Beruflich Dokumente
Kultur Dokumente
327
INTRODUCTION The occurrence of fungal infections that have been seen in the growing populations of immunocompromised hosts, such as individuals infected with HIV, transplant recipients and patients with cancer, has increased dramatically in the last few decades [1]. Of the thousands of recognized fungal species, practically a couple, are pathogenic and produce mycotic infections in humans and animals. In tropical and subtropical developing countries, dermatophytes and Candida sp. can cause infections in humans and other animals, especially in immunocompromised patients. In addition, the number of reported cases of immunocompromised patients which often develop opportunistic and superficial mycoses, such as candidiasis, cryptococcosis and aspergillosis, has increased dramatically in recent years, especially in those with AIDS [2] The yeast fungus, Cryptococcus neoformans, has been identified as the fourth most common cause of lifethreatening infection in AIDS patients. Potentially fatal infections with Candida albicans and other species of Candida are also known [3]. According to Sardari and Dezfulian, the fungi have varied susceptibility to different antifungal agents [4]. In other words, the dearth of wide-spectrum antifungal agents is of great alarm to medical mycologists and practitioners. Long-term treatment with the commonly used antifungals such as amphotericin B has toxic effects on the patient; flucytosine and echinocandins have a restricted spectrum, while azoles may result in strain resistance [5]. Therefore, in the search for a substitute form of treatment for fungal infections, the last decade has seen a rise in novel approaches, such as therapeutic antibodies and peptide molecules. Antimicrobial peptides have newly become the focus of considerable interest as a candidate for a new type of antibiotic, primarily due to their potency against pathogenic mi*Address correspondence to this author at the Drug Design and Bioinformatics Unit, Department of Biotechnology, Pasteur Institute of Iran, #69 Pasteur Ave., Tehran, Iran 13164; Tel: (98-21) 6640-5535; Fax: (98-21) 6646-5132; E-mail: ssardari@hotmail.com;sardari@pasteur.ac.ir 1871-5214/09 $55.00+.00
crobes that are resistant to conventional antibiotics, as well as their broad-spectrum activity [6]. Since the first identification of cecropin [7] and defensin [8] in insect hemolymph and human neutrophils, respectively, several antimicrobial peptides have been isolated from a wide variety of organisms, including vertebrates, invertebrates, and plants [9]. Other than pathogen-lytic activities, these peptides have other properties like anti-tumor, mutagen activity, or act as signaling molecules [10]. In addition, they have a number of biotechnological applications, e.g. in transgenic plants [11], in aquaculture, and as aerosol spray for patients of cystic fibrosis [12]. Among AMPs, there is a group with considerable fungicidal effect referred to as antifungal peptides (AFPs). Antifungal and antimicrobial peptides are becoming the focus of interesting molecules among scientists, as they are vital components of the innate defense of all species, they kill very quickly, do not simply select resistant mutants and are synergistic with potentially toxic conventional therapeutic agents against microbes. Some of these agents have reached clinical trials, while others are undergoing detailed preclinical testing [13]. Therefore, the search continues for new antibiotics that are active in vivo, fast acting and broadspectrum, do not induce fungal and bacterial resistance and have limited side effects. In addition to the properties described above, they have low MICs and broad-spectrum activity in both low and high ionic strength conditions [14], neutralize lipopolysacharides [15], encourage injury healing [16] and have synergistic activity with conventional antibiotics [17]. Very few side effects have been reported. Synthetic congeners of natural antimicrobial peptides are good candidates. Synthetic congeners including a different series of peptides truncated successively from the carboxyl-terminal end of larger monomer and some analogs, which have lysine residues in place of two internal histidines or have a lysine added to the amino terminus of the original molecule. Not only synthetic congeners benefit from natural properties of antifungal peptides, but also with alteration in structural
Moradi et al.
