A Novel Dimeric Short Peptide Derived from α-Defensin-Related Rattusin with Improved Antimicrobial and DNA-Binding Activities.

Rattusin, an α-defensin-related antimicrobial peptide isolated from the small intestine of rats, has been previously characterized through NMR spectroscopy to elucidate its three-dimensional structure, revealing a C2 homodimeric scaffold stabilized by five disulfide bonds. This study aimed to identify the functional region of rattusin by designing and synthesizing various short analogs, subsequently leading to the development of novel peptide-based antibiotics. The analogs, designated as F1, F2, F3, and F4, were constructed based on the three-dimensional configuration of rattusin, among which F2 is the shortest peptide and exhibited superior antimicrobial efficacy compared to the wild-type peptide. The central cysteine residue of F2 prompted an investigation into its potential to form a dimer at neutral pH, which is critical for its antimicrobial function. This activity was abolished upon the substitution of the cysteine residue with serine, indicating the necessity of dimerization for antimicrobial action. Further, we synthesized ß-hairpin-like analogs, both parallel and antiparallel, based on the dimeric structure of F2, which maintained comparable antimicrobial potency. In contrast to rattusin, which acts by disrupting bacterial membranes, the F2 dimer binds directly to DNA, as evidenced by fluorescence assays and DNA retardation experiments. Importantly, F2 exhibited negligible cytotoxicity up to 515 µg/mL, assessed via hemolysis and MTT assays, underscoring its potential as a lead compound for novel peptide-based antibiotic development.

Nanodefensin-encased hydrogel with dual bactericidal and pro-regenerative functions for advanced wound therapy.

Background: Host defense peptides (HDPs) have emerged as a novel therapeutic paradigm for wound management; however, their clinical applications remain a challenge owing to their poor pharmacological properties and lack of suitable pharmaceutical formulations. Nanodefensin (ND), a nanoengineered human α-defensin 5 (HD5), has shown improved pharmacological properties relative to the parent compound. In this study, we engineered a nanodefensin-encased hydrogel (NDEFgel), investigated the effects of NDEFgel on wound healing, and elucidated underlying mechanisms. Method: ND was chemically synthesized and tested functions by in vitro antimicrobial and scratch assays and western blotting. Different NDEFgels were evaluated by in vitro characterizations including degradation, drug release and antimicrobial activity. In full-thickness excisional murine models, the optimal NDEFgel was directly applied onto wound sites, and the efficacy was assessed. Moreover, the underlying mechanisms of pro-regenerative effect developed by NDEFgel were also explored. Results: Apart from bactericidal effects, ND modulated fibroblast behaviors by promoting migration and differentiation. Among the tested hydrogels, the Pluronic F127 (Plu) hydrogel represented the most desirable carrier for ND delivery owing to its favorable controlled release and compatibility with ND. Local treatment of NDEFgel on the wound bed resulted in accelerated wound regeneration and attenuated bacterial burden. We further demonstrated that NDEFgel therapy significantly upregulated genes related to collagen deposition and fibroblasts, and increased the expression of myofibroblasts and Rac1. We therefore found that Rac1 is a critical factor in the ND-induced modulation of fibroblast behaviors in vitro through a Rac1-dependent cytoskeletal rearrangement. Conclusion: Our results indicate that NDEFgel may be a promising dual-action therapeutic option for advanced wound management in the future.

Dynamics of human defensin 5 (HD5) self-assembly in solution: Molecular simulations/insights.

Human α -defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits broad-spectrum antimicrobial activity and plays an essential role in innate immunity in the human gut and other organ systems. Although its antimicrobial mechanism of action remains unclear, the high salt concentration seems to attenuate the antimicrobial function of HD5 via an unknown mechanism. In this work, we employ Molecular Dynamics (MD) simulations to analyse the oligomerization behaviour of HD5 when exposed to different salt concentration. We demonstrate that the presence of salt, such as sodium chloride (NaCl), promotes HD5 to form higher-order oligomers (up to heptamers) in our simulations. In addition, we also analyse the electrostatic interactions between the two Glu residues (E14 and E21) and their neighbouring residues. Our data confirm that the E14 residue is essential for the structural integrity, whereas the E21 residue contributes to the dimerization of HD5, suggesting that these Glu residues are important for the antimicrobial function of this peptide.

Human Defensin-5 Blocks Ethanol and Colitis-Induced Dysbiosis, Tight Junction Disruption and Inflammation in Mouse Intestine.

