Abstract
Antimicrobial peptides play important roles in the host innate defense mechanism of many plants, insects, and mammals. Most antimicrobial peptides do not target specific molecular receptors of the pathogens, but rather interact and permeabilize microbial membranes. With an increase of antibiotic resistance within bacteria, the potential for the development of antimicrobial peptides as novel therapeutic agents could overcome the problem of resistance A new type of Trp-rich peptide, Ac-KWRRWVRWI-NH2, designated as Pac-525, was found to possess improved activity against both Gram positive and negative bacteria. The variety of biophysical and biochemical experiments, including circular dichroism, fluorescence spectroscopy and microcalorimetry, were used to show that Pac-525 interacted strongly with negatively charged phospholipid vesicles and induced efficient dye release from these vesicles. We have determined the solution structures of Pac-525 bound to membrane-mimetic SDS micelles. The SDS micelle-bound structure of Pac-525 adopts an □-helical segment at residues Trp2, Arg3 and Arg4. The positively charged residues are clustered together to form a hydrophilic patch. The three hydrophobic residues including Trp2, Val6, and Ile9 form a hydrophobic core. The surface electrostatic potential map indicates the three tryptophan indole rings are packed against the peptide backbone and form an amphipathic structure. Moreover, the reverse sequence of Pac-525, Ac-IWRVWRRWK-NH2, designated as Pac-525rev, and two Pac-525 diastereomers, D-Pac-525 and D-Nal-Pac-525, derived from Pac-525 are also potent antibacterial agents against Gram-positive and Gram-negative bacteria; furthermore D-Nal-Pac-525 is highly active for antifungi, but displayed high haemolysis. The solution structure of D-Nal-Pac-525 bound to DPC micelles forms an □-helical segment at residues Lys1, Nal2, Arg3, Arg4, Nal5, Val6 and Arg7 and displayed an apparent amphipathic conformation. All hydrophobic residues including Nal2, Nal5, Val6, Nal8 and Ile9 formed a hydrophobic core. Visualized images of the peptides and bacteria on the electron microscopy present that Nal-substituted and Trp-containing peptides, D- Nal-Pac-525, Pac-525 and D-Pac-525, might proceed through membrane via the different modes and they indeed interacted with the membrane of bacteria. Our results indicate that the chirality of Pac-525 did not play a role in antibacterial activity and the aromatic chain of □-naphthylalanine makes the peptide to change and add the specificity of eukaryotic membrane. Src homology 2 (SH2) domains play an important role in transduction pathways and Grb2 is required for cell transformation by the neu and bcr-abl oncogenes. Therefore, the Grb2 SH2 domain has been chosen as the target for developing the potential agents of breast cancer and other human cancers. Here, the nonphosphorylated peptide inhibitors, linear tetrapeptides and thioether cyclized peptides for Grb2 SH2 domain are presented. The inhibitory effect of linear tetrapeptides with the sequence Fmoc-X-Y-Aib-N-amide on Grb2 SH2 domain were estimated by surface plasmon resonance technology. Using two-dimensional NMR and simulated annealing methods to determine the solution structure of G1TE, a cyclized nonphosphorylated peptides. From the solution structure of G1TE, we suggest that it may from a large circle-like binding surface than the BCR-Abl phosphopeptide in the bound. Further we want to solve the complex structure of the Grb2 SH2 domain and G1TE(Gla1), a analogue of G1TE, and realize the role of cyclized nonphosphorylated peptide in its interaction with the Grb2 SH2. Until now, we are still solving the complex structure of G1TE(Gla1) and Grb2 SH2 domain by NMR and X-ray. These results reveal that the nonphosphorylated linear tetrapeptides are effective antagonists and the structure of thioether cyclized peptide provides a molecular basis understanding, these peptides will serve as the lead structure for the further design of potential antagonists of Grb2 SH2.