characteristic of original antifungal peptides, their bioactivity would increase. ANTIFUNGAL PEPTIDES Classification of Antifungal Peptides There is a great diversity of anti-fungal peptides, with large variations in molecular mass, N-terminal sequence and antifungal specificity. Antifungal peptides are classified in several ways. Two major classification of antifungal peptide included classification by their mode of action and by the source. They will be described briefly here. According to the classification by the mode of action, AFPs are divided to the two groups. The first group acts by lysis, which occurs by means of several mechanisms. Lytic peptides can be amphipathic, that is, molecules with two faces, with one being positively charged and the other being neutral and hydrophobic. Some amphipathic peptides bind merely to the membrane surface and can disrupt the membrane structure without traversing the membrane. Others traverse membranes and interact distinctively with certain molecules. Lastly, other amphipathic peptides aggregate in a discriminating manner, forming aqueous pores of variable sizes, allowing passage of ions or other solutes [18]. The second peptide group interferes with cell wall synthesis or the biosynthesis of critical cellular components such as glucan or chitin [19]. An excellent review of lipopeptide antifungal agents affecting cell wall synthesis has been published previously [20]. Also Antifungal peptide can be classified according to their origin of isolation. Table 1 shows some antifungal peptide from different origin. Mode of Action of Antifungal Peptides The method of fungal cell lysis by peptides usually include non-specific interaction with the membrane phospholipids rather than binding to exact receptors on the cell membrane so that microorganisms develop resistance to antimicrobial peptides at rates that are orders of magnitude less than those observed for conventional antibiotics [21]. On the down side, the toxicities of many of the peptides and their fast rate of clearance from the circulation mean that topical or in vitro applications may be more appropriate than systemic administration [22]. Antifungal peptides are found in numerous organisms, ranging from fungi through mammals [23]. The majority of antifungal peptides constitute a simple structural motif; they are most commonly short (<40 residues) linear, cationic, amphipathic -helices. They exert antibacterial activity by cell membrane permeabilization and lysis, even though the accurate lytic mechanism has not been conclusively determined. Selectivity for the fungal cell appears to be mediated by favorable electrostatic interaction between positively charged peptides and the negatively charged cell surface [24]. However, an excessively hydrophobic peptide can bind arbitrarily to any cell membrane [25]. Antifungal peptide selectivity is dependent upon a precise balance of peptide hydrophobicity and electrostatic charge. In general, studies propose that the overall physicochemical parameters of antifungal peptides, rather than any specific receptorligand interactions, are responsible for antifungal activity [26]. As a result, antifungal peptides are attractive targets for biomimicry and peptidomimetic lead de-
velopment, as reproduction of critical peptide biophysical characteristics in an unnatural, sequence-specific mimicking the source oligomer should presumably be enough to donate antifungal efficacy, while circumventing the restrictions related to peptide pharmaceuticals [27]. Models of Peptide Interaction with Lipid Membranes According to the above-mentioned mode of action, a peptide which has lethality effect on fungi should be capable of forming ion channels in membrane by aggregation (pseudoionophores) and insertion in order to span cell membrane with 2.5-4.0 nm thickness (depending upon lipid composition) and should have at least 12 amino acids [28, 29]. Being laterally amphipathic meaning that one face of the helix displaying hydrophobic residues, while the opposite face displays hydrophilic residues, the peptide can form hydrophilic ion channels or pores and at the same time remain in contact with the hydrophobic components, e.g. fatty acyl moieties [30]. Based on these properties, two principal modes of action for membrane-perturbing peptides have been proposed: pore formation across the lipid bilayer or a "carpet" mechanism, lysing the membrane in a detergent-like manner. In the latter model, peptides "carpet" the surface of a target membrane and when sufficiently accumulated, create numerous pores [30]. The transmembrane model involves the peptides forming pores through the outer membrane: the "barrelstave" [31] and toroidal pore [32] mechanisms. In these models, the peptides oligomerize to form pores through the membrane. The pores act as non-selective channels for ions, toxins and metabolites, thus preventing the organism from maintaining homeostasis. Peptides with 20 or more amino acids lend themselves to these mechanisms, as they are able to span the lipid bilayer when in -helical conformation. A key difference between these two mechanisms is the positioning of the headgroup region of the lipid molecules with respect to the peptide. In the barrel-stave mechanism, the headgroups remain located along the membrane surface, while the pore is formed by the interaction of the peptide within the hydrophobic core of the membrane. The transmembrane pore is lined by the hydrophilic surface of the peptide. According to BSPM (Barrel-Stave Pore Model), AFPs should have distinct structure such as -helix or sheet or both and have hydrophobic interaction with the target membrane. By contrast, toroidal pores are formed when the peptides insert in such a way to cause the inner and outer membrane leaflets to curve and the lumen is lined by the hydrophilic surface of the peptide interspersed by the phospholipid headgroups. TPM (Toroidal Pore Model) is based on formation of several short-lived clusters of an undefined nature of secondary structure [75]. Preference for barrelstave vs. toroidal pore may depend on several factors including the peptide length and membrane thinning effect induced by the peptide [76]. Disadvantages of Antifungal Peptides The development of peptides as drugs is problematic as a result of poor oral and tissue absorption, rapid proteolytic cleavage and poor shelf-life or stability. Most proteins and small peptides are easily proteolyzed, rapidly excreted and poorly bioavailable. Much effort has been expended to find ways to replace portions of peptides with nonpeptide struc-
329
Table 1.
