Development of novel, daptomycin-inspired antimicrobial peptides using non-canonical amino acids - Abstract - Development of novel, daptomycin-inspired antimicrobial peptides using non-canonical amino acids The demand for novel antimicrobial compounds is a pressing challenge for society. However, due to bacterial resistance development and industrial challenges, the development of new antibacterial compounds has lagged far behind the current clinical needs. The proposed work aims to address this problem by developing novel, antimicrobial peptides based on an already clinically approved molecule, daptomycin, by improving the fundamental understanding of the sequence-structure-activity relationship and implementing this knowledge in novel molecules. Specifically, the goal is to develop peptide molecules which recapitulate the key components of daptomycin activity, i.e. calcium binding and lipid membrane disruption, while improving both the resistance to host degradation and synthetic processes required to create the molecules. This will be facilitated by incorporation of non-canonical amino acids into the novel sequences. Specifically, we will characterize the efficacy of novel peptides which mimic daptomycin’s calcium binding properties. The design of the peptides will start with a well characterized antimicrobial peptide and graft natural and de novo designed calcium binding sequences on to the backbone peptide. These experiments will entail the evaluation of antimicrobial activity using both laboratory and clinical isolate strains of bacteria. Using this information, we will investigate the response of different, clinically relevant bacteria to the peptides with the goal of optimizing efficacy. This will involve investigating bacterial transcriptional responses to the molecules and changes in membrane permeability. We will simultaneously characterize the biophysical properties of calcium binding and lipid interactions of the newly synthesized peptides. In parallel, we will also develop novel sequences that mimic the lipophilic tail of daptomycin, a key contributor to the membrane permeabilizing mechanism of action. These molecules will be characterized using similar approaches, with additional emphasis on evaluating the effects of these lipophilic modifications on peptide structure and aggregation in solution. We will screen a series of aromatic substitutions using non-canonical amino acids to improve protease resistance of the molecules. Finally, we will take the best performing sequences for calcium binding and membrane disruption and create and optimize hybrid peptides which can leverage the benefits realized in earlier stages. These sequences will be aggressively evaluated for efficacy against clinical isolates, potential for resistance development, and cytotoxicity in standard and co-culture models.
