The medicinal chemistry of short lactoferricin-based antibacterial peptides

This review discusses antibacterial peptides from the perspective of development into clinically useful chemotherapeutic drugs using short lactoferricin based peptides as examples. The review shows how important features for antibacterial activity can be identified and explored using the molecular properties of a range of natural and non-natural amino acids. The results have been further refined quantitatively using a "soft-modelling" approach where important structural parameters that influence the antibacterial activity of 15-residue model peptides were identified. The review describes how this knowledge is utilised to generate pharmacophores for antibacterial efficacy. These pharmacophores turn out to be surprisingly small and relatively consistent between typical Gram-negative and Gram-positive bacteria leading to the discovery of a novel class of short synthetic cationic antimicrobial peptides. These compounds are found to have high antibacterial activity against several bacterial strains that are resistant to commercial antibiotics, and are promising as future clinical candidates for treatment of infections caused by several clinically relevant pathogens.     

Tailoring an antibacterial peptide of human lysosomal cathepsin G to enhance its broad-spectrum action against antibiotic-resistant bacterial pathogens

Neutrophils contain several cationic antimicrobial proteins or peptides (CAPs) that exert antibiotic-like action against bacteria. These host-derived antibiotics kill susceptible bacteria by oxygen-independent mechanisms. Considerable interest in their activity has been generated in recent years due not only to their likely important role in innate host defense against infection, but also their possible use as therapeutic agents in treating infections caused by antibiotic-resistant pathogens. We have studied the antibacterial properties of human lysosomal cathepsin G (cat G). This highly cationic serine protease contains at least three antibacterial regions that by themselves can exert antibacterial action against Gram-negative bacteria, such as Pseudomonas aeruginosa. Only one of these peptides, defined by residues 117-136 of full-length cat G, has bactericidal action against Gram-positive pathogens, such as Staphylococcus aureus. Due to the broad-spectrum antibacterial action of this peptide, we have sought to define the amino acids within its primary sequence required for this activity and have developed variants with improved activity. This review emphasizes the importance of both cationicity and hydrophobicity as necessary characteristics for the antibacterial action of CAPs. It also proposes the strategy that naturally occurring large human CAPs can be dissected to smaller CAPs and then modified to enhance their activity in vitro. This approach could prove beneficial to those interested in developing antimicrobial peptides as therapeutic agents.     

Analogues of peptide SMAP-29 with comparable antimicrobial potency and reduced cytotoxicity

SMAP-29 (sheep myeloid antimicrobial peptide-29) is a peptide with potent antibacterial properties. However, it is also highly cytotoxic both to human red blood cells (hRBCs) and human embryonic kidney (HEK) cells. In this study, some of the amino acids of SMAP-29 were changed in an attempt to reduce haemolytic activity whilst maintaining high antibacterial efficacy. These analogues, plus other analogues described in the literature with potent antimicrobial activity against Gram-positive bacteria coupled with no or low haemolytic activity, were evaluated for their cytotoxicity (hRBCs and HEK cells) as well as antimicrobial efficacy against two Gram-positive (Bacillus anthracis and Bacillus globigii) and two Gram-negative bacteria (Escherichia coli and Burkholderia thailandensis). The analogues previously described in the literature were found to have low antibacterial and haemolytic activity. Two of the designed analogues had comparable antibacterial efficacy with SMAP-29 against B. anthracis but reduced haemolytic activity and therefore had a therapeutic index that was enhanced 2.3-2.6-fold over that of SMAP-29.     

Bacterial cell wall compounds as promising targets of antimicrobial agents II. Immunological and clinical aspects

The bacterial cell wall represents the primary target for antimicrobial agents. Microbial destruction is accompanied by the release of potent immunostimulatory membrane constituents. Both Gram-positive and Gram-negative bacteria release a variety of lipoproteins and peptidoglycan fragments. Gram-positive bacteria additionally provide lipoteichoic acids, whereas Gram-negative bacteria also release lipopolysaccharide (LPS, endotoxin), essential component of the outer leaflet of the bacterial cell wall and one of the most potent immunostimulatory molecules known. Immune activation therefore can be considered as an adverse effect of antimicrobial destruction and killing during anti-infective treatment. In contrast to antibiotics, the use of cationic amphiphilic antimicrobial peptides allows both effective bacterial killing and inhibition of the immunostimulatory effect of the released bacterial membrane constituents. The administration of antimicrobial peptides alone or in combination with antibiotic agents thus represents a novel strategy in the antiinfective treatment with potentially important beneficial aspects. Here, data are presented which describe immunological and clinical aspects of the use of antimicrobial peptides (AMPs) as therapeutic agents to treat bacterial infection and neutralize the immunostimulatory activity of released cell wall constituents.     

