Role of helicity of α-helical antimicrobial peptides to improve specificity

A major barrier to the use of antimicrobial peptides as antibiotics is the toxicity or ability to lyse eukaryotic cells. In this study, a 26-residue amphipathic α-helical antimicrobial peptide A12L/A20L (Ac-KWKSFLKTFKSLK KTVLHTLLKAISS-amide) was used as the framework to design a series of D- and L-diastereomeric peptides and study the relationships of helicity and biological activities of α-helical antimicrobial peptides. Peptide helicity was measured by circular dichroism spectroscopy and demonstrated to correlate with the hydrophobicity of peptides and the numbers of D-amino acid substitutions. Therapeutic index was used to evaluate the selectivity of peptides against prokaryotic cells. By introducing D-amino acids to replace the original L-amino acids on the non-polar face or the polar face of the helix, the hemolytic activity of peptide analogs have been significantly reduced. Compared to the parent peptide, the therapeutic indices were improved of 44-fold and 22-fold against Gram-negative and Grampositive bacteria, respectively. In addition, D- and L-diastereomeric peptides exhibited lower interaction with zwitterionic eukaryotic membrane and showed the significant membrane damaging effect to bacterial cells. Helicity was proved to play a crucial role on peptide specificity and biological activities. By simply replacing the hydrophobic or the hydrophilic amino acid residues on the non-polar or the polar face of these amphipathic derivatives of the parent peptide with D-amino acids, we demonstrated that this method could have excellent potential for the rational design of antimicrobial peptides with enhanced specificity.     

Improved proteolytic stability of chicken cathelicidin-2 derived peptides by D-amino acid substitutions and cyclization

A truncated version of host defense peptide chicken cathelicidin-2, C1-15, possesses potent, broad spectrum antibacterial activity. A variant of this peptide, F_2,5,12W, which contains 3 phenylalanine to tryptophan substitutions, possesses improved antibacterial activity and lipopolysaccharide (LPS) neutralizing activity compared to C1-15. In order to improve the proteolytic resistance of both peptides we engineered novel chicken cathelicidin-2 analogs by substitution of l- with d-amino acids and head-to-tail cyclization. Both cyclic and d-amino acid variants showed enhanced stability in human serum compared to C1-15 and F_2,5,12W. The d-amino acid variants were fully resistant to proteolysis by trypsin and bacterial proteases. Head-to-tail cyclization of peptide F_2,5,12W resulted in a 3.5-fold lower cytotoxicity toward peripheral blood mononuclear cells. In general, these modifications did not influence antibacterial and LPS neutralization activities. It is concluded that for the development of novel therapeutic compounds based on chicken cathelicidin-2 d-amino acid substitutions and cyclization must be considered. These modifications increase the stability and lower cytotoxicity of the peptides without altering their antimicrobial potency.     

Effects of single D-amino acid substitutions on disruption of beta-sheet structure and hydrophobicity in cyclic 14-residue antimicrobial peptide analogs related to gramicidin S.

Gramicidin S (GS) is a 10-residue cyclic beta-sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure-activity studies of de novo-designed 14-residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single L-amino acid with its corresponding D-enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274, 13181]. The diastereomer with a D-Lys substituted at position 4 caused the greatest improvement in specificity vs. other L to D substitutions within the cyclic 14-residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted beta-sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275, 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a D- or L-amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the D- and L-substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic D-amino acids D-Lys and D-Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the L-diastereomers, which demonstrated strong hemolysis. All of the L-substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the D-substituted peptides tended to improve based on the hydrophilicity of the residue. D-Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic D-amino acid substitutions had superior antimicrobial activity vs. the L-enantiomers although substitution of a hydrophobic D-amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted beta-sheet structure on antimicrobial activity.     

