θ-Defensins: Cyclic Peptides with Endless Potential

Abstract

-Defensins, the only cyclic peptides of animal origin, have been isolated from the leukocytes of rhesus macaques and baboons. Their biogenesis is unusual because each peptide is an 18-residue chimera formed by the head-to-tail splicing of nonapeptides derived from two separate precursors. -Defensins have multiple arginines and a ladder-like tridisulfide array spanning their two antiparallel ␤-strands. Human -defensin genes contain a premature stop codon that prevents effective translation of the needed precursors; consequently, these peptides are not present in human leukocytes. Synthetic -defensins with sequences that correspond to those encoded within the human pseudogenes are called retrocyclins. Retrocyclin-1 inhibits the cellular entry of HIV-1, HSV, and influenza A virus. The rhesus -defensin RTD-1 protects mice from an experimental severe acute respiratory syndrome coronavirus infection, and retrocyclin-1 protects mice from infection by Bacillus anthracis spores. The small size, unique structure, and multiple host defense activities of -defensins make them intriguing potential therapeutic agents.

The Cyclic Cystine Ladder in θ-Defensins Is Important for Structure and Stability, but Not Antibacterial Activity

“…The three-dimensional structures are consistent with the secondary H␣ chemical shifts (Fig. 2b) 3,5,7,12,14,16 ]BTD-2, the lack of angle, disulfide bond, and hydrogen bond restraints resulted in a poorly defined structure. The two turns of the acyclic analogue BTD-2 [3,4] are well defined in themselves, but the break in the backbone allows the two hairpin structures to rotate relative to each other.…”

“…2a shows the disulfide arrangements of the native BTD-2 -defensin, the disulfide mutants, and the acyclic analogue that were synthesized by solid phase peptide synthesis. In the disulfide mutants, ␣-aminobutyric acid was chosen to replace cysteine because it has steric properties similar to cysteine (33,34) 3,5,7,12,14,16 ]BTD-2 was cyclized using a peptide coupling reaction with HBTU and diisopropylethylamine in dimethylformamide (1), but the yield of cyclic peptide was lower than that of the peptides cyclized by native chemical ligation. The acyclic analogue BTD-2 [3,4] was synthesized by Fmoc solid phase peptide synthesis and oxidized in NH 4 HCO 3 buffer to form the three disulfide bonds.…”

“…The structures, stabilities, and antibacterial activities of the -defensin analogues were then compared to determine the role of the disulfide bonds and cyclic backbone. 3,5,7,12,14,16 ]BTD-2, and a linear analogue, l-BTD-2, were synthesized on a 0.25-mmol scale by manual solid phase peptide synthesis using tert-butyloxycarbonyl (Boc)-chemistry as described previously (8). The acyclic -defensin analogue, BTD-2 [3,4], in which the cyclic peptide backbone was broken between residues Cys 3 and Arg…”

“…Whereas ribosomally synthesized cyclic peptides from plants, fungi, and bacteria are products of single genes (5,6), -defensins are biosynthesized by the head-to-tail ligation of two separate translation products (1). Although the biosynthetic mechanism of -defensins has not yet been elucidated, several mutants have been chemically synthesized and are being developed as therapeutics (7).…”

“…Electrospray mass spectrometry was used to confirm the mass and purity of the peptides. Cyclization by native chemical ligation (23) 3,5,7,12,14,16 ]BTD-2 was achieved by stirring with 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and diisopropylethylamine in dimethylformamide overnight. All of the -defensin analogues were then further purified by RP-HPLC, using a 0.5%/min gradient of 90% acetonitrile in 0.05% aqueous TFA with absorbance detection at 215 nm, and lyophilized.…”