**3. Discussion**

Thiopeptides, or thiazolyl peptides, are highly modified sulfur-rich peptides of ribosomal origin. Over 100 chemical entities have been isolated in the last 50 years [9]. Of these entities, thiostrepton has been used as an FDA-approved active pharmaceutical ingredient for animals, and nosiheptide has been widely applied in veterinary antibiotics and food preservation. Their most characteristic feature is the central nitrogen-containing six-membered ring structure. Depending on the oxidation state of this central ring, thiopeptides can be classified into five different series [10]. The litoralimycins belong to the *d* series, which is the most numerous subgroup, due to their trisubstituted pyridine moiety.

Another option to group the thiopeptides is based on the ring size of the main macrocycle, since ring sizes of 26, 29, 32 and 35 atoms are found. Specifically, litoralimycins **1** and **2** belong to a small family of compounds with an oxazolyl-thiazolyl-pyridine fragment embedded in a 35-membered (13-residue) peptidyl macrocycle [11]. Other members of this family, which have highest structural similarity to **1** and **2**, are the berninamycins, sulfomycin, thioplabin and TP-1161A. Common variations between members of this family are the exchange of thiazole by oxazole moieties, di fferent methylation patterns and the size of the side chain (see Figure S3).

Most characterized thiopeptides display nanomolar potency toward Gram-positive bacteria by blocking protein translation, including the notorious pathogens methicillin-resistant *Staphylococcus aureus* (MRSA), vancomycin-resistant enterococci (VRE), and penicillin-resistant *Streptococcus pneumoniae* (PRSP). Their mechanism of action, acting as protein synthesis inhibitors, correlates with the size of the primary macrocycle: 29-atom macrocycles bind to elongation factor EF-TU, while 26- and 32-atom macrocycles bind to the interface of protein L11 and the 23S rRNA within the 50S ribosomal subunit. The general molecular target of compounds with the largest 35-membered macrocycle remains unknown [10], although berninamycin was reported to target the 50S ribosome, in a similar manner to thiostrepton [11].

Using NMR and biochemical assays, a three-dimensional interaction model was developed, identifying l-Thr as a preserved region for the interaction with the ribosome/L11 complex [12]. This residue is missing for the litoralimycins, since **1** and **2** bear a l-Val at this position instead. In conformity with **1** and **2**, radamycin also has a mutated residue with l-Val replacing l-Thr. In analogy to the litoralimycins, radamycin was devoid of any antibacterial activity in agar di ffusion assays [13,14]. Nevertheless, radamycin showed a strong *tipA* promoting activity. *tipA* gene promotion, encoding the two thiostrepton-induced proteins (Tip) TipAL and TipAS. The latter serves as a defense mechanism for bacteria against thiopeptides. Since the *tipA* promotion activity was identified to be dependent on a dehydroalanine-containing tail close to the six-membered central sca ffold [15], we expect the litoralimycins to be *tipA* activators.

Besides the aforementioned e ffects on bacteria, some thiopeptides show good anticancer activities [9]. For thiostrepton it was shown that this activity is based on its e ffect of reducing transcriptional activity of the forkhead box M1 (FOXM1). FOXM1 is an oncogenic transcription factor that is upregulated in a wide range of cancers. It is involved in the regulation of the cell cycle and promotes angiogenesis, as well as metastasis. Because treatment with thiostrepton had an e ffect on cell proliferation and cell-cycle progression in MCF-7 cells [16], and litoralimycins A (**1**) was strongly active against cell line MCF-7 in our test assay, FOXM1 might be the molecular target in common.