Peptide
Mammalian antifungal peptides Histatin 3 Halocidin Protegrins 1 Tritrptcin PAMP NP-3B SAMP29 Saliva of humans Halocynthia aurantium Human, porcine Human, porcine Human, porcine Rabbit Sheep 31 18 16 13 24 33 45 Lysis Lysis Lysis Lysis Unknown Lysis Lysis C. albicans C. albicans C. albicans A. flavus C. albicans A. fumigates C. albicans 75 10 3 250 23 100 4 [33] [34] [35] [36] [37] [38] [39]
Insect and amphibian antimicrobial peptides Melitin Cecropins B DSP Magainin 2 Drosomycin Tenecin Cicadin Esculentin-1 Brevin -2 Apis mellifera H. cecropia Leaf beetle Xenopus laevis D. melanogaster Inset defencin Juvenile cicadas Phyllomedusa sauvagii Phyllomedusa sauvagii 25 35 41 23 44 43 45 47 40 Lysis Lysis Unknown Lysis Lysis Lysis Lysis Lysis Lysis C. albicans A. fumigatus C. albicans C. albicans F. oxysporum C. albicans C. albicans C. albicans C. albicans 1.5 9.5 7 80 5 100 70 11 10 [40] [41] [42] [43] [44] [45,46] [47] [48] [49]
Bacterial and fungal antifungal peptides Anafp Fungicin M-4 HP Helioferin A Iturin A Leucinostatin A Nikkomycin X Polyoxin D Schizotrin A Trichopolyn A Pneumocandin A0 FR900403 CB-1 Aspergillus niger Bacillus licheniformis Helicobacter pylori Mycogone rosea Bacillus subtilis Phaeohelotium lilacinum Streptomyces tendae Streptomyces cacaoi Schizotrix sp. Trichoderma polysporum Zalerion arboricola 55 Cyclic peptide 11 Lipopeptide Lipopeptide Amino-lipopeptide Peptide-nucleoside Trinucleoside peptide Cyclic undecapeptide Amino-lipopeptide Lipopeptide Lysis Unknown Unknown Unknown Lysis Unknown Chitin synthesis Chitin synthesis Unknown Lysis Glucan synthesis C. albicans Mucor sp. C. albicans C. albicans S. cerevisiae C. neoformans C. immitis C. immitis C. albicans C. neoformans C. albicans 50 8.0 12.5 5.0 22. 0.5 0.125 0.125 0.02 0.78 0.1 [50] [51] [52] [53] [54] [55] [56] [57] [58] [59] [60]
Lipopeptide Lipopeptide
F. oxysporum F. oxysporum
0.4 50
[61] [62]
Plant antifungal peptides Zeamatin Zea mays 27 Lysis C. albicans 0.5 [63]
Moradi et al.