Rational design of alpha-helical antimicrobial peptides with enhanced activities and specificity/therapeutic index

In the present study, the 26-residue peptide sequence Ac-KWKSFLKTFKSAVKTVLHTALKAISS-amide (V681) was utilized as the framework to study the effects of peptide hydrophobicity/hydrophilicity, amphipathicity, and helicity (induced by single amino acid substitutions in the center of the polar and nonpolar faces of the amphipathic helix) on biological activities. The peptide analogs were also studied by temperature profiling in reversed-phase high performance liquid chromatography, from 5 to 80 degrees C, to evaluate the self-associating ability of the molecules in solution, another important parameter in understanding peptide antimicrobial and hemolytic activities. A higher ability to self-associate in solution was correlated with weaker antimicrobial activity and stronger hemolytic activity of the peptides. Biological studies showed that strong hemolytic activity of the peptides generally correlated with high hydrophobicity, high amphipathicity, and high helicity. In most cases, the D-amino acid substituted peptides possessed an enhanced average antimicrobial activity compared with L-diastereomers. The therapeutic index of V681 was improved 90- and 23-fold against Gram-negative and Gram-positive bacteria, respectively. By simply replacing the central hydrophobic or hydrophilic amino acid residue on the nonpolar or the polar face of these amphipathic derivatives of V681 with a series of selected D-/L-amino acids, we demonstrated that this method has excellent potential for the rational design of antimicrobial peptides with enhanced activities.     

Buforins: Histone H2A-derived antimicrobial peptides from toad stomach

Antimicrobial peptides (AMPs) constitute an important component of the innate immune system in a variety of organisms. Buforin I is a 39-amino acid AMP that was first isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. Buforin II is a 21-amino acid peptide that is derived from buforin I and displays an even more potent antimicrobial activity than its parent AMP. Both peptides share complete sequence identity with the N-terminal region of histone H2A that interacts directly with nucleic acids. Buforin I is generated from histone H2A by pepsin-directed proteolysis in the cytoplasm of gastric gland cells. After secretion into the gastric lumen, buforin I remains adhered to the mucous biofilm that lines the stomach, thus providing a protective antimicrobial coat. Buforins, which house a helix-hinge-helix domain, kill a microorganism by entering the cell without membrane permeabilization and thus binding to nucleic acids. The proline hinge is crucial for the cell penetrating activity of buforins. Buforins also are known to possess anti-endotoxin and anticancer activities, thus making these peptides attractive reagents for pharmaceutical applications. This review describes the role of buforins in innate host defense; future research paradigms; and use of these agents as human therapeutics.     

Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S

The cyclic beta-sheet structure possessed by the 10-residue antibiotic peptide gramicidin S was taken as the structural framework for the de novo design of biologically active peptides with membrane-active properties. We have shown from previous studies that gramicidin S is a broad-spectrum antibiotic effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, but is toxic to human red blood cells. We tested the effect of ring size on antimicrobial activity and hemolytic activity on peptides varying from 4 to 16 residues. Interestingly, we were able to dissociate hemolytic activity and antimicrobial activity by increasing the ring size of the peptide to 14 residues (peptide GS14). Furthermore, we increased specificity for microbial membranes while decreasing toxicity to red blood cells by substituting enantiomers (D-amino acids for L-amino acids and vice versa) into the GS14 sequence. The enantiomeric substitutions all disrupted beta-sheet structure in benign medium and decreased peptide amphipathicity. The least amphipathic peptide, produced by substituting a D-Lys at position 4 of GS14 (peptide GS14K4), also had the highest therapeutic index, i.e., highest degree of specificity for microbial cells over human cells. Solution structures of GS14 analogs solved by NMR spectroscopy showed that the D-amino acid side chain was located on the nonpolar face of GS14K4. Another analog, a beta-sheet peptide with reduced amphipathicity (peptide GS14 K3L4), also had a lysine (lysine 3) on the nonpolar face as determined by the NMR structure. Both GS14K4 and GS14 K3L4 had reduced amphipathicity relative to GS14 and much higher therapeutic indices. Finally, the alteration of the nonpolar face hydrophobicity of GS14K4 analogs provided a range of activities and specificities, where the peptides with the intermediate hydrophobicities among the series had the highest therapeutic indices. The optimal peptide hydrophobicities varied depending on the microorganism being tested, with higher hydrophobicity requirements against Gram-positive bacteria and yeast compared with Gram-negative microorganisms. The net result of these studies suggests that it is possible to rationally design a cyclic membrane-active antimicrobial peptide with high specificity towards prokaryotic (bacterial and fungal) membranes and minimal toxicity to eukaryotic (e.g., mammalian) membranes.     

