RESUMEN
Membrane disruptive α-helical antimicrobial peptides (AMPs) offer an opportunity to address multidrug resistance; however, most AMPs are toxic and unstable in serum. These limitations can be partly overcome by introducing D-residues, which often confers protease resistance and reduces toxicity without affecting antibacterial activity, presumably due to lowered α-helicity. Here, we investigated 31 diastereomers of the α-helical AMP KKLLKLLKLLL. Three diastereomers containing two, three, and four D-residues showed increased antibacterial effects, comparable hemolysis, reduced toxicity against HEK293 cells, and excellent serum stability, while another diastereomer with four D-residues additionally displayed lower hemolysis. X-ray crystallography confirmed that high or low α-helicity as measured by circular dichroism indicated α-helical or disordered structures independently of the number of chirality switched residues. In contrast to previous reports, α-helicity across diastereomers correlated with both antibacterial activity and hemolysis and revealed a complex relationship between stereochemistry, activity, and toxicity, highlighting the potential of diastereomers for property optimization.
Asunto(s)
Péptidos Antimicrobianos , Hemólisis , Humanos , Células HEK293 , Estructura Secundaria de Proteína , Antibacterianos/farmacología , Antibacterianos/química , Dicroismo Circular , Pruebas de Sensibilidad MicrobianaRESUMEN
The peptide α-helix is right-handed when containing amino acids with l-chirality, and left-handed with d-chirality, however mixed chirality peptides generally do not form α-helices unless a helix inducer such as the non-natural residue amino-isobutyric acid is used. Herein we report the first X-ray crystal structures of mixed chirality α-helices in short peptides comprising only natural residues as the example of a stapled bicyclic and a linear membrane disruptive amphiphilic antimicrobial peptide (AMP) containing seven l- and four d-residues, as complexes of fucosylated analogs with the bacterial lectin LecB. The mixed chirality α-helices are superimposable onto the homochiral α-helices and form under similar conditions as shown by CD spectra and MD simulations but non-hemolytic and resistant to proteolysis. The observation of a mixed chirality α-helix with only natural residues in the protein environment of LecB suggests a vast unexplored territory of α-helical mixed chirality sequences and their possible use for optimizing bioactive α-helical peptides.
RESUMEN
Herein, we report X-ray crystal structures of 11-13 residue antimicrobial peptides (AMPs) active against Pseudomonas aeruginosa as complexes of fucosylated d-enantiomeric sequences with the P. aeruginosa lectin LecB. These represent the first crystal structures of short AMPs. In 24 individual structures of eight different peptides, we found mostly α-helices assembled as two-helix or four-helix bundles with a hydrophobic core and cationic residues pointing outside. Two of the analogs formed an extended structure engaging in multiple contacts with the lectin. Molecular dynamics (MD) simulations showed that α-helices are stabilized by bundle formation and suggested that the N-terminal acyl group present in the linker to the fucosyl group can extend the helix by one additional H-bond and increase α-helix amphiphilicity. Investigating N-terminal acylation led to AMPs with equivalent and partly stronger antibacterial effects compared to the free peptide.
Asunto(s)
Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/química , Proteínas Bacterianas/metabolismo , Lectinas/metabolismo , Pseudomonas aeruginosa/efectos de los fármacos , Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/química , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Lectinas/química , Simulación de Dinámica Molecular , Unión ProteicaRESUMEN
We used the concept of chemical space to explore a virtual library of bicyclic peptides formed by double thioether cyclization of a precursor linear peptide, and identified an antimicrobial bicyclic peptide (AMBP) with remarkable activity against several MDR strains of Acinetobacter baumannii and Pseudomonas aeruginosa.