PEGylation of the antimicrobial peptide LyeTx I-b maintains structure-related biological properties and improves selectivity

Júlio César Moreira Brito; Lucas Raposo Carvalho; Amanda Neves de Souza; Guilherme Carneiro; Paula Prazeres Magalhães; Luiz Macêdo Farias; Natália Rocha Guimarães; Rodrigo Moreira Verly; Jarbas Magalhães Resende; Maria Elena de Lima

doi:10.3389/fmolb.2022.1001508

PEGylation of the antimicrobial peptide LyeTx I-b maintains structure-related biological properties and improves selectivity

Front Mol Biosci. 2022 Oct 13:9:1001508. doi: 10.3389/fmolb.2022.1001508. eCollection 2022.

Affiliations

¹ Fundação Ezequiel Dias, Diretoria de Pesquisa e Desenvolvimento, Belo Horizonte, MG, Brazil.
² Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
³ Departamento de Química, Instituto de Ciências Exatas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, MG, Brazil.
⁴ Departamento de Farmácia, Faculdade de Ciências Biológicas e da Saúde, Universidade Federal dos Vales do Jequitinhonha e Mucuri, Diamantina, MG, Brazil.
⁵ Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil.
⁶ Programa de Pós-graduação em Medicina e Biomedicina da Santa Casa de Belo Horizonte, Belo Horizonte, MG, Brazil.

Abstract

The biological activity of antimicrobial peptides and proteins is closely related to their structural aspects and is sensitive to certain post-translational modifications such as glycosylation, lipidation and PEGylation. However, PEGylation of protein and peptide drugs has expanded in recent years due to the reduction of their toxicity. Due to their size, the PEGylation process can either preserve or compromise the overall structure of these biopolymers and their biological properties. The antimicrobial peptide LyeTx I-b_cys was synthesized by Fmoc strategy and coupled to polyethylene glycol 2.0 kDa. The conjugates were purified by HPLC and characterized by MALDI-ToF-MS analysis. Microbiological assays with LyeTx I-b_cys and LyeTx I-bPEG were performed against Staphylococcus aureus (ATCC 33591) and Escherichia coli (ATCC 25922) in liquid medium. MIC values of 2.0 and 1.0 µM for LyeTx I-b_cys and 8.0 and 4.0 µM for LyeTx I-bPEG were observed against S. aureus and E. coli, respectively. PEGylation of LyeTx I-b_cys (LyeTx I-bPEG) decreased the cytotoxicity determined by MTT method for VERO cells compared to the non-PEGylated peptide. In addition, structural and biophysical studies were performed to evaluate the effects of PEGylation on the nature of peptide-membrane interactions. Surface Plasmon Resonance experiments showed that LyeTx I-b binds to anionic membranes with an association constant twice higher than the PEGylated form. The three-dimensional NMR structures of LyeTx I-b_cys and LyeTx I-bPEG were determined and compared with the LyeTx I-b structure, and the hydrodynamic diameter and zeta potential of POPC:POPG vesicles were similar upon the addition of both peptides. The mPEG-MAL conjugation of LyeTx I-b_cys gave epimers, and it, together with LyeTx I-bPEG, showed clear α-helical profiles. While LyeTx I-b_cys showed no significant change in amphipathicity compared to LyeTx I-b, LyeTx I-bPEG was found to have a slightly less clear separation between hydrophilic and hydrophobic faces. However, the similar conformational freedom of LyeTx I-b and LyeTx I-bPEG suggests that PEGylation does not cause significant structural changes. Overall, our structural and biophysical studies indicate that the PEGylation does not alter the mode of peptide interaction and maintains antimicrobial activity while minimizing tissue toxicity, which confirmed previous results obtained in vivo. Interestingly, significantly improved proteolytic resistance to trypsin and proteinase K was observed after PEGylation.

Keywords: LyeTx I-b; PEGylation; antimicrobial peptide; peptide-membrane interaction; post-translational modification; structure.