Source Allium cepa Passiflora edulis Red Bean Runner beans Green chickpea Yunnan bean Ceylon spinach seeds
Length 84 25 45 10 20 10 36
Synthetic and semisynthetic antifungal peptides LY303366 D4E1 MP1 Dhvar Cilofungin Lipopeptide Linear peptide Linear peptide Linear peptide Lipopeptide 6 cyclic 17 11 14 Lipopeptide Glucan synthesis Lysis Unknown Unknown Glucan synthesis Candida krusei A. flavus C. albicans C. albicans C. albicans 0.5 26 5 14 0.62 [70] [71] [72] [73] [74]
tures, called peptidomimetics, in the hope of obtaining more bioavailable entities [30]. Peptidomimetics can be seen as probes used in the transition pathway of small molecule drug design. Cyclization of the peptide backbone and its modification with aromatic residues constitutes an effective approach to mimic drug structures in the design process and circumvents obstacles associated with delivery and formulation of peptides. In the recent years [25] examples of mimicking pturn structures has led to combining design strategies with molecular libraries, demonstrating that peptidomimetics can provide valuable clues about receptor similarities not revealed by their endogenous ligands. PEPTIDOMIMETICS Recent efforts to reorganize disadvantageous peptide characteristics, and thus generate viable pharmaceutical therapies, have focused on the creation of non-natural peptide mimics. A peptidomimetic is a small protein-like chain designed to mimic a peptide which is often used in the literature to indicate a multitude of structural types that differ in fundamental ways. The term of peptidomimetic is often applied to highly modified analogs of peptides without distinguishing how these differ from classical analogs of peptides. These Non-natural, sequence-specific peptidomimetic oligomers are being designed to mimic bioactive peptides, with potential therapeutic application. These peptidomimetics can be based on any oligomer that mimics peptide primary structure through use of amide bond isosteres and/or modification of the native peptide backbone, including chain extension or heteroatom incorporation. Peptidomimetic oligomers are often protease-resistant, and may have reduced immunogenicity and improved bioavailability relative to peptide analogues [77]. In addition to primary structural mimicry, a select subset of the sequence-specific peptidomimetic oligomers, the so-called foldamers [78], exhibits well-defined
secondary structural elements such as helices, turns and small, sheet-like structures [79]. When peptide bioactivity is contingent upon a precise 3D structure, the capacity of a biomimetic oligomer to fold can be very important. They typically arise from modification of an existing peptide in order to alter the molecule's properties. For example, they may arise from modifications to change the molecule's stability or biological activity [80, 81]. This can have a role in the development of drug-like compounds from existing peptides. These modifications involve changes to the peptide that will not occur naturally (such as altered backbones and the incorporation of non-natural amino acids). Peptidomimetics are part of the wide effort by researchers, research labs and institutions to create cures for cancer by means of restoring or activating apoptotic pathways in specific cells. An example of peptidomimetics were those designed and synthesized with the purpose of binding to target proteins in order to induce cancer cells into a form of programmed cell death called apoptosis [82]. Basically, these work by mimicking key interactions that activate apoptotic pathway in the cell. The unfavorable pharmacokinetic properties associated with peptides when used as orally administered drugs can, in principle, be avoided by development of peptidomimetics. The general strategy when preparing peptidomimetics is to replace segments related to undesired properties with non-peptidic structures, while attempting to maintain the ability to elicit the same or improved biological response as the native peptide [83, 84]. Classification of Peptidomimetics Based on Rikpa and Rich classification [85], three most important classes of peptidomimetics have been described. The first type includes structures that mimic the local topography about an amide bond, i.e., amide bond isosteres, pyrrolinones or short portions of secondary structure ( turns).