Ceragenins (cationic steroid compounds), a novel class of antimicrobial agents

Ceragenins are a group of cholic acid derivatives that have been chemically modified to make them cationic amphiphiles. Several of these derivatives exhibit antimicrobial activity against a broad range of bacteria. These compounds have advantages over cationic amphipathic peptides in that they are resistant to proteolysis and they incorporate stably into membranes. Although some forms of ceragenins are effective against both Gram-negative and Gram-positive bacteria, they are generally more potent against Gram-positive bacteria. Surprisingly, it is not the cell wall, but the high content of phosphatidylethanolamine in most Gram-negative bacteria that endow them with resistance. Ceragenins have the unusual property of forming complexes with phospholipids. Factors contributing to the mechanism of action of these agents are discussed. The ceragenins are a class of agents with many properties to make them favorable for application as antiinfective agents.     

Screening of Ethanol, Petroleum Ether and Chloroform Extracts of Medicinal Plants, Lawsonia inermis L. and Mimosa pudica L. for Antibacterial Activity

Organic extracts (ethanol, petroleum ether and chloroform) of two medicinal plants Lawsonia inermis L. and Mimosa pudica L. were proven for antibacterial properties against 15 Gram-positive and Gram-negative human pathogenic bacteria. Among the three types of extracts tested, ethanol extract was found to possess maximum antibacterial activity. The diameter of the zone of inhibition of bacterial growth showed that Gram-negative bacteria are more sensitive than Gram-positive bacteria to plant extracts. Between the two plants species studied, Lawsonia inermis extract showed more antibacterial activity compared to Mimosa pudica extract.     

Identification of a Novel Cathelicidin from the Deinagkistrodon acutus Genome with Antibacterial Activity by Multiple Mechanisms

The abuse of antibiotics and the consequent increase of drug-resistant bacteria constitute a serious threat to human health, and new antibiotics are urgently needed. Research shows that antimicrobial peptides produced by natural organisms are potential substitutes for antibiotics. Based on Deinagkistrodon acutus (known as five-pacer viper) genome bioinformatics analysis, we discovered a new cathelicidin antibacterial peptide which was called FP-CATH. Circular dichromatic analysis showed a typical helical structure. FP-CATH showed broad-spectrum antibacterial activity. It has antibacterial activity to Gram-negative bacteria and Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The results of transmission electron microscopy (TEM) and scanning electron microscopy (SEM) showed that FP-CATH could cause the change of bacterial cell integrity, having a destructive effect on Gram-negative bacteria and inducing Gram-positive bacterial surface formation of vesicular structure. FP-CATH could bind to LPS and showed strong binding ability to bacterial DNA. In vivo, FP-CATH can improve the survival rate of nematodes in bacterial invasion experiments, and has a certain protective effect on nematodes. To sum up, FP-CATH is likely to play a role in multiple mechanisms of antibacterial action by impacting bacterial cell integrity and binding to bacterial biomolecules. It is hoped that the study of FP-CATH antibacterial mechanisms will prove useful for development of novel antibiotics.     

Isolation and characterisation of antimicrobial peptides from deer neutrophils

The New Zealand deer industry is the largest and most advanced in the world. Antimicrobial peptides have been isolated from a wide range of organisms, but as yet there have been no reports on any from deer. This work investigates the antimicrobial activity and characterisation of peptides isolated from Cervus elaphus blood. It was found that deer blood contains proline/arginine-rich cathelicidins, similar to Bac5 peptides isolated from cattle, sheep and goats. A beta-defensin was also isolated that had a conserved amino acid sequence and mass similar to bovine neutrophil beta-defensins. The cathelicidin displayed strong activity against Gram-negative bacteria, but lesser activity against Gram-positive bacteria and yeast, whilst the beta-defensin showed good activity against all three test organisms.     

Design and synthesis of cationic antimicrobial peptides with improved activity and selectivity against Vibrio spp

Extensive use of classical antibiotics has led to the growing emergence of many resistant strains of pathogenic bacteria. Evidence has suggested that cationic antimicrobial peptides (AMPs) are of greatest potential to represent a new class of antibiotics. The largest group of AMPs comprises peptides that fold into an amphipathic alpha-helical conformation when interacting with the target microorganism. In the current study, a series of cationic AMPs of 20 amino acids was designed and synthesised based on four structural parameters, including charge, polar angle, hydrophobicity and hydrophobic moment. The effect of these parameters on antimicrobial activity and selectivity was assessed by structural and biological analyses. Our results indicated that high hydrophobicity and amphipathicity (hydrophobic moment) were correlated with increased haemolytic activity, whilst antimicrobial activity was found to be less dependent on these factors. Three of the synthetic AMPs (GW-Q4, GW-Q6 and GW-H1) showed higher antimicrobial activity and selectivity against a broad spectrum of Gram-positive and Gram-negative bacteria compared with the naturally occurring AMPs magainin 2a and pleurocidin. This study also demonstrates that these rationally designed cationic and amphipathic helical AMPs exhibited high selectivity against several Vibrio spp. and are potential agents for future use in the treatment of these marine pathogens.     