Alcohol consumption has been shown to cause dysbiosis, but the mechanism involved in it is unknown. Recurrent colitis is known to induce expression of α-defensins in the colon, but the effect of alcohol consumption on it is not known. We investigated the effect of ethanol on α-defensin expression in the small intestine and colitis-induced expression in colon in mice. Furthermore, we evaluated the effect of human defensin-5 (HD5) on ethanol and colitis-induced gut barrier dysfunction and mucosal damage. Recurrent colitis was induced by feeding dextran sulfate sodium (DSS), 3 cycles of 5-days each with 15 days intervals, followed by 30-days remission. Ethanol was fed during the intervals and recovery in a liquid diet with or without HD5. Expression of α-defensins, tight junction (TJ) integrity and cytokine/chemokine expression were analyzed. Chronic ethanol feeding reduced α-defensin expression in the small intestine and colitis-induced defensin expression in the colon. HD5 attenuated the growth of enterotoxigenic Bacteriodes fragilis and E. coli, but had no effect on non-toxigenic Bacteriodes fragilis or probiotics, the Lactobacilli. Ethanol and colitis elevated Enterobacteriaceae, Firmicutes and Firmicutes to Bacteriodetes ratio in colonic mucosa. HD5 feeding attenuated ethanol and colitis-induced dysbiosis, disruption of intestinal epithelial TJ, mucosal inflammation, expression of pro-inflammatory cytokines and chemokines in the small intestine and colon, and endotoxemia. These results demonstrate that ethanol suppresses intestinal α-defensin expression, leading to dysbiosis, barrier dysfunction, inflammation and endotoxemia. HD5 feeding attenuates intestinal injury caused by ethanol and colitis, indicating that defensin expression is a potential target for treatment of alcoholic tissue injury and colitis.

Self-Assembling Myristoylated Human α-Defensin 5 as a Next-Generation Nanobiotics Potentiates Therapeutic Efficacy in Bacterial Infection.

The increasing prevalence of antibacterial resistance globally underscores the urgent need to the update of antibiotics. Here, we describe a strategy for inducing the self-assembly of a host-defense antimicrobial peptide (AMP) into nanoparticle antibiotics (termed nanobiotics) with significantly improved pharmacological properties. Our strategy involves the myristoylation of human α-defensin 5 (HD5) as a therapeutic target and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with its parent HD5, the C-terminally myristoylated HD5 (HD5-myr)-assembled nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro. Mechanistically, it selectively killed Escherichia coli ( E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) through disruption of the cell wall and/or membrane structure. The in vivo results further demonstrated that the HD5-myr nanobiotic protected against skin infection by MRSA and rescued mice from E. coli-induced sepsis by lowering the systemic bacterial burden and alleviating organ damage. The self-assembled HD5-myr nanobiotic also showed negligible hemolytic activity and substantially low toxicity in animals. Our findings validate this design rationale as a simple yet versatile strategy for generating AMP-derived nanobiotics with excellent in vivo tolerability. This advancement will likely have a broad impact on antibiotic discovery and development efforts aimed at combating antibacterial resistance.

A Simplified Derivative of Human Defensin 5 with Potent and Efficient Activity against Multidrug-Resistant Acinetobacter baumannii.

The increasing incidence of multidrug-resistant Acinetobacter baumannii (MDRAb) infections worldwide has necessitated the development of novel antibiotics. Human defensin 5 (HD5) is an endogenous peptide with a complex architecture and antibacterial activity against MDRAb In the present study, we attempted to simplify the structure of HD5 by removing disulfide bonds. We found that the Cys2-4 bond was most indispensable for HD5 to inactivate MDRAb, although the antibacterial activity of the derivative was significantly attenuated. We then replaced the noncationic and nonhydrophobic residues with electropositive Arg to increase the antibacterial activity of HD5 derivative that contains a Cys2-4 bond, obtaining another derivative termed HD5d5. The in vitro antibacterial assay and irradiation-wound-infection animal experiment both showed that HD5d5 was much more effective than HD5 at eliminating MDRAb Further investigations revealed that HD5d5 efficiently bound to outer membrane lipid A and penetrated membranes, leading to bacterial collapse and peptide translocation. Compared to HD5, more HD5d5 molecules were located in the cytoplasm of MDRAb, and HD5d5 was more efficient at reducing the activities of superoxide dismutase and catalase, causing the accumulation of reactive oxygen species that are detrimental to microbes. In addition, HD5 failed to suppress the pathogenic outer membrane protein A of Acinetobacter baumannii (AbOmpA) at concentrations up to 50 µg/ml, whereas HD5d5 strongly bound to AbOmpA and exhibited a dramatic toxin-neutralizing ability, thus expanding the repertoire of drugs that is available to treat MDRAb infections.

