Synthesis and antimicrobial evaluation of two peptide LyeTx I derivatives modified with the chelating agent HYNIC for radiolabeling with technetium-99m.
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 (99m)Tc.


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 (99m)Tc 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 (99m)Tc. The radiochemical purity analysis of LyeTx I-K-HYNIC-(99m)Tc 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 (99m)Tc 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.
Department of Clinical and Toxicological Analyses, School of Pharmacy, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901 Brazil.
Leonardo Lima
LL
Fuscaldi
Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG Brazil.
Daniel Moreira
DM
Dos Santos
Department of Chemistry, Institute of Exact Sciences, Federal University of Minas Gerais, Belo Horizonte, MG Brazil.
Natália Gabriela Silva
NG
Pinheiro
Department of Clinical and Toxicological Analyses, School of Pharmacy, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901 Brazil.
Raquel Silva
RS
Araújo
Department of Clinical and Toxicological Analyses, School of Pharmacy, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901 Brazil.
André Luís Branco
AL
de Barros
Department of Chemistry, Institute of Exact Sciences, Federal University of Minas Gerais, Belo Horizonte, MG Brazil.
Jarbas Magalhães
JM
Resende
Department of Clinical and Toxicological Analyses, School of Pharmacy, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901 Brazil.
Simone Odília Antunes
SO
Fernandes
Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG Brazil.
Maria Elena
ME
de Lima
Department of Clinical and Toxicological Analyses, School of Pharmacy, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901 Brazil.
Valbert Nascimento
VN
Cardoso
Antimicrobial peptides; Differential diagnosis; EDDA; HYNIC; LyeTx I derivatives; MALDI-ToF-MS; RP-HPLC; Septic and aseptic inflammation; Technetium-99m; Tricine