# Therapeutic Potential of Gramicidin S in the Treatment of Root Canal Infections
## 3 Discussion  
The cyclic decapeptide gramicidin S (S = \"Soviet\") was isolated by [PERSON] and [PERSON] from soil bacilli and described in _Nature_ and _Lancet_ in 1944 [24; 25]. The symmetric structure of GS is a double-repeated sequence of five amino acids, which are arranged in an antiparallel cyclic manner (cyclo[phe-Pro-Val-Orn-Leu]\({}_{2}\)) [26]. GS has an amphiphilic structure, consisting of the cationic non-canonical amino acid ornithine and the hydrophobic \({}^{D}\)phenylalanine, valine and leucine. As pharmaceuticals, antimicrobial peptides generally exhibit some unfavorable properties, such as instability, salt sensitivity, high cost of production, and a rather non-specific spectrum of activity, including hemolytic effects [27]. GS, on the other hand, is a very stable peptide. Its cyclic structure and the presence of the unusual amino acids ornithine and \({}^{D}\)phenylalanine avoid the typical proteolytic degradation by common proteases. Overall, GS has a high hemolytic activity, which limits its use to topical applications, though the addition of polyethylene glycol has been shown to prevent its hemolytic effects [28]. The production of GS via the fermentation of producing _Aneurinobacillus migulanus_ phenotypes [29] is less costly than the usual chemical synthesis of antimicrobial peptides of non-bacterial origin. GS is approved as the bioactive agent in Grammidin(r)Neo, a lozenge used against sore throat and mouth ulcers, produced by Russian JSC Valenta Pharmaceuticals. Notably, despite a long treatment history, no clinical cases of bacterial resistance against GS have been reported [30]. The promising observation that GS does not lead to bacterial resistance can be attributed to its ability to attack multiple targets in bacterial cells at the same time. The first target of GS is the prokaryotic plasma membrane; therefore, the peptide possesses enhanced activity against Gram-positive bacteria like _Staphylococcus_ spp., _Streptococcus_ spp., and _Enterococcus_ spp. [31]. In addition to depolarization, presumably via the formation of short-lived pores at sub-MICs [32], it also inhibits the respiratory enzymes NADH dehydrogenase and cytochrome _bd_ terminal oxidase in the bacterial membrane [33], and it detaches several vital peripheral membrane proteins, namely the cell-division regulator MinD, the Lipid II biosynthesis protein MurG, and cytochrome c [34]. Here, we have shown that GS does not only interact with the bacterial membrane, which causes structural and  
Figure 4: Molar 36 of a 25-year-old male patient before and after treatment. (**A**) The radiolucent area in the X-ray image indicates decay. (**B**) The sealed root canals after treatment with GS. The red arrowheads mark the decayed area and the sealed root canals, respectively. functional damage as described above. In fact, it can also penetrate into the cytoplasm, where it can bind to ppGpp, the intracellular regulator of biofilm growth [35], and to its precursors ATP and GTP, as well as the energy metabolites ADP and GDP (Figures S1-S4 in the Supplementary Materials). These new modes of activity broaden the spectrum of effects known so far for GS. It is especially important to realize that this multifaceted activity profile provides GS with unique bactericidal properties toward Gram-positive bacteria irrespective of their physiological state, virulence, resistance, tolerance and phenotypic variation. Generally, the stationary phase of a liquid bacterial culture contains a population of metabolically inactive non- or slow-growing reversible phenotypes, which are known as persister cells. They are tolerant to conventional antibiotics, even at 100 \(\times\) MIC [36], because these antibiotics target only growing cells. The ability of GS to disrupt membrane function and to form complexes with intracellular targets after its rapid penetration into the cell interior leads to a complete and rapid killing.