WAP-8294A2 (lotilibcin, 1) is a potent antibiotic with superior in vivo efficacy to vancomycin against methicillin-resistant Staphylococcus aureus (MRSA). Despite the great medical importance, its molecular mode of action remains unknown. Here we report the total synthesis of complex macrocyclic peptide 1 comprised of 12 amino acids with a β-hydroxy fatty-acid chain, and its deoxy analogue 2. A full solid-phase synthesis of 1 and 2 enabled their rapid assembly and the first detailed investigation of their functions. Compounds 1 and 2 were equipotent against various strains of Gram-positive bacteria including MRSA. We present evidence that the antimicrobial activities of 1 and 2 are due to lysis of the bacterial membrane, and their membrane-disrupting effects depend on the presence of menaquinone, an essential factor for the bacterial respiratory chain. The established synthetic routes and the menaquinone-targeting mechanisms provide valuable information for designing and developing new antibiotics based on their structures.

Lysocin E, a 37-membered natural depsipeptide, induces rapid bacteriolysis in methicillin-resistant Staphylococcus aureus via a unique menaquinone-dependent mechanism, presenting a promising therapeutic lead. Despite the great medical importance, exploring the potential utility of its derivatives as new platform structures for antibiotic development has remained a significant challenge. Here, we report a high-throughput strategy that enabled the preparation of thousands of analogues of lysocin E and large-scale structure-activity relationship analyses. We integrate 26-step total synthesis of 2401 cyclic peptides, tandem mass spectrometry-sequencing, and two microscale activity assays to identify 23 candidate compounds. Re-synthesis of these candidates shows that 11 of them ( A1 – A11 ) exhibit antimicrobial activity superior or comparable to that of lysocin E, and that lysocin E and A1 – A11 share l -Leu-6 and l -Ile-11. Therefore, the present strategy allows us to efficiently decipher biologically crucial residues and identify potentially useful agents for the treatment of infectious diseases.

Lysocin E is a lipopeptide with antibiotic activity against methicillin-resistant Staphylococcus aureus. For unclear reasons, the antibacterial activity of lysocin E in a mouse systemic infection model is higher than expected from in vitro results, and the in vitro activity is enhanced by addition of bovine serum. Here, we confirm that serum from various species, including humans, increases lysocin E antimicrobial activity, and identify apolipoprotein A-I (ApoA-I) as an enhancing factor. ApoA-I increases the antibacterial activity of lysocin E when added in vitro, and the antibiotic displays reduced activity in ApoA-I gene knockout mice. Binding of ApoA-I to lysocin E is enhanced by lipid II, a cell-wall synthesis precursor found in the bacterial membrane. Thus, the antimicrobial activity of lysocin E is potentiated through interactions with host serum proteins and microbial components.

The development of antimicrobials that exert therapeutic efficacy by novel mechanisms is crucial toward combatting multidrug-resistant bacteria. We previously reported a novel antibiotic, lysocin E, that is effective against methicillin-resistant Staphylococcus aureus (MRSA)1. Lysocin E exhibits more prominent therapeutic effects against S. aureus in a mouse systemic infection model compared with its basal antibacterial activity in vitro, but the detailed mechanism underlying this discrepancy was unclear. Here we show that serum or plasma from various species, including humans, increases the antimicrobial activity of lysocin E, and identified apolipoprotein A-I (ApoA-I) as an enhancing factor in the serum. Using gene knockout mice, we further revealed that ApoA-I contributes to the therapeutic effects of lysocin E. The binding capacity of lysocin E to ApoA-I was enhanced by lipid II, an intermediate component of S. aureus cell wall synthesis in the membrane, and ApoA-I enhanced the membrane-damaging activity of lysocin E at a sub-minimum inhibitory concentration (sub-MIC). Our results are the first to demonstrate that antimicrobial activity can be potentiated through interactions of host serum proteins with microbial components to enhance the therapeutic effect, which broadens the strategies for developing antimicrobials by taking advantage of host-microbe interactions.