331
The peptide backbone mimetics are also known as type-I mimetics. The first class is characterized by backbone changes, such as incorporation of amide bond isosteres and turn mimetics. The literature concerning peptidomimetics of class I including stabilized turn mimetics represented as bicycles [86, 87], aromatics [88, 89] and cyclic compounds [90]. These mimetics regularly match the peptide backbone atom-for-atom, while retaining functionality that makes significant contacts with binding sites. Latest studies paid attention to transition-state isosteres or collected substrate/product mimetics designed to mimic reaction pathway intermediates of the enzyme-catalyzed reactions. The reduced amide isostere developed by Szelke et al. [91] and the statine (hydroxylmethylene) isostere were early mimetics used to design inhibitors of a variety of aspartic proteases [92], and their success led to other tetrahedral intermediate mimics such as the hydroxylethylene and hydroxyethylamine isosteres. Cathepsin K, is new type-I peptidomimetic which is inhibitor of cysteine protease [86]. Two small-molecule inhibitors with an IC50 of 0.3 M and 0.013M have been designed lately, by Maibaum and co-workers [93, 94] which are orally active inhibitors of renin, the protease that catalyzes the first reaction in the renin-angiotensin system. The cysteine protease interleukin-lP-converting enzyme (ICE) inhibitor was designed by Dolle et al. [95] to generate potent aldehyde inhibitors of ICE. McKittrick et al. [96] designed a novel triple inhibitor of endothelin-converting enzyme (ECE), angiotensin-converting enzyme (ACE) and neutral endopeptidase (NEP) as a type-I peptidomimetic inhibitor. It has been shown that opportunistic fungi such as Candida albicans require N-myristoyltransferase (NMT) for viability. Brown et al. [97] created type-I mimetic compounds which are selective for C. albicans over human NMT. The second class of peptidomimetic is referred to as ligands exerting the same biological response as the native peptide ligand without any obvious structural resemblance (functional mimetics). The second type of mimetics is type-II or the functional mimetics, which are small nonpeptide molecules that binds to a peptide receptor. Initially these were presumed to be direct structural analogs of the natural peptide, but characterization of both the endogenous peptide and antagonist binding sites by site-directed mutagenesis [98] indicated that antagonists bind to subsites that are different from those used by the parent peptide for a large number of receptors. As a result, these functional mimetics do not essentially mimic the structure of the parent compound or hormone. The third class is represented by peptidomimetics with a nonpeptidic core structure, which position key functionalities for interactions with the receptor in a closely related way as the native peptide. Some examples from the vast literature [86] in the field are peptidomimetics of vasopressin [99], oxytocin [100], LHRH [101], somatostatin and angiotensin II [102, 103]. Type-III mimetics represent the idyllic peptidomimetics in that they possess novel templates which, though appearing unrelated to the original peptides, contain the necessary groups positioned on a novel nonpeptide scaffold to serve as topographical mimetics [104, 105]. At present, there is no systematic way to transform the structure of an enzyme-bound peptide substrate analog into a nonpeptide mimetic.
Type III peptidomimetics resemble the peptides, but employing a non-peptide scaffold to position key pharmacophores for receptor interaction, whereas type II mimetics, which are functional agonists or antagonists, do not structurally mimic the native peptide [106]. A wide variety of peptidomimetic building blocks have been developed in the past: e.g. peptoids [107], sulphonamides [108], ureapeptidomimetics [109], hydrazinopeptidomimetics [110], peptides [111], oligopyrrolinones [112], peptidosulfonamides [113], oligocarba-mates [114], oligoureas [110], azatides [111], and ethoxyformacetais [115] with unnatural oligoamide backbones which are self-organized at the molecular level to form stable helices useful to mimic protein secondary structure elements [116]. The predictability of folding of these oligomeric strands led to the development of molecules with functions including potent inhibitors of protein-protein interactions [117-119]. A set of novel type-III mimetics have been obtained and found to have activity towards Factor Xa with IC50=5 M [120]. Sall et al. and Chirgadze et al. [121, 122] designed Type-III peptidomimetic inhibitors of thrombin also. Another type-III mimetic was developed by a group that used pyranoses as templates to design thrombin inhibitors [98]. Some type-III mimetics of Inhibitors of Ras-farnesyltransferase (R-FT) have been developed by mimicking the carboxy-terminal CAAX motif which is the signal for farnesylation of Ras proteins [123125]. Such "unnatural biopolymers '' may have markedly different physicochemical proper-ties than natural peptides, including a superior pharmacological profile for development into therapeutic agents [126]. Finding unnatural oligoamide backbones can adopt well-defined and controlled helical secondary structures suggested that one could use them as scaffolds to distribute charged side chains in a predictable manner for de novo design of cationic amphiphilic molecules mimicking natural host defense -peptides. Antimicrobials based on either -peptides 314, 2.512, or 2.710,12 helical folds [127-131] or on peptoids polyproline type I-like [132] have been found to exhibit selective (non-hemolytic) and potent antibacterial activity against both Gram-positive and Gram-negative bacteria and fungi. Most antimicrobial peptidomimetics are typically discrete non-natural oligomers whose units are in many cases, connected via amide bonds. Much of the synthetic interest in peptidomimetics comes from the fact that these oligomers can present a wide variety of side chains which could be chemically identical to those found in natural peptides, but along an artificial backbone. The consequence of this hybrid structure is that peptidomimetics can mimic the conformation and functionality of biopolymers yet are not limited by the side chains of the main twenty naturally occurring -amino acid building blocks. Also the artificial backbone makes most peptidemimetics resistant to bio-degradation enzymes thus increasing the stability of peptidomimetic drugs in the body [133]. SPECIFICATIONS AND BIOACTIVITY OF PEPTIDOMIMETICS -Peptides -peptides consist of amino acids, which have their amino group bound to the carbon rather than the carbon
Moradi et al.
