Comparison of bactericidal and cytotoxic activities of trichogin analogs

Abstract

Peptaibiotics are a group of membrane active peptides of fungal origin. They typically contain α-aminoisobutyric acid (Aib; 1-letter code, U) and other non-coded residues (Toniolo and Brückner, 2009; Neumann et al., 2015; Benedett et al., 1982) [1], [2], [3] stabilizing their helical structure. Peptaibols are peptaibiotics carrying a 1, 2-aminoalcohol at the C-terminus. When a fatty acid chain (of 8–10 carbon atoms) is present at their N-terminus, they are called lipopeptaibols (Toniolo et al., 2001; Degenkolb et al., 2003) [4], [5]. We found (Tavano et al., 2015) [6] that the lipopeptaibol trichogin displays no antibacterial effects up to 64 µM, against both Gram− and Gram+ bacteria, but kills tumor and healthy human cells via a mechanism requiring both the C-terminal primary alcohol group and the N-terminal n-octanoyl moiety, with EC50s around 4–5 µM. However, the substitution of single Gly residues with Lys strongly improves anti-Gram+ activity (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion et al., 2012) [6], [7], [8]. To further characterize the activity of trichogin analogs as antibiotics and cytotoxic agents, we here manipulated the peptide helix amphipathicity by means of two different substitutions: (i) Aib to Leu (De Zotti et al., 2012) [7] or (ii) multiple Gly to Lys changes (Tavano et al., 2015; De Zotti, Biondi, Park et al., 2012; De Zotti, Biondi, Peggion, Formaggio et al., 2012; De Zotti, Biondi, Peggion, De Poli et al., 2012) [6], [7], [8], [9]. The antibacterial activity against four commensal or opportunistic bacterial species and the cytotoxicity against a panel of 9 healthy and tumor-derived eukaryotic cell types (including erythrocytes) are reported as MIC and EC50 (MTS - [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)]-2H-tetrazolium- reduction and LDH - lactate dehydrogenase - release assay).

Insights into peptide‐membrane interactions of newly synthesized, nitroxide‐containing analogs of the peptaibiotic trichogin GAIV using EPR

“…In summary, this work allowed us to verify that the overall orientation and insertion in model membranes of the native TG closely resemble those already published for its methyl ester analogs . Therefore, reminding that the C‐terminal 1,2‐amino alcohol is critical for the cytotoxic activity of TG and its derivatives towards eukaryotic cells, our new EPR data on different analogs suggest that this alcoholic function probably affects binding affinity, and/or partakes in a specific interaction with eukaryotic cells, rather than influencing peptide orientation in the membrane.…”

“…This study was inspired by the experimental observations that the C‐terminal Lol residue and the N‐terminal Oct group are both essential for the biological activity of TG and its derivatives, but the effects of these moieties on the orientation and dynamics in membranes of these peptides were not known. Therefore, we synthesized several TOAC‐labeled TG analogs and determined their membrane dynamics, orientation, and penetration depth by EPR.…”

“…Its primary structure is Oct‐Aib‐Gly‐Leu‐Aib‐Gly‐Gly‐Leu‐Aib‐Gly‐Ile‐Lol (Oct is 1‐octanoyl, Lol is the 1,2‐amino alcohol leucinol). To unravel the mechanism of action of TG and to possibly develop a drug from TG, in the last two decades we have been synthesizing selected TG analogs and investigating them by means of a variety of physicochemical techniques . The presence of the three helix‐inducing Aib residues allows TG to adopt a helical structure in the crystal state, in organic solvents, in membranes, and in membrane mimetic environments .…”

“…The presence of the three helix‐inducing Aib residues allows TG to adopt a helical structure in the crystal state, in organic solvents, in membranes, and in membrane mimetic environments . Both its N‐ and C‐ terminal moieties play a crucial role in the antimicrobial and cytotoxic activity of this peptide . In particular, we recently demonstrated the importance of its C‐terminal Lol amino alcohol, as its replacement with a methyl ester (‐Leu‐OMe) or a carboxylic acid (‐Leu‐OH) determines a significant inhibition of the cytotoxic action .…”

“…Both its N‐ and C‐ terminal moieties play a crucial role in the antimicrobial and cytotoxic activity of this peptide . In particular, we recently demonstrated the importance of its C‐terminal Lol amino alcohol, as its replacement with a methyl ester (‐Leu‐OMe) or a carboxylic acid (‐Leu‐OH) determines a significant inhibition of the cytotoxic action . This finding prompted us to revise some of the work previously performed on TG analogs not bearing an alcoholic function at the C‐terminus .…”