Structural determinants of host defense peptides for antimicrobial activity and target cell selectivity

Antimicrobial host defense peptides (HDPs) are a critical component of the innate immunity with microbicidal, endotoxin-neutralizing, and immunostimulatory properties. HDPs kill bacteria primarily through non-specific membrane lysis, therefore with a less likelihood of provoking resistance. Extensive structure–activity relationship studies with a number of HDPs have revealed that net charge, amphipathicity, hydrophobicity, and structural propensity are among the most important physicochemical and structural parameters that dictate their ability to interact with and disrupt membranes. A delicate balance among these factors, rather than a mere alteration of a single factor, is critically important for HDPs to ensure the antimicrobial potency and target cell selectivity. With a better understanding of the structural determinants of HDPs for their membrane-lytic activities, it is expected that novel HDP-based antimicrobials with minimum toxicity to eukaryotic cells can be developed for resistant infections, which have become a global public health crisis.     

Metformin promotes Mycobacterium tuberculosis killing and increases the production of human β-defensins in lung epithelial cells and macrophages

Diabetes has been associated with an increased risk of developing tuberculosis. The reasons related to the increased susceptibility to develop TB in type 2 diabetes mellitus (T2DM) individuals, has not been completely elucidated. However, this susceptibility has been attributed to several factors including failures and misfunctioning of the immune system. In the present study, we aimed to determine the role of anti-hyperglycemic drugs such as glyburide, insulin, and metformin to promote the killing of mycobacteria through the regulation of innate immune molecules such as host defense peptides (HDP) in lung epithelial cells and macrophages. Our results showed that metformin reduces bacillary loads in macrophages and lung epithelial cells which correlates with higher production of β-defensin-2, -3 and -4. Since β-defensins are crucial molecules for controlling Mycobacterium tuberculosis growth, the present results suggest that the use of metformin would be the first choice in the treatment for T2DM2, in patients within tuberculosis-endemic areas.     

Mapping the eosinophil cationic protein antimicrobial activity by chemical and enzymatic cleavage

The eosinophil cationic protein (ECP) is a human antimicrobial protein involved in the host immune defense that belongs to the pancreatic RNase A family. ECP displays a wide range of antipathogen activities. The protein is highly cationic and its bactericidal activity is dependant on both cationic and hydrophobic surface exposed residues. Previous studies on ECP by site-directed mutagenesis indicated that the RNase activity is not essential for its bactericidal activity. To further understand the ECP bactericidal mechanism, we have applied enzymatic and chemical limited cleavage to search for active sequence determinants.Following a search for potential peptidases we selected the Lys-endoproteinase, which cleaves the ECP polypeptide at the carboxyl side of its unique Lys residue, releasing the N-terminal fragment (0-38).Chemical digestion using cyanogen bromide released several complementary peptides at the protein N-terminus. Interestingly, ECP treatment with cyanogen bromide represents a new example of selective chemical cleavage at the carboxyl side of not only Met but also Trp residues. Recombinant ECP was denatured and carboxyamidomethylated prior to enzymatic and chemical cleavage. Irreversible denaturation abolishes the protein bactericidal activity.The characterization of the digestion products by both enzymatic and chemical approaches identifies a region at the protein N-terminus, from residues 11 to 35, that retains the bactericidal activity. The most active fragment, ECP(0-38), is further compared to ECP derived synthetic peptides. The region includes previously identified stretches related to lipopolysaccharide binding and bacteria agglutination. The results contribute to define the shortest ECP minimized version that would retain its antimicrobial properties. The data suggest that the antimicrobial RNase can provide a scaffold for the selective release of cytotoxic peptides.     

抗菌肽在宿主防御中作用

抗菌肽广泛地存在于自然界中,其中许多抗菌肽具有直接抗微生物活性,能作用于G-、 G+细菌、真菌、寄生虫甚至是包膜病毒,并且在宿主先天免疫和适应性反应中有重要的调节作用。近来,越来越多的证据表明抗菌肽是有效的免疫辅助因子,能够与其他的众多免疫效应子协同作用,从而起始适应性免疫,促进伤口愈合,抑制前炎症反应以及诱导和调节细胞因子和趋化因子的产生。另外,随着抗菌肽作用机理逐渐被揭开,将这些内源性肽及其衍生物制成抗感染治疗药剂将会有广阔的应用前景。     

Complement Activation Products C3a and C4a as Endogenous Antimicrobial Peptides

All vertebrate species are constantly challenged by infectious agents and pathogens. In order to fight these infectious agents the human host has developed a sophisticated and powerful immune defense. The complement system, which represents the first defense line of innate immunity is activated immediately, within seconds. The activated immune system recognizes and damages an invading microbe, coordinates the host immune response and further orchestrates the adaptive immune response. Activation of the complement system leads to a rapid and amplified response which includes the generation of small peptides like C3a and C4a that display antimicrobial, anti-fungal and anaphylactic activity. Here we report how these antimicrobial peptides are generated during the immune response and summarize the functional mechanisms of these intrinsically generated anti microbial peptides.     