Solution structure of the all L- and D-amino acid-substituted mucin 2 epitope peptides

High-molecular-weight mucin 2 (MUC2) glycoproteins show an aberrant glycosylation pattern when expressed in human colon carcinoma: the oligosaccharide chains are shorter and some are missing. In our ongoing effort of MUC2 vaccine development, we have solved the NMR structure of the all L-amino acid and various D-amino acid-substituted derivatives of the peptide TPTPTGTQTPT, previously identified as an epitope within the tandem repeat unit of the MUC2 glycoprotein. In the all L-amino acid containing peptide and in peptide tpTPTGTQtpt (where lowercase letters mark the position of D-amino acids) we identified a type I beta-turn spanning through residues (3)TPTG(6) and (5)TGTQ(8), respectively. Our structural findings are in good agreement with the antibody recognition properties of the investigated peptides and demonstrate that peptides with good stability against enzymatic degradation can be designed with good antibody binding characteristics.     

A left-handed alpha-helix containing both L- and D-amino acids: the solution structure of the antimicrobial lipodepsipeptide tolaasin

The 18-amino acid cytolytic lipodepsipeptide tolaasin, produced in culture by virulent strains of Pseudomonas tolaasii, is the causal agent of the brown blotch disease of the cultivated mushroom. Tolaasin has a sequence of D-amino acids in its N-terminal region, then alternates L- and D-amino acids, and bears a C-terminal lactone macrocycle composed of 5-residues. The solution structure of tolaasin in sodium dodecyl sulfate was studied by 2D-NMR spectroscopy and molecular dynamics simulated annealing calculations. Tolaasin forms an amphipathic left-handed alpha-helix in the regionDPro2-DalloThr14 comprising the sequence of seven D-amino acids and the adjacent L-D-L-D-D-region. To the best of our knowledge, this is the first recognized example of a left-handed alpha-helix including both D- and L-amino acids. The lactone macrocycle adopts a "boat-like" conformation and is shifted from the helical axis as to form a "golf-club" overall conformation. These structural features will be of importance in understanding, and preventing, tolaasin's role in the bacterial colonization of the host plant, and its toxic action on cells. Furthermore, the observed antimicrobial activity together with the potential resistance to enzymatic degradation and the increased antigenicity (both due to the presence of L- and D-amino acids) strongly suggests for tolaasin a potential role as a template model for the design of new therapeutic antibacterial molecules.     

Structure-activity relationship study: short antimicrobial peptides.

Many short antimicrobial peptides (< 18mer) have been identified for the development of therapeutic agents. However, Structure-activity relationship (SAR) studies about short antimicrobial peptides have not been extensively performed. To investigate the relationship between activity and structural parameters such as an alpha-helical structure, a net positive charge and a hydrophobicity, we synthesized and characterized diastereomers, scramble peptides and substituted peptides of the short antimicrobial peptide identified by combinatorial libraries. Circular dichroism (CD) spectra and in vitro activity indicated that an alpha-helical structure correlated with the antimicrobial activity and a beta-sheet structure also satisfied a structural requirement for antimicrobial activity. Most peptides consisting of L-amino acids lost antifungal activity in the presence of heat-inactivated serum, while active diastereomers and a scramble peptide with the beta-sheet structure retained antifungal activity in the same condition.     

