Optimizing the synthesis of dimeric peptides: influence of the reaction medium and effects that modulate kinetics and reaction yield.

Peptides are remarkably interesting alternatives to several applications. In particular, antimicrobial sequences have raised major interest of the scientific community due to the resistance acquired by commonly used antibiotics. Amongst these, some dimeric peptides have shown very promising characteristics as strong biological activities and resistance against degradation by peptidases. However, despite such promising characteristics, a relatively small number of studies address dimeric peptides, mainly due to the synthesis-related obstacles in their production, whereas the well-implemented routines of solid phase peptide synthesis-which includes the possibility of automation-makes life significantly easier. Here, we present kinetic investigations of the dimerization of a cysteine-containing sequence to obtain the homodimeric antimicrobial peptide homotarsinin. Based on the structural and membrane interaction data already available for the dimer and its monomeric chain, we have proposed distinct dimerization protocols in selected environments, namely, aqueous buffer, TFE:H2O and micellar solutions. The experimental results were adjusted by a theoretical model. Both the kinetic profiles and the reaction yields are dependent on the reaction medium, clearly indicating that aggregation, peptide structure, and peptide-membrane interactions play major roles in the formation of the disulfide bond. Finally, the rationalization of the different aspects addressed here is expected to contribute to research and applications that demand the obtainment of dimeric peptides.

A Novel Dimeric Conotoxin, FrXXA, from the Vermivorous Cone Snail Conus fergusoni, of the Eastern Pacific, Inhibits Nicotinic Acetylcholine Receptors.

We isolated a new dimeric conotoxin with inhibitory activity against neuronal nicotinic acetylcholine receptors. Edman degradation and transcriptomic studies indicate a homodimeric conotoxin composed by two chains of 47 amino acid in length. It has the cysteine framework XX and 10 disulfide bonds. According to conotoxin nomenclature, it has been named as αD-FrXXA. The αD-FrXXA conotoxin inhibited the ACh-induced response on nAChR with a IC50 of 125 nM on hα7, 282 nM on hα3ß2, 607 nM on α4ß2, 351 nM on mouse adult muscle, and 447 nM on mouse fetal muscle. This is first toxin characterized from C. fergusoni and, at the same time, the second αD-conotoxin characterized from a species of the Eastern Pacific.

Peptides Derived from (RRWQWRMKKLG)2-K-Ahx Induce Selective Cellular Death in Breast Cancer Cell Lines through Apoptotic Pathway.

The effect on the cytotoxicity against breast cancer cell lines of the substitution of 26Met residue in the sequence of the Bovine Lactoferricin-derived dimeric peptide LfcinB (20-30)2: (20RRWQWRMKKLG30)2-K-Ahx with amino acids of different polarity was evaluated. The process of the synthesis of the LfcinB (20-30)2 analog peptides was similar to the original peptide. The cytotoxic assays showed that some analog peptides exhibited a significant cytotoxic effect against breast cancer cell lines HTB-132 and MCF-7, suggesting that the substitution of the Met with amino acids of a hydrophobic nature drastically enhances its cytotoxicity against HTB-132 and MCF-7 cells, reaching IC50 values up to 6 µM. In addition, these peptides have a selective effect, since they exhibit a lower cytotoxic effect on the non-tumorigenic cell line MCF-12. Interestingly, the cytotoxic effect is fast (90 min) and is maintained for up to 48 h. Additionally, through flow cytometry, it was found that the obtained dimeric peptides generate cell death through the apoptosis pathway and do not compromise the integrity of the cytoplasmic membrane, and there are intrinsic apoptotic events involved. These results show that the obtained peptides are extremely promising molecules for the future development of drugs for use against breast cancer.

PET-Based Human Dosimetry of the Dimeric αvß3 Integrin Ligand 68Ga-DOTA-E-[c(RGDfK)]2, a Potential Tracer for Imaging Tumor Angiogenesis.

UNLABELLED: Peptides containing the Arg-Gly-Asp (RGD) sequence have high affinity for αvß3 integrin receptors overexpressed in tumor cells. The objective of this research was to determine the biodistribution and estimate the radiation dose from (68)Ga-DOTA-E-[c(RGDfK)]2 using whole-body PET scans in humans. METHODS: Five healthy volunteers (2 women, 3 men; mean age ± SD, 37.2 ± 15.6 y; range, 28-65 y; mean weight, 79.2 ± 21.0 kg; range, 64-115 kg) were included. After intravenous injection of the tracer (198.3 ± 3.3 MBq), 3 successive whole-body (vertex to mid thigh) PET/CT scans at 3 time points (30, 60, and 120 min) were obtained on a 16-slice PET/CT scanner. The subjects did not void the bladder until the entire series of images was completed. Low-dose CT without contrast agent was used for anatomic localization and attenuation correction. OLINDA/EXM software was applied to calculate human radiation doses using the reference adult model. RESULTS: The highest uptake was in the urinary bladder, followed by the liver, kidneys, and spleen, in descending order. The critical organ was the urinary bladder wall. The mean effective doses (all subjects, men and women) were 34.1 ± 4.9, 31.0 ± 2.4, and 20.9 ± 5.2 µSv/MBq for the no-voiding, 2.5-h-voiding, and 1-h-voiding models, respectively. CONCLUSION: Of particular interest in this research was the visualization of the choroid plexus and ventricular system, which seems to be a characteristic of RGD-dimeric peptides. Measured absorbed doses and effective doses are comparable to other previously reported RGD-based radiopharmaceuticals labeled with (68)Ga and (18)F. Therefore, (68)Ga-DOTA-E-[c(RGDfK)]2 can safely be used for imaging integrin αVß3 expression.