as in the 20 standard biological amino acids. The only commonly naturally occurring amino acid is -alanine. Although it is used as a component of larger bioactive molecules, -peptides in general do not appear in nature Fig. (1). Due to this reason, -peptide-based antibiotics are being explored as ways of evading antibiotic resistance. In amino acids, both the carboxylic acid group and the amino group are bound to the same carbon, termed the carbon (C ) because it is one atom away from the carboxylate group. In amino acids, the amino group is bound to the carbon (C ), which is found in most of the 20 standard amino acids. Only glycine lacks a carbon, which means that there is no glycine molecule. The chemical synthesis of amino acids can be challenging, especially given the diversity of functional groups bound to the carbon and the necessity of maintaining chirality. In alanine molecule, the carbon is achiral, however, larger amino acids have a chiral C atom. The -peptides are by far the best well-studied peptidomimetics. Because the backbones of -peptides are longer than those of peptides that consist of -amino acids, peptides form different secondary structures. The alkyl substituent at both the and positions in a amino acid favor a gauche conformation about the bond between the carbon and -carbon. This also affects the thermodynamic stability of the structure. -peptides are stable against proteolytic degradation in vitro and in vivo, an important advantage age over natural peptides in the preparation of peptide-based drugs [8]. -peptides have been used to mimic natural peptide-based antibiotics such as magainins, which are extremely powerful but difficult to use as drugs because they are degraded by proteolytic enzymes in the body [9].
H H N+ H CH2 H H C O O-
CH3 H H H C N+ H C O O-
Many types of helix structures consisting of -peptides have been reported [6]. These conformation types are distinguished by the number of atoms in the hydrogen-bound ring that is formed in solution; 8-helix, 10-helix, 12-helix, 14helix, and 10/12-helix. -peptides have more conformational freedom than -peptides, because of an additional methylene unit present in the polymer backbone. Consequently, whereas -peptide helices most commonly adopt the -helix conformation, -peptide sequences have been shown to adopt several distinct helical conformations, the choice of which depends largely upon the substitution pattern at backbone C and C atoms [134-136]. Of these, the -peptide 12helix and the 14-helix have been successfully employed as magainin mimics. The terms 12-helix and 14-helix correspond to the number of atoms participating in a ring created by intrachain hydrogen bonds. Recent studies in which peptides were designed to mimic the magainin have helped to illustrate which physical characteristics are critical for ideal antibacterial efficacy and biocompatibility in nonnatural oligomers. DeGrado and co-workers [137] first reported the de novo design of amphipathic, cationic, monosubstituted -peptide 14-helices as antibacterial compounds against the Gram-negative bacterium K91 Escherichia coli. Their study led to design of non-hemolytic analogues such as 1 in Fig. (3) [133]. Although potent antibiotics, these peptides displayed poor selectivity for bacteria, as indicated by extensive mammalian red blood cell lysis (haemolysis). Assuming that excessive side-chain hydrophobicity was responsible for the poor selectivity observed, Liu and colleagues [138] modified their original 14-helix designs to substitute a valine-like ( -HVal) residue with a less hydrophobic alanine-like ( -HAla) residue. In support of their original hypothesis, this modification abolished haemolytic activity, while retaining good antibacterial efficacy in both 12- and 15-residue oligomers. Generally, compounds that are not hemolytic at relative concentrations of their antimicrobial activity are typically deemed selective and have the potential for promising therapeutics that may kill microbial but not mammalian cells. In addition, hemolysis, by far the most common measure of mammalian toxicity, may not be the best predictor [139]. Simultaneously and independently, Seebach and Mathews [140] studied quite similar mono-substituted peptides, also designed to adopt the -peptide 14-helix for antimicrobial and hemolytic activity and demonstrated that "