Chemical-biology based approaches to discovering and characterizing antimicrobial peptides

Abstract

As more and more bacteria become resistance against the drugs currently used in the clinic we are in dire need of novel antimicrobial compounds. To prevent resistance to occur against novel compounds it is important to carefully select the target against which such novel compounds act. Lipid II is an essential component in the synthesis of the bacterial cell wall and nature itself has proven that it is an effective target for antibacterial purposes. Several naturally occurring antibiotics bind to lipid II and are often not susceptible to resistance. However, these naturally occurring compounds are often not suitable for use in humans. The complex structure of such compounds prevents optimization of the molecule for therapeutic use. Therefore we set out to identify novel peptides that bind to lipid II which can potentially be developed into novel drugs. By using carbohydrate and peptide chemistry we synthesized analogues of lipid II that were suitable for use in phage display experiments. Phage display is an established and potent technique to identify binding peptides from a vast peptide library. As lipid II is a small molecule compared to the proteins that are commonly used in phage display, optimization of the screening conditions was first required. When the optimal conditions were found we analyzed the output of the phage display experiment by high-throughput DNA analysis. The obtained peptide sequences were synthesized and tested for their antibiotic activity. The activity assays led to the identification of three lead peptides that showed a lipid II mediated antibiotic effect against indicator organisms. To further optimize the potency of our lead peptides we modified them either N- or C-terminally with a lipid. The modification led to a significant increase in potency (8-64 fold) and more interestingly one of the compounds was now also effective against clinically relevant resistant enterococci. The lead peptide was further analyzed to prove its mode of action was through sequestration of lipid II. The phage display strategy we designed allowed the identification of novel lipid II binding antibiotic peptides from large libraries. By changing the library or make modifications to the target a large chemical space can quickly be searched for novel antibiotics.