Effect of multiple aliphatic amino acids substitutions on the structure, function, and mode of action of diastereomeric membrane active peptides

The initial stages leading to the binding and functioning of membrane-active polypeptides including hormones, signal sequences, and lytic peptides are mainly governed by electrostatic attraction and hydrophobic partitioning between water and lipid bilayers. Antimicrobial peptides serve as an important model for studying the details of these initial steps. However, a systematic analysis of the contribution of multiple hydrophobic amino acids to these steps have been hindered by the propensity of many peptides to aggregate and become inactivated in solution. To this end, we synthesized a series of model amphipathic all L-amino acid peptides and their diastereomers with the sequence KX(3)KWX(2)KX(2)K, where X = Gly, Ala, Val, Ile, or Leu. The effect of the aliphatic amino acids on the biological activity, binding, structure, membrane localization, and mode of action of these peptides was investigated. Most of the L-amino acid peptides oligomerized and adopted distinct structures in solution and in a membrane mimetic environment. Among this group only the Leu containing peptide was hemolytic and highly active on most bacteria tested. The Val- and Leu-containing peptides were hemolytic but inactive toward most bacteria tested. In contrast, the diastereomeric peptides were monomeric and unstructured in solution, but they adopted distinct structures upon membrane binding. While hemolytic activity was drastically reduced, the spectrum of antibacterial activity was preserved or increased. Importantly, we found a direct correlation with the diastereomers between hydrophobicity and propensity to form a helical/distorted-helix and activity (induced membrane leakage and antibacterial activity), despite the fact that they contained 30% D-amino acids. Furthermore, efficient increase in membrane permeability can proceed through different mechanisms. Specifically, the Leu-containing diastereomeric peptide micellized vesicles and possibly bacterial membranes while the Ile-containing diastereomeric peptide fused model membranes and irregularly disrupted bacterial membranes.     

Effect of Sequence and Stereochemistry Reversal on p53 Peptide Mimicry

Peptidomimetics effective in modulating protein-protein interactions and resistant to proteolysis have potential in therapeutic applications. An appealing yet underperforming peptidomimetic strategy is to employ D-amino acids and reversed sequences to mimic a lead peptide conformation, either separately or as the combined retro-inverso peptide. In this work, we examine the conformations of inverse, reverse and retro-inverso peptides of p53(15–29) using implicit solvent molecular dynamics simulation and circular dichroism spectroscopy. In order to obtain converged ensembles for the peptides, we find enhanced sampling is required via the replica exchange molecular dynamics method. From these replica exchange simulations, the D-peptide analogues of p53(15–29) result in a predominantly left-handed helical conformation. When the parent sequence is reversed sequence as either the L-peptide and D-peptide, these peptides display a greater helical propensity, feature reflected by NMR and CD studies in TFE/water solvent. The simulations also indicate that, while approximately similar orientations of the side-chains are possible by the peptide analogues, their ability to mimic the parent peptide is severely compromised by backbone orientation (for D-amino acids) and side-chain orientation (for reversed sequences). A retro-inverso peptide is disadvantaged as a mimic in both aspects, and further chemical modification is required to enable this concept to be used fruitfully in peptidomimetic design. The replica exchange molecular simulation approach adopted here, with its ability to provide detailed conformational insights into modified peptides, has potential as a tool to guide structure-based design of new improved peptidomimetics.     

Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library

The purpose of the present study was to improve the antifungal activity against selected phytopathogenic fungi of the previously identified hexapeptide PAF19. We describe some properties of a set of novel synthetic hexapeptides whose D-amino acid sequences were obtained through screening of a synthetic peptide combinatorial library in a positional scanning format. As a result of the screening, 12 putative bioactive peptides were identified, synthesized, and assayed. The peptides PAF26 (Ac-rkkwfw-NH(2)), PAF32 (Ac-rkwhfw-NH(2)), and PAF34 (Ac-rkwlfw-NH(2)) showed stronger activity than PAF19 against isolates of Penicillium digitatum, Penicillium italicum, and Botrytis cinerea. PAF26 and PAF32, but not PAF34, were also active against Fusarium oxysporum. Penicillium expansum was less susceptible to all four PAF peptides, and only PAF34 showed weak activity against it. Assays were also conducted on nontarget organisms, and PAF26 and PAF32 showed much-reduced toxicity to Escherichia coli and Saccharomyces cerevisiae, demonstrating selectivity towards certain filamentous fungi. Thus, the data showed distinct activity profiles for peptides differentiated by just one or two residue substitutions. Our conclusion from this observation is that a specificity factor is involved in the activity of these short peptides. Furthermore, PAF26 and PAF32 displayed activities against P. digitatum, P. italicum, and B. cinerea similar to that of the hemolytic 26-amino acid melittin, but they did not show the high toxicity of melittin towards bacteria and yeasts. The four peptides acted additively, with no synergistic interactions among them, and PAF26 was shown to have improved activity over PAF19 in in vivo orange fruit decay experiments.     

