Structural determinants for the membrane interaction of novel bioactive undecapeptides derived from gaegurin 5.

Gaegurin 5 is a 24-residue, membrane-active antimicrobial peptide isolated from the skin of an Asian frog, Rana rugosa. We recently reported the antimicrobial activities of two novel undecapeptides derived from an inactive N-terminal fragment (residues 1-11) of gaegurin 5 (Won, et al. J. Biol. Chem. 2004, 279, 14784-14791). In the present work, the anticancer activities of the two antimicrobial undecapeptide analogues were additionally identified. The relationships between their structural properties and biological activities were assessed by characterizing the fundamental structural determinant for the basic membrane interaction. The circular dichroism and nuclear magnetic resonance results revealed that in a membrane-mimetic environment, the active peptides adopt a more stabilized helical conformation than that of the inactive fragment, and this conformation conferred an overall amphipathicity to the active peptides. Therefore, the most decisive factor responsible for the activity and selectivity could be the intramolecular amphipathic cooperativity, rather than the amphipathicity itself. Especially, the tryptophan residue of the active peptides seems to play a crucial role at the critical amphipathic interface that promotes and balances the amphipathic cooperativity by stabilizing both the hydrophilic and hydrophobic interactions with the membrane. Altogether, the present results suggest that the two novel undecapeptides are worthy of therapeutic development as new antibiotic and anticancer agents and provide structural information about their action mechanism.

Column-switching high-performance liquid chromatographic analysis of fluvastatin in rat plasma by direct injection.

A column-switching high-performance liquid chromatographic (HPLC) method has been developed and validated for quantification of fluvastatin in rat plasma. Plasma samples were diluted with an equal volume of mobile phase, i.e. acetonitrile-5 mM potassium phosphate buffer (pH 6.8) (15:85, v/v), and the mixture was directly injected onto the HPLC system. The analyte was enriched in a pre-treatment column, while endogenous components were eluted to waste. The analyte was then back-flushed onto an analytical column and quantified with fluorescence detection (lambdaex=305 nm; lambdaem=390 nm). The standard curve for the drug was linear in the range 0.5-100 ng mL(-1) in rat plasma. The limit of quantitation for plasma was found to be 0.5 ng mL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple and rapid because of a minimized sample preparation and appears to be useful for the pharmacokinetic study of fluvastatin.

Prediction of the permeability of drugs through study on quantitative structure-permeability relationship.

This study is to research the quantitative structure-permeability relationship of 20 drugs having similar structure. Permeability was determined by using the Caco-2 cell in vitro model. The apparent permeability coefficient (Papp) of each drug both of apical to basolateral side and basolateral to apical side was measured at the concentration corresponding to 0.1 times the highest dose strength of 250 mL dissolved buffer. In order to test the permeability system suitability, we measured the Papp of 19 model drugs out of 20 which presented in 'Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms based on the Biopharmaceutics Classification System' of FDA guidance. Also, we demonstrated the functional expression of efflux systems (e.g., p-gp) by bi-directional transport studies with rhodamine 123. Also, as a result of the study on quantitative structure-permeability relationship by using the partial least square method, it was possible to predict the permeability of drugs from their 3D structure. The quantitative structure-permeability relationship provided a cross-validated q2=0.789, a normal r2=0.998. Considering all of above results, analysis on this quantitative structure-permeability relationship appears to be a very useful tool to predict the permeability.

Column-switching high-performance liquid chromatographic method for the determination of zaltoprofen in rat plasma.

A direct injection column-switching high-performance liquid chromatography (HPLC) method was developed and validated for quantification of zaltoprofen in rat plasma. Following dilution with mobile phase A, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (12:88, v/v) samples were directly injected to the pre-column without sample pre-purification step. After endogenous plasma components were eluted to waste, the system was switched and the analyte was eluted to the trap column. Zaltoprofen was then back-flushed to the analytical column for separation with mobile phase B, i.e. acetonitrile-10mM potassium phosphate buffer (pH 6.8) (35:65, v/v) and quantification with an ultraviolet detector at 230 nm. The calibration curve was linear in the concentration range of 40-5000 ngmL(-1). This method has been fully validated and shown to be specific, accurate and precise. The method is simple, rapid and the sample preparation is minimal and appears to be useful for the pharmacokinetic study of zaltoprofen.

