Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m
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Universidade Federal de Minas Gerais
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Background: Current diagnostic methods and imaging techniques are not able to differentiate septic and aseptic
inflammation. Thus, reliable methods are sought to provide this distinction and scintigraphic imaging is an interesting
option, since it is based on physiological changes. In this context, radiolabeled antimicrobial peptides have been
investigated as they accumulate in infectious sites instead of aseptic inflammation. The peptide LyeTx I, from the
venom of Lycosa erythrognatha, has potent antimicrobial activity. Therefore, this study aimed to synthesize LyeTx I
derivatives with the chelating compound HYNIC, to evaluate their antimicrobial activity and to radiolabel them
with 99mTc.
Methods: Two LyeTx I derivatives, HYNIC-LyeTx I (N-terminal modification) and LyeTx I-K-HYNIC (C-terminal
modification), were synthesized by Fmoc strategy and purified by RP-HPLC. The purified products were assessed
by RP-HPLC and MALDI-ToF-MS analysis. Microbiological assays were performed against S. aureus (ATCC® 6538)
and E. coli (ATCC® 10536) in liquid medium to calculate the MIC. The radiolabeling procedure of LyeTx I-K-HYNIC
with 99mTc was performed in the presence of co-ligands (tricine and EDDA) and reducing agent (SnCl2
.
2H2O), and
standardized taking into account the amount of peptide, reducing agent, pH and heating. Radiochemical purity
analysis was performed by thin-layer chromatography on silica gel strips and the radiolabeled compound was
assessed by RP-HPLC and radioactivity measurement of the collected fractions. Data were analyzed by ANOVA,
followed by Tukey test (p-values < 0.05).
Results: Both LyeTx I derivatives were suitably synthesized and purified, as shown by RP-HPLC and MALDI-ToF-MS
analysis. The microbiological test showed that HYNIC-LyeTx I (N-terminal modification) did not inhibit bacterial growth,
whereas LyeTx I-K-HYNIC (C-terminal modification) showed a MIC of 5.05 μmol.
L−1 (S. aureus) and 10.10 μmol.
L−1
(E. coli). Thus, only the latter was radiolabeled with 99mTc. The radiochemical purity analysis of LyeTx I-K-HYNIC-99mTc
showed that the optimal radiolabeling conditions (10 μg of LyeTx I-K-HYNIC; 250 μg of SnCl2
.
2H2O; pH = 7; heating for
15 min) yielded a radiochemical purity of 87 ± 1 % (n = 3). However, RP-HPLC data suggested 99mTc transchelation from
LyeTx I-K-HYNIC to the co-ligands (tricine and EDDA). Conclusions: The binding of HYNIC to the N-terminal portion of LyeTx I seems to affect its activity against bacteria.
Nevertheless, the radiolabeling of the C-terminal derivative, LyeTx I-K-HYNIC, must be better investigated to
optimize the radiolabeled compound, in order to use it as a specific imaging agent to distinguish septic and
aseptic inflammation.
Abstract
Assunto
Química
Palavras-chave
Septic and aseptic inflammation, Differential diagnosis, Antimicrobial peptides, LyeTx I derivatives, MALDI-ToF-MS, RP-HPLC, Technetium-99m, HYNIC, EDDA, Tricine
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Endereço externo
https://www.scielo.br/j/jvatitd/a/bR6xtMwsLbjrR9CY6XrrzKN/?lang=en#