Correlation between simulated physicochemical properties and hemolycity of protegrin-like antimicrobial peptides: predicting experimental toxicity

The therapeutic, antibiotic potential of antimicrobial peptides can be prohibitively diminished because of the cytotoxicity and hemolytic profiles they exhibit. Quantifying and predicting antimicrobial peptide toxicity against host cells is thus an important goal of AMP related research. In this work, we present quantitative structure activity relationships for toxicity of protegrin-like antimicrobial peptides against human cells (epithelial and red blood cells) based on physicochemical properties, such as interaction energies and radius of gyration, calculated from molecular dynamics simulations of the peptides in aqueous solvent. The hypothesis is that physicochemical properties of peptides, as manifest by their structure and interactions in a solvent and as captured by atomistic simulations, are responsible for their toxicity against human cells. Protegrins are beta-hairpin peptides with high activity against a wide variety of microbial species, but in their native state are toxic to human cells. Sixty peptides with experimentally determined toxicities were used to develop the models. We test the resulting relationships to determine their ability to predict the toxicity of several protegrin-like peptides. The developed QSARs provide insight into the mechanism of cytotoxic action of antimicrobial peptides. In a subsequent blind test, the QSAR correctly ranked four of five protegrin analogues newly synthesized and tested for toxicity.     

The antimicrobial peptide cathelicidin interacts with airway mucus

Antimicrobial peptides (AMPs) and mucins are components of airway secretions and both contribute to the innate host defense system. At neutral pH, AMPs are positively charged, mucins negatively. It was the aim of the study to test whether these opposite charges result in interactions between AMPs and mucins. We measured binding of mucins isolated from porcine gastric mucosa to the cathelicidin LL-37 coated to multiwell plates and found that LL-37 electrostatically interacts with mucins. Circular dichroism spectra of the peptide revealed the induction of -helical conformation by mucins. Addition of mucins to solutions of LL-37 significantly decreased the antimicrobial activity of the peptide against Pseudomonas aeruginosa and Streptococcus pneumoniae. We then tested whether LL-37 is bound to mucins in airway secretions from human subjects and found that a significant proportion of the peptide and its propeptide are bound to high molecular weight components. Together these data show that cationic AMPs interact with anionic mucins in airway secretions. Functions of AMPs are modulated by this interaction.     

The salmonid cathelicidins: A gene family with highly varied C-terminal antimicrobial domains

The cathelicidin family of host defence peptides is regarded as an important component of the host innate immune system. Its members have been found in mammals, birds, primitive vertebrate Atlantic hagfish and, most recently, also in ray-finned fish such as rainbow trout and Atlantic salmon. By using genomic PCR amplifications and RT-PCR tissue analyses we have here investigated and characterized the cathelicidin gene family in three salmonids: brown trout (Salmo trutta fario), brook trout (Salvelinus fontinalis) and grayling (Thymallus thymallus). One or two different genes were found in each species coding for almost identical cathelin-like domains and largely varied cationic C-terminal regions. Multiple alignment of the amino acid sequences let us recognize two distinctive hallmarks of these peptides: the presence of a high number of serine and glycine residues, which may collocate them in a new class of antimicrobial peptides, and of the six-amino-acid repeated sequence RPGGGS detected in a variable number of copies among different cathelicidins. The high variation in length and sequence of this region suggests the existence of a genetically unstable region similar to that found in some mammalian cathelicidins.     

Structural congruence among membrane-active host defense polypeptides of diverse phylogeny