Backbone 1H, 15N, and 13C resonance assignment of HP1242 from Helicobacter pylori.

One of the small proteins from Helicobacter pylori, HP1242, was investigated by the solution nuclear magnetic resonance (NMR) spectroscopy. HP1242 is known as a 76-residue conserved hypothetical protein and its function cannot be identified based on sequence homology. Here, the results of the backbone (1)H, (15)N, and (13)C resonance assignments of the HP1242 are reported using double- and triple-resonance techniques. About 95 % of all of the (1)HN, (15)N, (13)CO, (13)Calpha, and (13)Cbeta resonances that cover 75 non-Proline residues of the 76 residues are clarified through sequential- and specific- assignments. In addition, three helical regions were clearly identified on the basis of the resonance assignments.

Structure-activity relationships of antimicrobial peptides from the skin of Rana esculenta inhabiting in Korea.

The anuran (frogs and toads) skin is a rich source of antimicrobial peptides that can be developed therapeutically. We searched the skin secretions of Korean Rana esculenta for antimicrobial peptides, and isolated two cationic peptides with antimicrobial activity and little hemolytic activity: a 46-residue peptide of the esculentin-1 family and a 24-residue peptide of the brevinin-1 family. Their sequences showed some differences from the esculentins-1 and brevinins-1 of European Rana esculenta, indicating that sequence diversification of anuran skin antimicrobial peptides can arise from differences in habitat as well as from species differences. The 46-residue peptide named esculentin-1c had broad antimicrobial activity, while the 24-residue peptide named brevinin-1Ed exhibited limited activity. The solution structure of brevinin-1Ed was in good agreement with that of other brevinin-1-like peptides, with an amphipathic alpha-helix spanning residues 3-20, stabilized in membrane-mimetic environments. The weak bioactivity of brevinin-1Ed was attributable to the unusual presence of an anionic amino acid in the middle of the helical hydrophilic face. This report contributes to world-wide investigations of the structure-activity relationships and evolutional diversification of anuran-skin antimicrobial peptides.

Systematic peptide engineering and structural characterization to search for the shortest antimicrobial peptide analogue of gaegurin 5.

As part of an effort to develop new, low molecular mass peptide antibiotics, we searched for the shortest bioactive analogue of gaegurin 5 (GGN5), a 24-residue antimicrobial peptide. Thirty-one kinds of GGN5 analogues were synthesized, and their biological activities were analyzed against diverse microorganisms and human erythrocytes. The structural properties of the peptides in various solutions were characterized by spectroscopic methods. The N-terminal 13 residues of GGN5 were identified as the minimal requirement for biological activity. The helical stability, the amphipathic property, and the hydrophobic N terminus were characterized as the important structural factors driving the activity. To develop shorter antibiotic peptides, amino acid substitutions in an inactive 11-residue analogue were examined. Single tryptophanyl substitutions at certain positions yielded some active 11-residue analogues. The most effective site for the substitution was the hydrophobic-hydrophilic interface in the amphipathic helical structure. At this position, tryptophan was the most useful amino acid conferring favorable activity to the peptide. The introduced tryptophan played an important anchoring role for the membrane interaction of the peptides. Finally, two 11-residue analogues of GGN5, which exhibited strong bactericidal activity with little hemolytic activity, were obtained as property-optimized candidates for new peptide antibiotic development. Altogether, the present approach not only characterized some important factors for the antimicrobial activity but also provided useful information about peptide engineering to search for potent lead molecules for new peptide antibiotic development.