טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentMarynka Keren
SubjectStructure Activity Relationship of Acylated Dermaseptin
S4 Derivatives
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Amram Mor


Abstract

To better understand the mechanism of action of antimicrobial peptides I designed and investigated a library of truncated and/or acylated analogs of the well characterized frog-derived antimicrobial peptide, dermaseptin S4. Truncation and acylation strategies were previously shown to individually improve various properties of antimicrobial peptides. In this study, I investigated the option of combining both strategies through gradual replacement of N-terminal residues of the 15-mer derivative K4S4(1-15) with various acyl chains differing in their length and hydrophobicity (heptanoic (C7) and dodecanoic (C12) acids as well as their aminated counterparts).

The parent peptide exhibited potent bactericidal activity as well as significant hemolytic activity. N-terminal truncation alone rapidly reduced all activities but acylation of the truncated peptides recovered activities even after deletion of up to seven residues. However, acylation significantly affected selectivity profiles. Decreasing hydrophobicity by conjugation of amino-acyls decreased hemolytic activity but also led to activity loss for the shortest derivatives. Interestingly, whereas amino acid sequence substitutions with acyl chains revealed short potent but unselective compounds, an 8-mer dodecanoyl derivative exhibited selectivity and specificity towards pseudomonas aeruginosa. Namely, out of 40 bacterial strains tested, the 8-mer acylated derivative C12-S4(8-15) displayed similar MICs as K4S4(1-15) only against pseudomonas aeruginosa sp. (identical MIC90 = 25 mM), but was practically inactive against most other bacteria. Moreover, hemolytic activity of C12-S4(8-15) was significantly reduced (e.g., 0.03% versus 12.45% hemolysis at 10 multiples of the MIC value). SPR experiments detected significant differences in binding properties to idealized membranes, indicating comparable binding affinity to a negatively charged membrane (Kapp 2.6 versus 8.6 x 105 M-1) whereas in presence of zwiterionic and cholesterol containing membrane, binding of K4S4(1-15) has increased (Kapp 1.8 x 108 M-1) while that of C12-S4(8-15) was reduced (Kapp 6.3 x 104 M-1). Additional mechanistic studies revealed that C12-S4(8-15) maintained secondary structure, bactericidal kinetics and ability to perturb cytoplasmic membrane, pointing to a similar mode of action as K4S4(1-15).

Overall, this combination strategy revealed economic derivatives with selective or wide-spectrum bactericidal activities. Similar studies may lead to safe compounds for systemic treatments of infectious diseases.