Vibrational analysis of peptides, polypeptides, and proteins. XVIII. Conformational sensitivity of the α‐helix spectrum: α<sub>I</sub>‐ and α<sub>II</sub>‐Poly(<scp>L</scp>‐alanine)

Shahar

Eisenbach

et al. 2003

145

113

The high toxicity of most chemotherapeutic drugs and their inactivation by multidrug resistance phenotypes motivated extensive search for drugs with new modes of action. We designed a short cationic diastereomeric peptide composed of D-and L-leucines, lysines, and arginines that has selective toxicity toward cancer cells and significantly inhibits lung metastasis formation in mice (86%) with no detectable side effects. Its ability to depolarize the transmembrane potential of cancer cells at the same rate (within minutes) and concentration (3 M), at which it shows biological activity, suggests a killing mechanism that involves plasma membrane perturbation. Confocal microscopy experiments verified that the cells died as a result of acute injury, swelling, and bursting, suggesting necrosis. Biosensor binding experiments and attenuated total reflectance-Fourier transform infrared spectroscopy using model membranes have substantiated its high selectivity toward cancer cells. Although this is an initial study that looked at tumor formation rather than the ability of the peptides to reduce established tumors, the simple sequence of the peptide, its high solubility, substantial resistance to degradation, and inactivation by serum components might make it a good candidate for future anticancer treatment.Current anticancer chemotherapies that are based on alkylating agents, antimetabolites, and natural products respond incompletely; this is probably related to the development of drug resistance. Most chemotherapeutic agents also affect normal cells and consequently cause severe side effects (1). Thus, there is an urgent need to develop new classes of anticancer drugs with new modes of action that selectively target the cancer cells. A promising group under investigation includes cationic antimicrobial peptides, which are known to play an important role in the innate immunity of a diverse range of organisms, including insects, amphibians, and mammals (2). Most of these peptides have an amphipathic structure, and they preferentially bind and insert into negatively charged cell membranes. Consequently, destabilization of the membrane disturbs the electrolyte balance and induces the intracellular contents to leak, leading to cell death. In contrast to normal eukaryotic cells, which generally have low membrane potentials and whose outer leaflet almost exclusively consists of zwitterionic phospholipids, the prokaryotic and cancer cell membranes maintain large transmembrane potentials and have a higher content of anionic phospholipids on their outer leaflet. Many antimicrobial peptides therefore preferentially disrupt prokaryotic and cancer cell membranes rather than eukaryotic membranes (3).Several in vitro studies were conducted with native all L-amino acid antimicrobial peptides with defined ␣-helical or ␤-sheet secondary structures (3). However, the use of native antimicrobial peptides in vivo is mainly limited due to the loss of their function in serum, partially because of enzymatic degradation and binding to serum compone...

Section: Anticancer Activity Insupporting

confidence: 74%

A Novel Lytic Peptide Composed of dl-Amino Acids Selectively Kills Cancer Cells in Culture and in Mice

Shahar

Eisenbach

et al. 2003

145

113

“…In support of this, the amide I region from 1656 to 1670 cm Ϫ1 is characteristic of a 3 10 -helix or dynamic/distorted ␣-helix, as was previously suggested in a study that examined the structural changes in phospholipase A 2 (69). The assignment of the amide I region from 1656 to 1670 cm Ϫ1 to the dynamic helix is further supported by studies that examined distorted ␣-helical structures (a II -helices) in bacteriorhodopsin and other proteins (70,71). Similar structures were reported previously for a model amphipathic ␣-helical peptide and its diastereomer (25).…”

Section: Resultssupporting

confidence: 54%

A Molecular Mechanism for Lipopolysaccharide Protection of Gram-negative Bacteria from Antimicrobial Peptides

Shai

2005

223

218

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic ␣-helical antimicrobial peptide (K 5 L 7 ) and its diastereomer (4D-K 5 L 7 ) (containing four D-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K 5 L 7 binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K 5 L 7 is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection of bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.

“…The amide I region from 1656 to 1670 cm Ϫ1 is characteristic of 3 10 -helix or dynamic/distorted ␣-helix, as was previously suggested in a study that examined structural changes in phospholipase A 2 (48). The assignment of the amide I region from 1656 to 1670 cm Ϫ1 to dynamic helix is further supported by studies that examined distorted ␣-helical structures (␣ II -helices) in bacteriorhodopsin and other proteins (49,50). These studies demonstrated that distorted ␣-helical structures are characterized by increased amide I frequencies.…”

Section: Figsupporting

confidence: 65%

The Consequence of Sequence Alteration of an Amphipathic α-Helical Antimicrobial Peptide and Its Diastereomers

Ziv

Pag

et al. 2002

204

176

The search for antibiotics with a new mode of action led to numerous studies on antibacterial peptides. Most of the studies were carried out with L-amino acid peptides possessing amphipathic ␣-helix or ␤-sheet structures, which are known to be important for biological activities. Here we compared the effect of significantly altering the sequence of an amphipathic ␣-helical peptide (15 amino acids long) and its diastereomer (composed of both L-and D-amino acids) regarding their structure, function, and interaction with model membranes and intact bacteria. Interestingly, the effect of sequence alteration on biological function was similar for the L-amino acid peptides and the diastereomers, despite some differences in their structure in the membrane as revealed by attenuated total reflectance Fourier-transform infrared spectroscopy. However, whereas the all L-amino acid peptides were highly hemolytic, had low solubility, lost their activity in serum, and were fully cleaved by trypsin and proteinase K, the diastereomers were nonhemolytic and maintained full activity in serum. Furthermore, sequence alteration allowed making the diastereomers either fully, partially, or totally protected from degradation by the enzymes. Transmembrane potential depolarization experiments in model membranes and intact bacteria indicate that although the killing mechanism of the diastereomers is via membrane perturbation, it is also dependent on their ability to diffuse into the inner bacterial membrane. These data demonstrate the advantage of the diastereomers over their all L-amino acid counterparts as candidates for developing a repertoire of new target antibiotics with a potential for systemic use.The widespread use of antibiotics led to the development of numerous antibiotic-resistant strains, resulting in an urgent need for new antibiotics (1-4). The search for antibiotics with a new mode of action has increased the interest in antibacterial peptides as potential therapeutic agents. Isolation from biological sources (bacteria, insects, amphibians, mammals, etc.) has served as a common means for discovering novel antibacterial peptides such as gramicidins, magainins, defensins, cecropins, dermaseptins, and indolicidins (5-9). The amphipathic ␣-helix or ␤-sheet structures and a net positive charge are common characteristics found to be crucial for most native antibacterial peptides that act by perturbing the barrier function of membranes (3, 10 -12). These features were used as building blocks to modify native sequences as well as to create many de novo designed antimicrobial peptides (13)(14)(15)(16)(17)(18)(19)(20). Another important group of peptide antibiotics composed of both L-and D-amino acids includes gramicidins, actinomycins, bacitracin, lantibiotics, and bombinins H 3-5 (21-24). The coexistence of L-and D-amino acids causes the formation of unique structures and properties completely different from all L-amino acid peptides. Unfortunately, these peptide antibiotics are not cell-selective and are highly toxic to normal euk...