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

 

Leonardo Lima Fuscaldi¹, Daniel Moreira dos Santos², Natália Gabriela Silva Pinheiro³, Raquel Silva Araújo¹, André Luís Branco de Barros¹, Jarbas Magalhães Resende³, Simone Odília Antunes Fernandes¹, Maria Elena de Lima², Valbert Nascimento Cardoso¹

 

1 Department of Clinical and Toxicological Analyses, School of Pharmacy, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte, MG 31270-901, Brazil.

2 Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.

3 Department of Chemistry, Institute of Exact Sciences, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil.

 

ABSTRACT

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.

 

Key words: Septic and aseptic inflammation; Differential diagnosis; Antimicrobial peptides LyeTx I derivatives; MALDI-ToF-MS RP-HPLC; Technetium-99m; HYNIC EDDA Tricine

 

Funding

The authors would like to thank the Toxinology Network sponsored by the Coordination for the Improvement of Higher Education Personnel (CAPES) for the PhD fellowship, as well as National Council for Scientific and Technological Development (CNPq), the State of Minas Gerais Research Foundation (FAPEMIG) and the Office of the Dean for Research of the Federal University of Minas Gerais (PRPq/UFMG) for their grants. Thanks are also due to the Center for the Study of Venoms and Venomous Animals (CEVAP) of UNESP for enabling the publication of this paper (CAPES, grant no. 23038.006285/2011-21, AUXPE - Toxinologia – 1219/2011).

 

Received: January 15, 2016.

Accepted: April 12, 2016.

Accepted: April 22, 2016.

 

Correspondence: valbertncardoso@gmail.com

 

Competing interests

The authors declare that they have no competing interests.

 

Authors’ contributions

NGSP synthesized both peptide LyeTx I derivatives. LLF and DMS purified synthetic crude peptide LyeTx I derivatives and performed RP-HPLC and MALDI-ToF-MS analysis. LLF assessed in vitro antimicrobial activity, radiolabeled LyeTx I-K-HYNIC derivative with ^99mTc and performed radiochemical purity analysis. LLF, RSA and ALBB evaluated the stability of LyeTx I-K-HYNIC-^99mTc. LLF worked on the statistical analysis. JMR, SOAF, MEL and VNC participated in the design and guidance of the study. LLF wrote the manuscript. All authors read and approved the final manuscript.

 

doi: 10.1186/s40409-016-0070-y