Stereoselectivity and structural determinants in molecular recognition by the ACC transport system in isolated maize mesophyll vacuoles

The ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), is actively transported across the tonoplast of plant cells, impacting cellular compartmentation of ACC and ethylene biosynthesis. In the present study, the effects of ACC and amino acid analogs on ACC uptake into isolated maize (Zea mays L. cv. Golden Cross Bantam) mesophyll vacuoles were investigated to identify the stereospecific and structural features that are important in molecular recognition by the ACC transport system. Of the four stereoisomers of l-amino-2-ethylcyclopropane-l-carboxylic acid (AEC), (1S, 2R)-(–)-AEC having a configuration corresponding to an L-amino acid was the preferred substrate for the ACC transport system, competitively inhibiting ACC transport with a Ki of 18 μM. Of 11 neutral amino acid stereoisomers, L-isomers were stronger inhibitors of ACC transport than corresponding D-isomers. Neutral L-amino acids with nonpolar side chains generally were more inhibitory than those with polar side chains, whereas several cationic and anionic L-amino acids were ineffective antagonists of ACC transport. These observations suggest that the ACC transport system is stereospecific for relatively nonpolar, neutral L-amino acids. This conclusion was supported by the observation that group additions, substitutions, or deletions at the carboxyl. α-amino and the Pro- (R) methylene or hydrogen moieties (analogous to D-amino acids) of ACC and other neutral amino acids and analogs essentially eliminated transport inhibition. In contrast, L-amino acid analogs with variable substitutions at the distal end of the molecule remained antagonists. The relative activity of analogs was influenced by the length and degree of unsaturation of the side chain and by the location of side chain branching. Increasing the ring size of ACC analogs reduced antagonism whereas incorporating the α-amino group into the ring structure as an L-amino acid increased antagonism. The kinetics of L-methoxyvinylglycine, L-methionine. p-nitro-L-phenylalanine and 1-aminocyclobutane-l-carboxylic acid were competitive with Ki values of 3, 13, 16 and 19 μM, respectively. These results indicate that the ACC transport system can be classifie as a neutral L-amino acid carrier having a relatively high affinity for ACC and other nonpolar amino acids. The results also suggest that the carrier interacts with the carboxyl, α-amino and Pro-(R) groups and with other less restricted side chain substituents of substrate amino acids.     

Total hydrolysis of proteins with strongly reduced racemization of amino acids

A new method has been devised for the complete hydrolysis of proteins with an extremely low level of racemization of amino acids. Proteins are incubated in 10 M HCl at a low temperature to obtain partial hydrolysis. They are then incubated with pronase and finally with leucine aminopeptidase and peptidyl-D-amino-acid hydrolase from Loligo vulgaris. The proposed method ensures the total hydrolysis of either purified proteins or proteins contained in a crude homogenate of animal or vegetable tissue. In both cases, the racemization of amino acids (expressed as rate of D form/D + L form X 100) was lower than 0.015% for aspartic acid and lower than 0.01% for other amino acids. D-Amino acids released from peptides or proteins were estimated with enzymatic methods based on the use of octopus D-aspartate oxidase or hog kidney D-amino acid oxidase; with these enzymes, 0.05 nmol of a D-amino acid was determined in the presence of up to 20 mumols of a mixture of L-amino acids (ratio %D/D + L = 0.00025). The method allows the determination of D-amino acids either in tissues in which they are present in high concentrations (as human cataract lenses, tooth enamel, etc.) or in those with low enantiomer content (as brain, erythrocytes, etc.). Using the method described, we hydrolyzed several synthetic peptides consisting of D- and L-amino acids and determined the amount of D-amino acids. In addition, we totally hydrolyzed all the nuclear proteins of human cataractous lenses. The amount of D-aspartic acid was 0.026 mumols/mg in lenses of women aged between 71 and 76 years and 0.0256 mumols/mg in lenses of men aged between 55 and 72 years. The D-aspartic acid measured corresponds to about 12% with respect to total aspartic acid.     