A requisite for efficacious host defense against pathogens and predators has prioritized evolution of effector molecules thereof. A recent multidimensional analysis of physicochemical properties revealed a novel, unifying structural signature among virtually all classes of cysteine-containing antimicrobial peptides. This motif, termed the γ-core, is seen in host defense peptides from organisms spanning more than 2.6 billion years of evolution. Interestingly, many toxins possess the γ-core signature, consistent with discoveries of their direct antimicrobial activity. Many microbicidal chemokines (kinocidins) likewise contain iterations of the γ-core motif, reconciling their antimicrobial efficacy. Importantly, these polypeptide classes have evolved to target and modulate biomembranes in protecting respective hosts against unfavorable interactions with potential pathogens or predators. Extending on this concept, the current report addresses the hypothesis that antimicrobial peptides, kinocidins, and polypeptide toxins are structurally congruent and share a remarkably close phylogenetic relationship, paralleling their roles in host-pathogen relationships. Analyses of their mature amino acid sequences demonstrated that cysteine-stabilized antimicrobial peptides, kinocidins, and toxins share ancient evolutionary relatedness stemming from early precursors of the γ-core signature. Moreover, comparative 3-D structure analysis revealed recurring iterations of antimicrobial peptide γ-core motifs within kinocidins and toxins. However, despite such congruence in γ-core motifs, the kinocidins diverged in overall homology from microbicidal peptides or toxins. These findings are consistent with observations that chemokines are not toxic to mammalian cells, in contrast to many antimicrobial peptides and toxins. Thus, specific functions of these molecular effectors may be governed by specific configurations of structural modules associated with a common γ-core motif. These concepts are consistent with the hypothesis that the γ-core is an archetype determinant in polypeptides that target or regulate with biological membranes, with specific iterations optimized to unique or cognate host defense contexts. Quantitative and qualitative data suggest these protein families emerged through both parallel and divergent processes of modular evolution. Taken together, the current and prior findings imply that the γ-core motif contributes to conserved structures and functions of host defense polypeptides. The presence of this unifying molecular signature in otherwise diverse categories of membrane-active host defense peptides implies an ancient and essential role for such a motif in effector molecules governing host-pathogen relationships.     

Simple oligomers as antimicrobial peptide mimics

New approaches to antibiotic design are desperately needed. The design of simple oligomers that capture the shape and biological function of natural antimicrobial peptides could prove to be versatile and highly successful. We discuss the use of aromatic backbones to design facially amphiphilic (FA) -sheet like structures which are potently antimicrobial. These oligomers capture the physiochemical properties of peptides like the Magainins and Defensins, which fold into specific conformations that are amphiphilic resulting in antimicrobial activity. However, natural peptides are expensive to prepare and difficult to produce on large scale. The design of polymers and oligomers that mimic the complex structures and remarkable biological properties of proteins is an important endeavor and provides attractive alternatives to the difficult synthesis of natural peptides. We therefore have designed a series of FA oligomers that are easy to prepare from inexpensive monomers. They adopt structures very reminiscent of amphiphilic -sheets and have significant activity with minimal inhibitory concentrations at 6 h in the low microgram per ml range (M to nM). They are active against a broad spectrum of bacteria including gram-positive and gram-negative as well as antibiotic resistant strains.     

Structure-activity studies of dermorphin. The role of side chains of amino acid residues on the biological activity of dermorphin

Seventeen analogues of dermorphin were synthesized and bio-assayed to determine the influence of side chains of the individual amino acid residues forming the sequence of dermorphin on the biological activity of this opioid peptide. Syntheses were carried out using solid-phase procedure, and the analogues obtained were purified by gel filtration on Sephadex G-10. Biological activities determined in guinea pig ileum (GPI) and mouse vas deferens (MVD) tests showed that the N-terminal tetrapeptide is responsible for the activity of dermorphin. Substitutions in the C-terminal fragment, particularly in position 5, for other amino acid residues results in substantial differentiation towards mu and delta receptors.     

Discovery and synthesis of 6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-one-based novel chemotype CCR2 antagonists via scaffold hopping strategy

The chemokine CC receptor subtype 2 (CCR2) has attracted intensive interest for drug development in diverse therapeutic areas, including chronic inflammatory diseases, diabetes, neuropathic pain, atherogenesis and cancer. By employing a cut-and-sew scaffold hopping strategy, we identified an active scaffold of 3,4-dihydro-2,6-naphthyridin-1(2H)-one as the central pharmacophore to derive novel CCR2 antagonists. Systematic structure–activity relationship study with respect to the ring size and the substitution on the naphthyridinone ring gave birth to 1-arylamino-6-alkylheterocycle-6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-ones as a brand new chemotype of CCR2 antagonists with nanomolar inhibitory activity. The best antagonism activity in this series was exemplified by compound 13a, which combined the optimal substitutions of 3,4-dichlorophenylamino at C-1 and 3-(4-(N-methylmethylsulfonamido)piperidin-1-yl)propyl at N-6 position, leading to an IC₅₀ value of 61?nM and 10-fold selectivity for CCR2 over CCR5. Efficient and general synthesis was established to construct the innovative core structure and derive the compound collections. This is the first report on our designed 6,7,8,9-tetrahydro-5H-pyrido[4,3-c]azepin-5-one as novel CCR2 antagonist scaffold and its synthesis.     

