Investigation and application of heterochiral proteins enabled by flow-based peptide synthesis

Abstract

Natural proteins are comprised primarily of (L)-amino acids. (D)-amino acids are rare in protein structures. Solid-phase peptide synthesis (SPPS) and native chemical ligation enable the total chemical synthesis of proteins. Using these techniques, it is possible to design and study polypeptides foreign to Nature. Herein, I describe the investigation and application of proteins simultaneously comprised of (L)- and (D)-amino acids. SPPS has traditionally been a time intensive endeavor. Recently, the Pentelute laboratory described a flow-based system that reduces the time required to synthesize a polypeptide by over an order of magnitude. We have systematically studied variables that influence peptide quality with this system. From these efforts, we established protocols used in the synthesis of heterochiral polypeptides. We proceeded to study two different heterochiral systems. In the first system, we examined folding of (L)-proteins containing loops of (D)-amino acids, and vice-versa. Protein loops are important structural features that mediate protein-protein interactions. Using Ecballium elaterium trypsin inhibitor II (EETI-ll), we discovered that strategic incorporation of linkers such as P-alanine or glycine can facilitate efficient folding of heterochiral proteins. We used NMR spectroscopy and molecular dynamic simulations to interrogate the structure and folding pathway of one such protein that possesses a (D)-amino acid core and a loop with 5 (L)-amino acids, 2 P-alanine, and 1 glycine. We also determined that our heterochiral proteins were more resistant to proteolysis than natural proteins. In the second system, we examined heterochiral antibody-drug conjugates (ADCs) for the treatment of P. aeruginosa infection. New therapeutic modalities are needed to address bacterial resistance to conventional antibiotics. We developed a biologically expressed antibody that targets P. aeruginosa and bears antimicrobial peptides chemically synthesized from (D)-amino acids. In vivo studies demonstrated that heterochiral ADCs are effective at reducing the bacterial burden in a murine lung infection model. These ADCs bind a conserved glycan of P. aeruginosa lipopolysaccharide and directly kill the pathogen through a mechanism of membrane disruption. Our lead heterochiral ADC candidate is being advanced through additional pre-clinical studies.