Microdetermination of serum D-amino acids

A method for the quantitative determination of serum D-amino acids in the range 0.5-20 nmol is described. In the method alpha-keto acids, derived from D-amino acids by D-amino acid oxidase, are measured as hydrazones. The method is unresponsive to the presence of a large excess of L-amino acids. It allows a fast assay in a small amount of specimen (0.1 ml), with good reproducibility and accuracy.     

Quantitation of the tumor-targeting properties of antibody fragments conjugated to cell-permeating HIV-1 TAT peptides

Human monoclonal antibodies are promising agents for the development of more selective anticancer therapeutics. However, the tumor-targeting efficiency of most anticancer antibodies is severely limited by their poor penetration into the tumor mass. Recent studies have shown that a peptide derived from the HIV TAT protein could improve the distribution of cytoplasmic reporter proteins when administered systemically as fusion proteins or cross-linked chimeras. In this article, we tested by quantitative biodistribtution analysis whether conjugation to TAT peptides could improve the tumor targeting properties of scFv(L19)-Cys: an engineered human antibody fragment specific for the ED-B domain of fibronectin, a marker located in the modified extracellular matrix surrounding tumor neovasculature. Our results show that TAT peptides, consisting either of L-amino acids or D-amino acids, can efficiently transduce target cells when conjugated to fluorophores and/or antibody fragments, suggesting a receptor-independent cell entry mechanism. However, conjugation of scFv(L19)-Cys to TAT peptides resulted in a severely reduced tumor targeting performance compared to the unconjugated antibody, as measured in murine F9 teratocarcinoma-bearing mice, after intravenous injection of the radiolabeled antibody preparations. Our results outline the usefulness of TAT peptides for the efficient in vitro transduction of cells with globular proteins. In particular, the use of TAT peptides composed of D-amino acids may significantly reduce proteolytic degradation. At the same time, the poor biodistribution properties of antibody-TAT conjugates cast doubts over the applicability of this methodology for the delivery of biopharmaceuticals in vivo.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

Development of protegrins for the treatment and prevention of oral mucositis: structure-activity relationships of synthetic protegrin analogues

Protegrin antimicrobial peptides possess activity against gram-positive and gram-negative bacteria and yeasts. An extensive structure-activity relationship (SAR) study was conducted on several hundred protegrin analogues to gain understanding of the relationship between the primary and secondary structure of the protegrins and their antimicrobial activities, and to identify a protegrin analogue for clinical development. Native sequence protegrins are cationic, amphiphilic peptides that are characterized by the presence of a beta-sheet structure that is maintained by two disulfide bridges. The presence of the beta-sheet is key to the stability of the protegrin structure; linearized analogues or analogues that have amino acid substitutions that eliminate hydrogen bonding across the beta-sheet have reduced activity, especially in the presence of physiological concentrations of NaCl. Also, maintaining amphiphilicity of the beta-sheet is key; analogues with substitutions of polar amino acids in the hydrophobic face have reduced activity. Analogues with reduced positive charge tend to be less active, an observation that is more marked for gram-negative than gram-positive bacteria, and may implicate binding to lipopolysaccharide as a key mechanistic step in the killing of gram-negative bacteria. A very large number of amino acid substitutions are tolerated by the protegrin structure, implying that overall structural features such as amphiphilicity, charge, and shape are more important to activity than the presence of specific amino acids. This lack of importance of specific stereochemistry is supported by the fact that completely D-amino acid substituted protegrins are fully potent. Based on the SAR studies, and on the microbiological data from an animal model, one protegrin analogue, IB-367, was selected for clinical development as a topical agent to prevent the oral mucositis associated with cancer therapy.