Human α-Defensin 6: A Small Peptide That Self-Assembles and Protects the Host by Entangling Microbes.

Human α-defensin 6 (HD6) is a 32-residue cysteine-rich peptide that contributes to innate immunity by protecting the host at mucosal sites. This peptide is produced in small intestinal Paneth cells, stored as an 81-residue precursor peptide named proHD6 in granules, and released into the lumen. One unusual feature of HD6 is that it lacks the broad-spectrum antimicrobial activity observed for other human α-defensins, including the Paneth cell peptide human α-defensin 5 (HD5). HD6 exhibits unprecedented self-assembly properties, which confer an unusual host-defense function. HD6 monomers self-assemble into higher-order oligomers termed "nanonets", which entrap microbes and prevent invasive gastrointestinal pathogens such as Salmonella enterica serovar Typhimurium and Listeria monocytogenes from entering host cells. One possible advantage of this host-defense mechanism is that HD6 helps to keep microbes in the lumen such that they can be excreted or attacked by other components of the immune system, such as recruited neutrophils. In this Account, we report our current understanding of HD6 and focus on work published since 2012 when Bevins and co-workers described the discovery of HD6 nanonets in the literature. First, we present studies that address the biosynthesis, storage, and maturation of HD6, which demonstrate that nature uses a propeptide strategy to spatially and temporally control the formation of HD6 nanonets in the small intestine. The propeptide is stored in Paneth cell granules, and proteolysis occurs during or following release into the lumen, which affords the 32-residue mature peptide that self-assembles. We subsequently highlight structure-function studies that provide a foundation for understanding the molecular basis for why HD6 exhibits unusual self-assembly properties compared with other characterized defensins. The disposition of hydrophobic residues in the HD6 primary structure differs from that of other human α-defensins and is an important structural determinant for oligomerization. Lastly, we consider functional studies that illuminate how HD6 contributes to mucosal immunity. We recently discovered that in addition to blocking bacterial invasion into host epithelial cells by Gram-negative and Gram-positive gastrointestinal pathogens, HD6 suppresses virulence traits displayed by the opportunistic human fungal pathogen Candida albicans. In particular, we found that C. albicans biofilm formation, which causes complications in the treatment of candidiasis, is inhibited by HD6. This observation suggests that HD6 may contribute to intestinal homeostasis by helping to keep C. albicans in its commensal state. We intend for this Account to inspire further biochemical, biophysical, and biological investigations that will advance our understanding of HD6 in mucosal immunity and the host-microbe interaction.

Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins.

Human defensins are innate immune defense peptides with a remarkably broad repertoire of anti-pathogen activities. In addition to modulating immune response, inflammation, and angiogenesis, disintegrating bacterial membranes, and inactivating bacterial toxins, defensins are known to intercept various viruses at different stages of their life cycles, while remaining relatively benign towards human cells and proteins. Recently we have found that human defensins inactivate proteinaceous bacterial toxins by taking advantage of their low thermodynamic stability and acting as natural "anti-chaperones", i.e. destabilizing the native conformation of the toxins. In the present study we tested various proteins produced by several viruses (HIV-1, PFV, and TEV) and found them to be susceptible to destabilizing effects of human α-defensins HNP-1 and HD-5 and the synthetic θ-defensin RC-101, but not ß-defensins hBD-1 and hBD-2 or structurally related plant-derived peptides. Defensin-induced unfolding promoted exposure of hydrophobic groups otherwise confined to the core of the viral proteins. This resulted in precipitation, an enhanced susceptibility to proteolytic cleavage, and a loss of viral protein activities. We propose, that defensins recognize and target a common and essential physico-chemical property shared by many bacterial toxins and viral proteins - the intrinsically low thermodynamic protein stability.

Synthesis and Macrodomain Binding of Mono-ADP-Ribosylated Peptides.

Mono-ADP-ribosylation is a dynamic posttranslational modification (PTM) with important roles in signaling. Mammalian proteins that recognize or hydrolyze mono-ADP-ribosylated proteins have been described. We report the synthesis of ADP-ribosylated peptides from the proteins histone H2B, RhoA and, HNP-1. An innovative procedure was applied that makes use of pre-phosphorylated amino acid building blocks. Binding assays revealed that the macrodomains of human MacroD2 and TARG1 exhibit distinct specificities for the different ADP-ribosylated peptides, thus showing that the sequence surrounding ADP-ribosylated residues affects the substrate selectivity of macrodomains.

