lupus and, hence, indicated as putative therapeutic target. Although multiple CXCR4 antagonists have been developed, there is only one marketed drug, plerixafor, indicated for stem cell mobilization in poor mobilizer patients. In this work, we have designed and synthesized two peptides, six and seven residues long, using as template the N-terminal region of CXCL12; analyzed their conformations by CD, NMR, and molecular dynamics simulations; simulated their complexes with CXCR4 by docking methods; and validated these data by in vitro studies. The results showed that the two peptides are rather flexible in aqueous solution lacking ordered secondary structure elements and present a promising affinity for CXCR4. This affinity is not revealed for CXCR7, indicating a specificity for CXCR4. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

In this work, we have designed and synthesized two peptides, six and seven residues long, using as template the N-terminal region of CXCL12; analyzed their conformations by CD, NMR, and molecular dynamics simulations; simulated their complexes with CXCR4 by docking methods; and validated these data by in vitro studies. The results showed that the two peptides are rather flexible in aqueous solution lacking ordered secondary structure elements and present a promising affinity for CXCR4.

A range of inorganic and organoelement halides was evaluated as acidic promoters of direct Nⁱⁿ-formylation of tryptophan. In addition to Me₃SiBr, the less expensive PBr₃ was found to be highly efficient and was selected for further optimization. A convenient and reproducible synthetic procedure for Nⁱⁿ-formyltryptophan hydrobromide developed in this way was scaled to 150 mmol and successfully extended to some derivatives of Trp and closely related indoles as detailed in the present paper. The scope of the method seems to be restricted to indoles substituted at C-3. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

The bromides indicated earlier efficiently promote selective formylation of indole nitrogen in tryptophan, its derivatives and related 3-functionalized indoles. Multigram scale procedure for Trp(For) is detailed.

A procedure for obtaining isotopically labeled peptides, by combining affinity chromatography, urea-equilibrated gel filtration, and hydrophobic chromatography procedures, is presented using the Disabled-2 (Dab2) sulfatide-binding motif (SBM) as a proof of concept. The protocol is designed to isolate unstructured, membrane-binding, recombinant peptides that co-purify with bacterial proteins (e.g., chaperones). Dab2 SBM is overexpressed in bacteria as an isotopically labeled glutathione S-transferase (GST) fusion protein using minimal media containing [¹⁵N] ammonium chloride as the nitrogen source. The fusion protein is purified using glutathione beads, and Dab2 SBM is released from GST using a specific protease. It is then dried, resuspended in urea to release the bound bacterial protein, and subjected to urea-equilibrated gel filtration. Urea and buffer reagents are removed using an octadecyl column. The peptide is eluted with acetonitrile, dried, and stored at −80 °C. Purification of Dab2 SBM can be accomplished in 6 days with a yield of ~2 mg/l of culture. The properties of Dab2 SBM can be studied in the presence of detergents using NMR spectroscopy. Although this method also allows for the purification of unlabeled peptides that co-purify with bacterial proteins, the procedure is more relevant to isotopically labeled peptides, thus alleviating the cost of peptide production. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Recombinant Disabled-2 sulfatide-binding motif (SBM) can be efficiently isolated in an intact and active form for NMR studies. The reported approach can be easily applied to the isolation of recombinant peptides for structural studies. The figure displays ¹H, ¹⁵N-HSQC overlaid spectra of ¹⁵N-labeled Disabled-2 SBM in the absence (red) and presence of dodecylphosphocholine micelles (black). Addition of micelles improved resolution of the spectrum of the peptide, suggesting that this region is responsible for Disabled-2 membrane targeting.

Scolopendra subspinipes mutilans, also known as Chinese red-headed centipede, is a venomous centipede from East Asia and Australasia. Venom from this animal has not been researched as thoroughly as venom from snakes, snails, scorpions, and spiders. In this study, we isolated and characterized SsmTx-I, a novel neurotoxin from the venom of S. subspinipes mutilans. SsmTx-I contains 36 residues with four cysteines forming two disulfide bonds. It had low sequence similarity (<10%) with other identified peptide toxins. By whole-cell recording, SsmTx-I significantly blocked voltage-gated K⁺ channels in dorsal root ganglion neurons with an IC₅₀ value of 200 nM, but it had no effect on voltage-gated Na⁺ channels. Among the nine K⁺ channel subtypes expressed in human embryonic kidney 293 cells, SsmTx-I selectively blocked the Kv2.1 current with an IC₅₀ value of 41.7 nM, but it had little effect on currents mediated by other K⁺ channel subtypes. Blockage of Kv2.1 by SsmTx-I was not associated with significant alteration of steady-state activation, suggesting that SsmTx-I might act as a simple inhibitor or channel blocker rather than a gating modifier. Our study reported a specific Kv2.1-blocker from centipede venom and provided a basis for future investigations of SsmTx-I, for example on structure–function relationships, mechanism of action, and pharmacological potential. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

SsmTx-I, a novel peptide toxin from the venom of the centipede S subspinipes mutilans, specifically inhibits voltage-gated K⁺ channel subtype Kv2.1.