Dataset Information

A Synthetic Strategy for Conjugation of Paromomycin to Cell-Penetrating Tat(48-60) for Delivery and Visualization into Leishmania Parasites.

ABSTRACT: A successful approach to deliver paromomycin, a poorly absorbed aminoglycoside antibiotic, to parasite cells is reported, based on selective protection of amino and hydroxyl groups followed by conjugation to a fluorolabeled, PEG-functionalized cell-penetrating Tat(48-60) peptide. The resulting construct is efficiently internalized into Leishmania cells, evidencing the fitness of cell-penetrating peptides as vectors for efficiently transporting low-bioavailability drugs into cells.

SUBMITTER: Defaus S

PROVIDER: S-EPMC5329690 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Publications

A Synthetic Strategy for Conjugation of Paromomycin to Cell-Penetrating Tat(48-60) for Delivery and Visualization into Leishmania Parasites.

Defaus Sira S Gallo Maria M Abengózar María A MA Rivas Luis L Andreu David D

International journal of peptides 20170214

A successful approach to deliver paromomycin, a poorly absorbed aminoglycoside antibiotic, to parasite cells is reported, based on selective protection of amino and hydroxyl groups followed by conjugation to a fluorolabeled, PEG-functionalized cell-penetrating Tat(48-60) peptide. The resulting construct is efficiently internalized into Leishmania cells, evidencing the fitness of cell-penetrating peptides as vectors for efficiently transporting low-bioavailability drugs into cells. ...[more]

PMID: 28286529

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Project description:Conjugates based on cell-penetrating peptides (CPPs) are scientifically relevant owing to their structural complexity; their ability to enter cells and deliver drugs, labels, antioxidants, bioactive compounds, or DNA fragments; and, consequently, their potential for application in research and biomedicine. In this study, carboxyamidated fluorescently labeled conjugates FAM-GG-TAT(47-57)-NH2 and FAM-PEG6-TAT(47-57)-NH2 and photosensitizer-labeled conjugate Chk-PEG6-TAT(47-57)-NH2 [where TAT(47-57) is the CPP, 5(6)-carboxyfluorescein is the (FAM) fluorophore, chlorin k (Chk) is the photosensitizer, and the dipeptide glycyl-glycine (GG) or hexaethylene glycol (PEG6) is the spacer] were originally designed, prepared, and fully characterized. Practically, all chemical reactions of the synthetic steps (peptide synthesis, spacer incorporation, and conjugation) were microwave-assisted at 60 °C using optimized protocols to give satisfying yields and high-quality products. Detailed analyses of the conjugates using spectrofluorimetry and singlet oxygen detection showed that they display photophysical properties typical of FAM or Chk. Anticandidal activity assays showed that not only this basic property of TAT(47-57) was preserved in the conjugates but also that the minimal inhibitory concentration was slightly reduced for cells incubated with PS-bearing conjugate Chk-PEG6-TAT(47-57)-NH2. Overall, these results indicated that the synthetic approach on-resin assisted by microwaves at 60 °C is simple, straightforward, selective, metal-free, sufficiently fast, cleaner, and more cost-effective than those previously used for preparing this type of macromolecule. Furthermore, such new data show that microwaves at 60 °C and/or conjugation did not harm the integrity of the conjugates' constituents. Therefore, FAM-GG-TAT(47-57)-NH2, FAM-PEG6-TAT(47-57)-NH2, and Chk-PEG6-TAT(47-57)-NH2 have high potential for practical applications in biochemistry, biophysics, and therapeutics.

Project description:BackgroundPatients suffering from cutaneous leishmaniasis (CL) caused by New World Leishmania (Viannia) species are at high risk of developing mucosal (ML) or disseminated cutaneous leishmaniasis (DCL). After the formation of a primary skin lesion at the site of the bite by a Leishmania-infected sand fly, the infection can disseminate to form secondary lesions. This metastatic phenotype causes significant morbidity and is often associated with a hyper-inflammatory immune response leading to the destruction of nasopharyngeal tissues in ML, and appearance of nodules or numerous ulcerated skin lesions in DCL. Recently, we connected this aggressive phenotype to the presence of Leishmania RNA virus (LRV) in strains of L. guyanensis, showing that LRV is responsible for elevated parasitaemia, destructive hyper-inflammation and an overall exacerbation of the disease. Further studies of this relationship and the distribution of LRVs in other Leishmania strains and species would benefit from improved methods of viral detection and quantitation, especially ones not dependent on prior knowledge of the viral sequence as LRVs show significant evolutionary divergence.Methodology/principal findingsThis study reports various techniques, among which, the use of an anti-dsRNA monoclonal antibody (J2) stands out for its specific and quantitative recognition of dsRNA in a sequence-independent fashion. Applications of J2 include immunofluorescence, ELISA and dot blot: techniques complementing an arsenal of other detection tools, such as nucleic acid purification and quantitative real-time-PCR. We evaluate each method as well as demonstrate a successful LRV detection by the J2 antibody in several parasite strains, a freshly isolated patient sample and lesion biopsies of infected mice.Conclusions/significanceWe propose that refinements of these methods could be transferred to the field for use as a diagnostic tool in detecting the presence of LRV, and potentially assessing the LRV-related risk of complications in cutaneous leishmaniasis.

Dataset Information

A Synthetic Strategy for Conjugation of Paromomycin to Cell-Penetrating Tat(48-60) for Delivery and Visualization into Leishmania Parasites.

Publications

A Synthetic Strategy for Conjugation of Paromomycin to Cell-Penetrating Tat(48-60) for Delivery and Visualization into Leishmania Parasites.

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