Paneth cell α-defensin 6 (HD-6) is an antimicrobial peptide.

Defensins protect human barriers from commensal and pathogenic microorganisms. Human α-defensin 6 (HD-6) is produced exclusively by small intestinal Paneth cells but, in contrast to other antimicrobial peptides (AMPs) for HD-6, no direct antibacterial killing activity has been detected so far. Herein, we systematically tested how environmental factors, like pH and reducing conditions, affect antimicrobial activity of different defensins against anaerobic bacteria of the human intestinal microbiota. Remarkably, by mimicking the intestinal milieu we detected for the first time antibacterial activity of HD-6. Activity was observed against anaerobic gut commensals but not against some pathogenic strains. Antibiotic activity was attributable to the reduced peptide and independent of free cysteines or a conserved histidine residue. Furthermore, the oxidoreductase thioredoxin, which is also expressed in Paneth cells, is able to reduce a truncated physiological variant of HD-6. Ultrastructural analyses revealed that reduced HD-6 causes disintegration of cytoplasmic structures and alterations in the bacterial cell envelope, while maintaining extracellular net-like structures. We conclude that HD-6 is an antimicrobial peptide. Our data suggest two distinct antimicrobial mechanisms by one peptide: HD-6 kills specific microbes depending on the local environmental conditions, whereas known microbial trapping by extracellular net structures is independent of the reducing milieu.

Discovery and characterization of a disulfide-locked C(2)-symmetric defensin peptide.

We report the discovery of HD5-CD, an unprecedented C2-symmetric ß-barrel-like covalent dimer of the cysteine-rich host-defense peptide human defensin 5 (HD5). Dimerization results from intermonomer disulfide exchange between the canonical α-defensin Cys(II)-Cys(IV) (Cys(5)-Cys(20)) bonds located at the hydrophobic interface. This disulfide-locked dimeric assembly provides a new element of structural diversity for cysteine-rich peptides as well as increased protease resistance, broad-spectrum antimicrobial activity, and enhanced potency against the opportunistic human pathogen Acinetobacter baumannii.

Potential of host defense peptide prodrugs as neutrophil elastase-dependent anti-infective agents for cystic fibrosis.

Host defense peptides (HDPs) are short antimicrobial peptides of the innate immune system. Deficiencies in HDPs contribute to enhanced susceptibility to infections, e.g., in cystic fibrosis (CF). Exogenous HDPs can compensate for these deficiencies, but their development as antimicrobials is limited by cytotoxicity. Three HDP prodrugs were designed so their net positive charge is masked by a promoiety containing a substrate for the enzyme neutrophil elastase (NE). This approach can confine activation to sites with high NE levels. Enzyme-labile peptides were synthesized, and their activation was investigated using purified NE. Susceptibilities of Pseudomonas aeruginosa to parent and prodrug peptides in the presence and absence of NE-rich CF human bronchoalveolar lavage (BAL) fluid and different NaCl concentrations were compared. The effect of the HDP promoiety on cytotoxicity was determined with cystic fibrosis bronchial epithelial (CFBE41o-) cells. NE in CF BAL fluids activated the HDP prodrugs, restoring bactericidal activity against reference and clinical isolates of P. aeruginosa. However, activation also required the addition of 300 mM NaCl. Under these conditions, the bactericidal activity levels of the HDP prodrugs differed, with pro-P18 demonstrating the greatest activity (90% to 100% of that of the parent, P18, at 6.25 µg/ml). Cytotoxic effects on CFBE41o- cells were reduced by the addition of the promoiety to HDPs. We demonstrate here for the first time the selective activation of novel HDP prodrugs by a host disease-associated enzyme at in vivo concentrations of the CF lung. This approach may lead to the development of novel therapeutic agents with low toxicity that are active under the challenging conditions of the CF lung.

An optimized Fmoc synthesis of human defensin 5.

