Search

The quest for new approaches for generating novel bioactive designer proteins/peptides has continued with their success in various biomedical applications. Previously, we designed a 14-mer α-helical peptide with antimicrobial and antimycobacterial activities by employing a tandem repeat of the 7-mer, “KVLGRLV” human chemerin segment. Herein, we devised a new method of “sliding framework” with this segment to create amino acid scaffolds of varying sizes and sequences and explored the design of a peptide library with antibacterial and antimycobacterial activities. By utilizing 2 to 7 repeats of these 2 to 6-residue scaffolds, we designed and synthesized 30 peptides of 10–16 residue lengths. Thus, we identified novel AMPs with α-helical, β-sheet, and random coil structures, membrane-destabilizing, and intracellular modes of action, and 9 of them showed therapeutic indices between 100 and 750. Three and two of these nine peptides showed in vivoantibacterial and antitubercular efficacies against Escherichia coliATCC 25922 and Mycobacterium bovisBCG infections, respectively, in a mouse model.

To design novel antimicrobial peptides by utilizing the sequence of the human host defense protein, chemerin, a seven-residue amphipathic stretch located in the amino acid region, 109–115, was identified, which possesses the highest density of hydrophobic and positively charged residues. Although this 7-mer peptide was inactive toward microorganisms, its 14-mer tandem repeat (Chem-KVL) was highly active against different bacteria including methicillin-resistant Staphylococcus aureus, a multidrug-resistant Staphylococcus aureusstrain, and slow- and fast-growing mycobacterial species. The selective enantiomeric substitutions of its two l-lysine residues were attempted to confer cell selectivity and proteolytic stability to Chem-KVL. Chem-8dK with a d-lysine replacement in its middle (eighth position) showed the lowest hemolytic activity against human red blood cells among Chem-KVL analogues and maintained high antimicrobial properties. Chem-8dK showed in vivoefficacy against Pseudomonas aeruginosainfection in BALB/c mice and inhibited the development of resistance in this microorganism up to 30 serial passages and growth of intracellular mycobacteria in THP-1 cells.

Naturally occurring cationic antimicrobial peptides (AMPs) mostly adopt α-helical structures in bacterial membrane mimetic environments. To explore the design of novel β-sheet AMPs, we identified two short cationic amphipathic β-strand segments from the crystal structure of the innate immune protein, MyD88. Interestingly, of these, the 10-residue arginine–valine-rich synthetic MyD88-segment, KRCRRMVVVV (M3), exhibited β-sheet structure when bound to the outer membrane Gram-negative bacterial component, LPS. Isothermal titration calorimetric data showed that M3 bound to LPS with high affinity, and the interaction was hydrophobic in nature. Supporting these observations, computational studies indicated strong interactions of multiple and consecutive valine residues of M3 with the acyl chain of LPS. Moreover, M3 adopted nanosheet and nanofibrillar structure in 25% acetonitrile/water and isopropanol, respectively. M3 showed substantial antibacterial activities against both Gram-positive and Gram-negative bacteria which it appreciably retained in the presence of human serum and physiological salts. M3 was non-hemolytic against human red blood cells and non-cytotoxic to 3T3 cells up to 200 μM and to mice in vivo at a dose of 40 mg/kg. Furthermore, M3 neutralized LPS-induced pro-inflammatory responses in THP-1 cells and rat bone marrow-derived macrophages. Consequently, M3 attenuated LPS-mediated lung inflammation in mice and rescued them (80% survival at 10 mg/kg dose) against a lethal dose of LPS. The results demonstrate the identification of a 10-mer LPS-interacting, β-sheet peptide from MyD88 with the ability to form nanostructures and in vivo activity against LPS challenge in mice. The identified M3-template provides scope for designing novel bioactive peptides with β-sheet structures and self-assembling properties.

Bovine cathelicidin, BMAP‐28 exhibits potent antimicrobial activity without any cell‐selectivity. Considering the role of its C‐terminus amino acid residues in its cytotoxic activities, we envisioned to rearrange the amino acids at its C‐terminus without altering its composition for designing new cell‐selective BMAP‐28‐analog. Thus an isoleucine residue at 20th position, also an “a” position of its previously identified isoleucine zipper sequence, was exchanged with a proline residue at 19th position. As a result, a remarkable reduction in the hemolytic activity of BMAP‐28 toward human red blood cells (hRBCs) was observed without hampering its antibacterial activity in liquid media, and in presence of salts and serum. BMAP‐28‐analog, BMAP‐28M depolarized S. aureusmembrane and bacterial membrane‐mimetic, PE/PG lipid vesicles similar to BMAP‐28 but showed weaker efficacy to depolarize hRBC membrane and mammalian membrane‐mimetic, PC/Chol vesicles. Differences in the localization of tryptophan residues of BMAP‐28 and BMAP‐28M were observed only in PC/Chol vesicles. BMAP‐28 induced pores of ~3.4 nm diameter onto hRBCs whereas BMAP‐28M showed minor perturbation onto these cells. BMAP‐28 and BMAP‐28M induced the entry of calcein (size, ~1 nm) and 4.4 kDa FITC‐dextran (size, ~2.8 nm) into B. megateriumbut not of higher sized fluorescent‐dextrans indicating toroidal pore formation onto these bacterial membranes.