In vitro evaluation of the immunogenic potential of gramicidin S and its photocontrolled analogues†

Three hallmarks of ICD (immunogenic cell death), release of adenosine triphosphate (ATP), release of high mobility group box 1 protein, and calreticulin exposure on the cell surface, were studied upon treatment of mammalian cells with small cyclic peptides, namely, the natural antibiotic gramicidin S (GS) and two photocontrolled GS analogues (LMB002 and LMB033). The analogues contained a photoisomerizable diarylethene fragment, and they exhibited different bioactivities in their “open” and “closed” photoisomeric forms. The data (obtained from cell cultures and spheroids) were collected in a concentration-dependent manner to assess cytotoxicity. Results showed that treatment with all peptides induced ICD at sub-IC₅₀ and higher concentrations, indicating that GS and its derivatives have promising immunogenic potential. The “open” photoisomers of the photoswitchable GS analogues generated using visible light were as efficient as ICD inducers and the parent GS, while the UV-generated “closed” photoforms induced ICD only at higher concentrations. Herein, the cell specificity and time dependency of the observed effects are presented.