Human α-defensin 5 (DEF5), expressed by the Paneth cells of human small intestine, plays an important role in host defense against microbial infections. DEF5, a 32-residue peptide adopting a three-stranded ß-sheet fold stabilized by three internal disulfide bonds, is not efficiently produced by recombinant expression techniques and is, therefore, an interesting goal for chemical synthesis. While DEF5 production by Boc-based solid-phase synthesis has been described, to date no synthetic account by the more convenient Fmoc method has been published. Herein, we report an optimized solid-phase synthesis of DEF5 using the Fmoc strategy. Starting from a rather problematic initial synthesis using standard Wang resin and coupling protocols, the sequence elongation process has been monitored by mini-cleavage and MS analysis at strategic points, to identify problematic spots and act accordingly. For expediency, some of the optimization rounds have been run on defensin 5 amide. Main modifications have included the ChemMatrix(®) resin, known to decrease chain aggregation, and the use of pseudoproline dipeptide units at selected positions. Combination of some of these improvements results in a significantly purer product, to the extent that it can undergo in situ anaerobic oxidative folding to the native form without the need of an intermediate purification step. A typical synthesis run yielded about 15 mg of >95 % pure material. This approach should facilitate production of DEF5 and of selected analogs for structure-activity studies and other applications.

Sub-inhibitory concentrations of human α-defensin potentiate neutralizing antibodies against HIV-1 gp41 pre-hairpin intermediates in the presence of serum.

Human defensins are at the forefront of the host responses to HIV and other pathogens in mucosal tissues. However, their ability to inactivate HIV in the bloodstream has been questioned due to the antagonistic effect of serum. In this study, we have examined the effect of sub-inhibitory concentrations of human α-defensin HNP-1 on the kinetics of early steps of fusion between HIV-1 and target cells in the presence of serum. Direct measurements of HIV-cell fusion using an enzymatic assay revealed that, in spite of the modest effect on the extent of fusion, HNP-1 prolonged the exposure of functionally important transitional epitopes of HIV-1 gp41 on the cell surface. The increased lifetime of gp41 intermediates in the presence of defensin was caused by a delay in the post-coreceptor binding steps of HIV-1 entry that correlated with the marked enhancement of the virus' sensitivity to neutralizing anti-gp41 antibodies. By contrast, the activity of antibodies to gp120 was not affected. HNP-1 appeared to specifically potentiate antibodies and peptides targeting the first heptad repeat domain of gp41, while its effect on inhibitors and antibodies to other gp41 domains was less prominent. Sub-inhibitory concentrations of HNP-1 also promoted inhibition of HIV-1 entry into peripheral blood mononuclear cells by antibodies and, more importantly, by HIV-1 immune serum. Our findings demonstrate that: (i) sub-inhibitory doses of HNP-1 potently enhance the activity of a number of anti-gp41 antibodies and peptide inhibitors, apparently by prolonging the lifetime of gp41 intermediates; and (ii) the efficiency of HIV-1 fusion inhibitors and neutralizing antibodies is kinetically restricted. This study thus reveals an important role of α-defensin in enhancing adaptive immune responses to HIV-1 infection and suggests future strategies to augment these responses.

Synthesis, structure, and activities of an oral mucosal alpha-defensin from rhesus macaque.

The oral cavity is an environment challenged by a large variety of pathogens. Consequently, the antimicrobial peptides expressed in that environment are interesting as they evolved to defend against a broad spectrum of bacteria and fungi. Here we report the discovery of new alpha-defensins from rhesus macaque oral mucosa and determine the first alpha-defensin structure from that species. The new peptides were identified by sequencing of reverse transcriptase-PCR products obtained from oral mucosal tissues, disclosing three mucosal alpha-defensins, termed rhesus macaque oral alpha-defensins (ROADs). The peptide corresponding to fully processed ROAD-1 was synthesized, subjected to folding/oxidation conditions, and purified. ROAD-1 was active against Staphylococcus aureus, Escherichia coli, and Candida albicans in a concentration-dependent manner. We determined the structure of ROAD-1 using NMR spectroscopy and find that the synthetic peptide adopts the canonical disulfide pairing and alpha-defensin fold. The antimicrobial mechanism of defensins has been correlated with their ability to disrupt and permeabilize the cell envelope, activities that depend on the surface features of the folded peptide. Although ROAD-1 maintains the defensin fold, the oral defensin displays distinct surface features when compared with other alpha-defensin structures.

Kinetics of cryptdin-4 translocation coupled with peptide-induced vesicle leakage.