Dual assay for MCLV3 activity reveals structure-activity relationship of CLE peptides

The dodecapeptide MCLV3 is a functional peptide, derived from the CLV3 precursor protein, which is a candidate ligand of the CLV1/CLV2 receptor complex that restricts the stem cell population in the shoot apical meristem (SAM). MCLV3 can induce shoot and root meristem consumption, the typical phenotype of transgenic plants overexpressing CLV3. We investigated the bioactivities of a series of alanine-substituted MCLV3 and related peptides on the root growth of Arabidopsis. The structure-activity relationship (SAR) of MCLV3 had high similarity with that of tracheary element differentiation inhibitory factor (TDIF). We also evaluated the binding activities of the peptides by a competitive receptor binding assay using tritiated MCLV3 and the membrane fraction of a tobacco BY-2 cell line overexpressing the MCLV3 ectodomain. This dual assay, combining a biological and receptor binding assay for evaluating the activities of MCLV3-related peptides, uncovered the SAR of MCLV3, and indicated that the terminal residues play critical roles in exerting its activity and are important for specific binding to the receptor, CLV1.     

Action mechanism and structural requirements of the antimicrobial peptides, gaegurins

Gaegurins (GGNs) are a family of cationic, α-helical, antimicrobial peptides that were isolated from a Korean frog, Glandirana emeljanovi (formerly classified as Rana rugosa) and represent one of the structurally well-characterized groups. Among six gaegurins, gaegurin 4 (renamed herein esculentin-2EM), gaegurin 5 (brevinin-1EMa), and gaegurin 6 (brevinin-1EMb) have been investigated comprehensively in terms of structure-activity relationships. In this paper, we first suggest renaming of gaegurins according to a recently raised rule of systematic nomenclature. Then, the current understanding of gaegurins is reviewed by summarizing their structure-activity relationships. In particular competing arguments on gaegurins are synthetically inspected. Finally their action mechanism and structural requirements will be discussed.     

Synthesis,in vitro β-glucuronidase inhibitory activity and in silico studies of novel (E)-4-Aryl-2-(2-(pyren-1-ylmethylene)hydrazinyl)thiazoles

Current research is based on the synthesis of novel (E)-4-aryl-2-(2-(pyren-1-ylmethylene)hydrazinyl)thiazole derivatives (3–15) by adopting two steps route. First step was the condensation between the pyrene-1-carbaldehyde (1) with the thiosemicarbazide to afford pyrene-1-thiosemicarbazone intermediate (2). While in second step, cyclization between the intermediate (2) and phenacyl bromide derivatives or 2-bromo ethyl acetate was carried out. Synthetic derivatives were structurally characterized by spectroscopic techniques such as EI-MS, ¹H NMR and ¹³C NMR. Stereochemistry of the iminic double bond was confirmed by NOESY analysis. All pure compounds 2–15 were subjected for in vitro β-glucuronidase inhibitory activity. All molecules were exhibited excellent inhibition in the range of IC₅₀ = 3.10 ± 0.10–40.10 ± 0.90 μM and found to be even more potent than the standard d-saccharic acid 1,4-lactone (IC₅₀ = 48.38 ± 1.05 μM). Molecular docking studies were carried out to verify the structure-activity relationship. A good correlation was perceived between the docking study and biological evaluation of active compounds.     

Intein-mediated expression is an effective approach in the study of beta-defensins

Mammalian beta-defensins are an important family of host defense peptides with diverse functions. Surprisingly most of the mammalian beta-defensin genes are revealed preferentially expressed in the male organs. There is a pressing need to understand how the ample defensin repertoires work in both host defense and fertility with an aim to overcome antibiotic resistance of pathogens and reproductive problems. The biggest obstacle is the production of beta-defensin peptides as beta-defensins are small, antimicrobial and multi-disulfide molecules. In this study, the well documented HBD2, function-unknown RBD1 and function-partly-known rBin1b are successfully expressed and assayed. This approach overcomes the difficulties in beta-defensin production and provides a convenient and economical peptide-production platform to elucidate the antimicrobial activities and clinical prospects of beta-defensins. In the strategy of recombinant expression, this approach may be the best to develop the "natural" peptide pools for both host defense and fertility in a cost-effective manner.     

Examination of α-exosite inhibitors against Botulinum neurotoxin A protease through structure-activity relationship studies of chicoric acid

Botulinum neurotoxins (BoNT) are among the most toxic known substances and currently there are no effective treatments for intraneuronal BoNT intoxication. Chicoric acid (ChA) was previously reported as a BoNT/A inhibitor that binds to the enzyme’s α-exosite. Herein, we report the synthesis and structure-activity relationships (SARs) of a series of ChA derivatives, which revealed essential binding interactions between ChA and BoNT/A. Moreover, several ChA-based inhibitors with improved potency against the BoNT/A were discovered.