Selective phenylalanine to proline substitution for improved antimicrobial and anticancer activities of peptides designed on phenylalanine heptad repeat

• Published in: Acta biomaterialia 2017-07, Vol.57, p.170-186
• Main Authors: Tripathi, Amit Kumar, Kumari, Tripti, Tandon, Anshika, Sayeed, Mohd, Afshan, Tayyaba, Kathuria, Manoj, Shukla, P.K., Mitra, Kalyan, Ghosh, Jimut Kanti
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Amino Acid Substitution; Animals; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - pharmacology; Antimicrobial Cationic Peptides - chemistry; Antimicrobial Cationic Peptides - pharmacology; Antimicrobial peptides; Antineoplastic Agents - chemistry; Antineoplastic Agents - pharmacology; Apoptosis; Breast Neoplasms - drug therapy; Breast Neoplasms - metabolism; Breast Neoplasms - pathology; Escherichia coli - growth & development; Female; Humans; Intrinsic pathway of apoptosis; Lipopolysaccharide; Mice; Phenylalanine - chemistry; Phenylalanine - genetics; Phenylalanine zipper; Proline - chemistry; Proline - genetics; RAW 264.7 Cells; Repetitive Sequences, Amino Acid; THP-1 Cells
• ISSN: 1742-7061; 1878-7568
• DOI: 10.1016/j.actbio.2017.05.007

[Display omitted] Introducing cell-selectivity in antimicrobial peptides (AMPs) without compromising the antimicrobial and anti-endotoxin properties is a crucial step towards the development of new antimicrobial agents. A peptide designed on phenylalanine heptad repeat possesses significant cytotoxicity along with desired antimicrobial and anti-endotoxin properties. Amino acid substitutions at ‘a’ and/or ‘d’ positions of heptad repeats of AMPs could alter their helical structure in mammalian membrane-mimetic environments and cytotoxicity towards mammalian cells. Since proline is a helix breaker, effects of selective proline substitution(s) at ‘a’ and/or ‘d’ positions of a 15-residue peptide designed on phenylalanine heptad repeat (FR-15) were investigated. Proline-substituted FR-15 variants were highly selective toward bacteria and fungi over hRBCs and murine 3T3 cells and also retained their antibacterial activities at high salt, serum and elevated temperatures. These non-cytotoxic variants also inhibited LPS-induced production of pro-inflammatory cytokines/chemokines in human monocytes, THP-1, RAW 264.7 and in BALB/c mice. The two non-cytotoxic variants (FR8P and FR11P) showed potent anti-cancer activity against highly metastatic human breast cancer cell line MDA-MB-231 with IC50 values less than 10μM. At sub-IC50 concentrations, FR8P and FR11P also showed anti-migratory and anti-invasive effects against MDA-MB-231 cells. FR8P and FR11P induced cellular apoptosis by triggering intrinsic apoptotic pathway through depolarization of mitochondrial membrane potential and activation of caspases. Overall the results demonstrated the utilization of selective phenylalanine to proline substitution in a heptad repeat of phenylalanine residues for the design of cell-selective, broad-spectrum AMPs with significant anti-cancer properties. We have demonstrated a methodology to design cell-selective potent antimicrobial and anti-endotoxin peptides by utilizing phenylalanine zipper as a template and replacement of phenylalanine residue(s) from “a” and/or “d” position(s) with proline residue(s) produced non-cytotoxic AMPs with improved antibacterial properties against the drug-resistant strains of bacteria. The work showed that the ‘a’ and ‘d’ positions of the phenylalanine heptad repeat could be replaced by an appropriate amino acid to control cytotoxicity of the peptide without compromising its potency in antimicrobial and anti-endotoxin properties. The direct bacterial