The antimicrobial peptide cryptdin-4 (Crp4), a member of the alpha-defensin family, is shown to translocate cooperatively across phospholipid bilayers. The cooperativity of the process is manifested by translocation kinetics which vary with the peptide to lipid molar ratio. A simple association model suggests dimerization. Black lipid membrane experiments reveal that Crp4 translocation does not create well-defined aqueous pores, as is often common among peptides exhibiting cooperative translocation. Still, the efflux induced by Crp4 upon its interaction with fluorophore-loaded vesicles is shown to be a direct result of the membrane perturbation resulting from the translocation process. Leakage can be predicted by relating membrane permeability to the fraction of peptide translocated. Crp4 translocation has implications for its antimicrobial activity as internalized peptide would be available to attack intracellular targets.

Binding orientation and activity determinants of the antimicrobial peptide cryptdin-4 revealed by potency of mutants.

Cryptdin-4 is a beta-sheet antimicrobial peptide of the defensin family that is found in the immune system of mice. Several structure-activity studies of this peptide have previously been conducted, but none have been based on residue-membrane interactions as part of an overall hypothesis on the peptide's orientation in the membrane. We pursue this valuable approach by first using previously reported NMR structural data to propose a membrane-bound orientation of the peptide. Four mutants are then strategically designed to modulate membrane perturbative activity in a manner consistent with the proposed binding orientation. Membrane perturbation is evaluated using a simple fluorescence-based vesicle leakage assay using POPG to form the model membrane. Effects of peptide mutations are found to be consistent with the suggested binding orientation. This approach is successfully used to create synthetic peptides with enhanced or diminished ability to perturb membranes and also yields insights on the nature of peptide-membrane interactions.

Structure-dependent functional properties of human defensin 5.

The mucosal epithelium secretes a variety of antimicrobial peptides that act as part of the innate immune system to protect against invading microbes. Here, we describe the functional properties of human defensin (HD) 5, the major antimicrobial peptide produced by Paneth cells in the ileum, in relation to its structure. The antimicrobial activity of HD-5 against Escherichia coli proved to be independent of its structure, whereas the unstructured peptide showed greatly reduced antimicrobial activity against Staphylococcus aureus. We find that HD-5 binds to the cell membrane of intestinal epithelial cells and induced secretion of the chemokine interleukin (IL)-8 in a concentration- and structure-dependent fashion. Incubation of HD-5 in the presence of tumor necrosis factor alpha further increased IL-8 secretion synergistically, suggesting that HD-5 may act as a regulator of the intestinal inflammatory response.

Antibacterial activity and specificity of the six human {alpha}-defensins.

We developed a kinetic, 96-well turbidimetric procedure that is capable of testing the antimicrobial properties of six human alpha-defensins concurrently on a single microplate. The defensins were prepared by solid-phase peptide synthesis and tested against gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Enterobacter aerogenes and Escherichia coli). Analysis of the growth curves provided virtual lethal doses (vLDs) equivalent to conventional 50% lethal doses (LD(50)s), LD(90)s, LD(99)s, and LD(99.9)s obtained from colony counts. On the basis of their respective vLD(90)s and vLD(99)s, the relative potencies of human myeloid alpha-defensins against S. aureus were HNP2 > HNP1 > HNP3 > HNP4. In contrast, their relative potencies against E. coli and E. aerogenes were HNP4 > HNP2 > HNP1 = HNP3. HD5 was as effective as HNP2 against S. aureus and as effective as HNP4 against the gram-negative bacteria in our panel. HD6 showed little or no activity against any of the bacteria in our panel, including B. cereus, which was highly susceptible to the other five alpha-defensins. The assay described provides a quantitative, precise, and economical way to study the antimicrobial activities of host-defense peptides. Its use has clarified the relative potencies of human alpha-defensins and raised intriguing questions about the in vivo function(s) of HD6.

Synthesis and characterization of human alpha-defensins 4-6.

Human alpha-defensins are small, Cys-rich, cationic proteins expressed predominantly in neutrophils and intestinal epithelia. They play important roles in innate and adaptive immunity against infection. Progress in studying these molecules can be accelerated by access to large quantities of high-quality materials, which have been obtained mainly from natural sources. Here, we report total synthesis of human alpha-defensins 4, 5, and 6, also known as HNP4, HD5, and HD6, using the optimized N,N-diisopropylethylamine (DIEA) in situ neutralization/2-(1 H-benzotriazolyl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (HBTU) activation protocol for solid-phase Boc chemistry. Oxidative folding/disulfide formation was achieved directly using crude peptides, resulting in an overall synthetic yield of 10-16% with high purity. Antimicrobial activity assays were performed with Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213, using colony-counting methods, and the results demonstrated differential activity against these strains. Our report describes a highly efficient synthetic approach that enables thorough structural and functional studies of these three important immunologic molecules.