Antipathogenic peptides and compositions comprising them

Details Antipathogenic peptides and compositions comprising them Antipathogenic peptides and compositions comprising them

1998-12-08

2001-01-09

Russel, Jeffrey E. (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S012200, C514S013800, C514S014800, C514S021800, C530S300000, C530S324000, C530S326000, C530S327000, C530S345000

Reexamination Certificate

active

06172038

ABSTRACT:

FIELD OF THE INVENTION
The present invention concerns novel non-hemolytic cytolytic peptides, compositions comprising them and their use in the treatment of diseases or disorders and in agriculture.
BACKGROUND OF THE INVENTION
In the text below, reference is being made to prior art documents, the complete particulars of which can be found in the “References” section at the end of the specification before the claims.
The increasing resistance of microorganisms to the available antimicrobial drugs has resulted in extensive studies focused on developing alternative antimicrobial compounds.
In addition, or complementary, to the highly specific cell-mediated irmune response, vertebrates and other organisms have a defense system made up of distinct groups of broad spectrum cytolytic, e.g., antibacterial, peptides.
Studies on lipid-peptide interactions of such cytolytic peptides, also known as cytolysins, tend to emphasize the importance of the amphipathic &agr;-helical structure for their cytolvtic activity. This conclusion is based mainly on studies with cytolysins that act on either mammalian cells or bacteria alone or on both types of cells. A major group of cytolytic peptides in this family are host-defense short linear peptides (≦40 amino acids), which are devoid of disulfide bridges (Boman, 1995). These peptides vary considerably in chain length, hydrophobicity and overall distribution of charges, but share a common structure upon association with lipid bilayers, namely, an amphipathic &agr;-helix structure (Segrest et al., 1990).
Examples of known cytolysins include: (i) antibacterial peptides that are cytolytic to bacteria only, e.g. cecropins, isolated from the cecropia moth (Steiner et al., 1981), magainins (Zasloff, 1987) and dermaseptins (Mor et al., 1991) isolated from the skin of frogs; (ii) cytolysins that are selectively cytotoxic to mammalian cells, such as &dgr;-hemolysin isolated from
Staphylococcus aureus
(Dhople and Nagaraj, 1993); and (iii) cytolysins that are not cell-selective, such as the bee venom melittin (Habermann and Jentsch, 1967) and the neurotoxin pardaxin (Shai et al., 1988) that lyse both mammalian cells and bacteria.
Antibacterial peptides were initially discovered in invertebrates, and subsequently in vertebrates, including humans. As a complementary or additional defense system, this secondary, chemical immune system provides organisms with a repertoire of small peptides that are synthesized promptly upon induction, and which act against invasion by occasional and obligate pathogens as well as against the uncontrolled proliferation of commensal microorganisms (Boman, 1995). So far, more than 100 different antibacterial peptides have been isolated and characterized. The largest family, and probably the most studied, includes those peptides that are positively charged and adopt an amphipathic &agr;-helical structure. Numerous studies conducted on various native antibacterial peptides tend to emphasize the importance of an amphipathic &agr;-helical structure and a net positive charge for cytolytic activity. The positive charge facilitates interaction of the peptides with the negatively-charged membranes (Andreu et al., 1985) found in higher concentrations in the pathogenic cell membrane as compared to normal eukaryotic cells, and the amphipathic &agr;-helical structure is essential for lytic activity (Chen et al., 1988). Such interactions have been proposed to destroy the energy metabolism of the target organism by increasing the permeability of energy-transducing membranes (Okada and Natori, 1984). Because of their amphipathic structure, it has been suggested that these antibacterial peptides permeate the membrane by forming ion channels/pores via a “barrel-stave” mechanism (Rizzo et al., 1987). According to this model transmembrane amphiphilic &agr;-helices form bundles in which outwardly-directed hydrophobic surfaces interact with the lipid constituents of the membrane, while inwardly facing hydrophilic surfaces produce a pore. Alternatively, the peptides bind parallel to the surface of the membrane, cover the surface of the membrane in a “carpet”-like manner and dissolve it like a detergent (Shai, 1995).
Despite extensive studies, the exact mode of action of short linear non cell-selective peptides, such as pardaxin and melittin, is not known yet, and it is not clear whether similar structural features are required for their cytbtoxicity towards mammalian cells and bacteria.
Pardaxin, a 33-mer peptide, is an excitatory neurotoxin that has been purified from the Red Sea Moses Sole
Pardachirus marmoratus
(Shai et al., 1988) and from the Peacock Sole of the western Pacific
Pardachirus pavoninus
(Thompson et al., 1986). Pardaxin possesses a variety of biological activities depending upon its concentration (reviewed in Shai, 1994). At concentrations below 10
−7
M, pardaxin induces the release of neurotransmitters in a calcium-dependent manner. At higher concentrations of 10
−7
M to 10
−5
M, the process is calcium-independent, and above 10
−5
M cytolysis is induced. Pardaxin also affects the activities of various physiological preparations in vitro. Its biological roles have been attributed to its interference with the ionic transport of the osmoregulatory system in epithelium and to presynaptic activity by forming ion channels that are voltage dependent and slightly selective to cations. A “barrel-stave” mechanism for insertion of pardaxin into membranes was proposed on the basis of its structure and various biophysical studies (reviewed in Shai, 1994). Pardaxin has a helix-hinge-helix structure: the N-helix includes residues 1-11 and the C-helix includes residues 14-26. The helices are separated by a proline residue situated at position 13. This structural motif is found both in antibacterial peptides that can act specifically on bacteria (e.g., cecropin), and in cytotoxic peptides that can lyse a variety of cells (e.g., melittin).
Melittin, a 26-mer amphipathic peptide, is the major component of the venom of the honey bee
Apis mellifera
(Habermann and Jentsch, 1967) and is one of the most studied membrane-seeking peptides (Dempsey, 1990). Melittin is highly cytotoxic for mammalian cells, but is also a highly potent antibacterial agent (Steiner et al., 1981). Numerous studies have been undertaken to determine the nature of the interaction of melittin with membranes, both with the aim of understanding the molecular mechanism of melittin-induced hemolysis and as a model for studying the general features of structures of membrane proteins and interactions of such proteins with phospholipid membranes. Much of the currently described evidence indicates that different molecular mechanisms may underlie different actions of melittin. Nevertheless, the amphipathic &agr;-helical structure has been shown to be a prerequisite for its various activities (Perez et al., 1994).
The structure of melittin has been investigated using various techniques. The results of X-ray crystallography and NMR in methanolic solutions indicate that the molecule consists of two &agr;-helical segments (residues 1-10 and 13-26) that intersect at an angle of 120°. These segments are connected by a hinge (11-12) to form a bent &agr;-helical rod with the hydrophilic and hydrophobic sides facing opposite directions. Four such monomeric melittin molecules cluster together, through hydrophobic interactions, to form a tetramer (Anderson et al., 1980; Bazzo et al., 1988; Terwilliger and Eisenberg, 1982; Terwilliger and Eisenberg, 1982). Upon initial interaction with membrane surfaces, it has been found that the tetramer dissociates to monomers, which retain &agr;-helical conformation prior to insertion into the membrane (Altenbach and Hubbell, 1988).
Melittin shares some similarities with pardaxin. Both pardaxin and melittin are composed of two helices with a proline hinge between them. Furthermore, they exhibit significant homology in their N-helices, which are mostly hydrophobic (Thompson et al., 1986). However, pardaxin (net charge +1) contains

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