An investigation on the antibacterial, cytotoxic, and antibiofilm efficacy of starch-stabilized silver nanoparticles

The increased emergence of drug resistant microbes creates a major challenge to the scientific community for successful development of effective therapeutics. The antimicrobial activities of silver ions are well known, but limited information is available on the effects of green silver-nanoparticles (AgNPs) on human pathogens. In this article, we evaluated the antibacterial activity of starch-stabilized AgNPs against a panel of human pathogens commonly associated with air, water and food borne infections. The shape and size distribution of AgNPs were characterized by transmission electron microscopy. We showed that AgNPs were more effective against Gram-positive and Gram-negative pathogens as compared with acid-fast bacteria. AgNPs were not cytotoxic to macrophages at the bactericidal concentration and can augment intracellular killing potential of macrophages. Furthermore, we showed that AgNPs disrupt biofilm formation and exhibit better antibacterial activity compared to human cationic antimicrobial peptide LL-37. In summary, our data suggest AgNPs as a promising template for the design of novel antibacterial agents.     

Biological properties of low molecular mass peptide dendrimers

A series of new, low molecular mass, lysine-based peptide dendrimers with varying distribution of cationic and aromatic groups in the structure were synthesized. They expressed antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria as well as against fungal pathogens (Candida albicans). Their cytotoxic, haematotoxic, and genotoxic effects were studied. It appears that degree of branching and steric distribution and types of hydrophobic (aromatic) groups and cationic centres are important components of dendrimeric structure and influence both antimicrobial potency and toxicity. Such 3D structure of our dendrimers mimics that of the natural antimicrobial peptides and can be achieved by application of dendrimer chemistry.     

Antimicrobial peptides in animals and their role in host defences

Domesticated animals have a large variety of antimicrobial peptides that serve as natural innate barriers limiting microbial infection or, in some instances, act as an integral component in response to inflammation or microbial infection. These peptides differ in size, composition, mechanisms of activity and range of antimicrobial specificities. They are expressed in many tissues, polymorphonuclear leukocytes, macrophages and mucosal epithelial cells. There is a small group of anionic antimicrobial peptides found in ruminants and a much larger group of cationic antimicrobial peptides found in all domesticated animals. The cationic peptides include linear, helical peptides, linear peptides rich in proline and cysteine-stabilized peptides with a beta-sheet and are commonly referred to as cathelicidins and defensins. These peptides are generally broad-spectrum for Gram-positive bacteria, Gram-negative bacteria and fungi (e.g. myeloid antimicrobial peptides, alpha-, beta-defensins, and protegrins) or are specific to one of these groups (e.g. porcine cecropin P1, Bac5, Bac7, PR-39 and prophenin).     

In vitro antibacterial and hemolytic activities of crotamine, a small basic myotoxin from rattlesnake Crotalus durissus

Crotamine, a myotoxin from the venom of South American rattlesnake, is structurally related to β-defensins, antimicrobial peptides (AMPs) found in vertebrate animals. Here, we tested the antibacterial properties of crotamine and found that it killed several strains of Escherichia coli, with the MICs ranging from 25 to 100 μg ml?1. Time-kill and bacterial membrane permeabilization assays revealed that killing of bacteria by crotamine occurred within 1 h and reached the maximum by 2 h. Additionally, the anti-E. coli activity of crotamine was completely abolished with 12.5 mM NaCl. Furthermore, the three intramolecular disulfide bonds of crotamine appeared dispensable for its antibacterial activity. The reduced form of crotamine was active against E. coli as well. However, crotamine showed no or weak activity up to 200 μg ml?1 against other species of Gram-negative and Gram-positive bacteria. Crotamine showed no appreciable hemolytic activity to erythrocytes. Our studies revealed that crotamine is also an AMP that kills bacteria through membrane permeabilization. However, crotamine appears to have a narrow antibacterial spectrum, distinct from many classical β-defensins, reinforcing the notion that crotamine originated from the β-defensin gene lineage, but has undergone significant functional diversification.     

Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. These new antimicrobials should possess both novel modes of action as well as different cellular targets compared with the existing antibiotics. Lysozyme, muramidase, and aprotinin, a protease inhibitor, both exhibit antimicrobial activities against different microorganisms, were chosen as model proteins to develop more potent bactericidal agents with broader antimicrobial specificity. The antibacterial specificity of lysozyme is basically directed against certain Gram-positive bacteria and to a lesser extent against Gram-negative ones, thus its potential use as antimicrobial agent in food and drug systems is hampered. Several strategies were attempted to convert lysozyme to be active in killing Gram-negative bacteria which would be an important contribution for modern biotechnology and medicine. Three strategies were adopted in which membrane-binding hydrophobic domains were introduced to the catalytic function of lysozyme, to enable it to damage the bacterial membrane functions. These successful strategies were based on either equipping the enzyme with a hydrophobic carrier to enable it to penetrate and disrupt the bacterial membrane, or coupling lysozyme with a safe phenolic aldehyde having lethal activity toward bacterial membrane. In a different approach, proteolytically tailored lysozyme and aprotinin have been designed on the basis of modifying the derived peptides to confer the most favorable bactericidal potency and cellular specificity. The results obtained from these strategies show that proteins can be tailored and modelled to achieve particular functions. These approaches introduced, for the first time, a new conceptual utilization of lysozyme and aprotinin, and thus heralded a great opportunity for potential use in drug systems as new antimicrobial agent.     

Alignment-based design and synthesis of new antimicrobial Aurein-derived peptides with improved activity against Gram-negative bacteria and evaluation of their toxicity on human cells

Considering the worldwide increasing prevalence of resistance to traditional antibiotics, it is necessary to find new antibiotics to deal with this issue. Recently, antimicrobial peptides (AMPs) have been proposed as new antimicrobial agents. Aureins are a family of AMPs that are isolated from Green and Golden Bell Frogs. These peptides have a favorable antibacterial activity against Gram-positive bacteria. We designed two peptides derived from natural Aurein enjoying alignment-based design method. After synthesis of the peptides, their secondary structure was checked by circular dichroism. Consequently, the antibacterial effects of these peptides were investigated by determining the minimum inhibitory concentration (MIC) and bactericidal concentration. Eventually, the toxicity of these peptides was determined by MTT (3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide) assay on normal human skin cells (Hu02 cell line). Natural Aurein1.2 was used as a natural control to compare the properties in all stages. The results indicated that these new peptides had medium-upward antimicrobial activity against Escherichia coli, Pseudomonas aeruginosa, and Bacillus subtilis (MIC of 8–64 μg/mL) and weak bactericidal activity against Staphylococcus aureus (MIC of 128–256 μg/mL). Also, MTT assays results showed that AureinN2 is less toxic than AureinN1 and Aurein1.2. Toxicity of AureinN2 for Hu02 cell lines was between 20 and 40% at the concentration of 8–500 μg/mL. In this study, we were able to improve antimicrobial activity of two synthetic derivatives of the Aurein family against Gram-negative bacteria by using machine-learning algorithm and other in silico methods.     

Therapeutic potential of the bactericidal/permeability-increasing protein

Innate immune mechanisms respond rapidly to bacterial infection. A key cellular component of the innate immune response is the neutrophil, whose cytoplasmic granules contain a variety of antimicrobial proteins and peptides. Among these is the bactericidal/permeability-increasing protein (BPI), a cationic 55 kDa protein whose selective anti-infective action against Gram-negative bacteria is based on its high (nM) affinity for lipopolysaccharide (LPS, or "endotoxin"). Binding of BPI to Gram-negative bacteria results in growth inhibition, serves as an opsonin that enhances phagocytosis of bacteria and inhibits bacteria-induced inflammatory responses by blocking the interaction of LPS with host pro-inflammatory pathways. Expression of BPI appears to be developmentally regulated as human newborns apparently have lower neutrophil BPI levels than adults. BPI expression has also recently been demonstrated in human epithelial cells where it appears to be inducible by endogenous anti-inflammatory lipids (lipoxins). BPI's potent anti-endotoxic activity against a broad range of Gram-negative bacterial pathogens is manifest in biological fluids and renders it an attractive template for pharmaceutical development. Indeed, rBPI(21), an active recombinant protein derived from human BPI, has proven safe in Phase I human trials, shown promise in Phase II trials and has recently completed a Phase III trial for severe meningococcaemia with apparent benefit. Identification and evaluation of additional disease entities characterised by Gram-negative bacteraemia and/or endotoxaemia as possible targets for BPI therapy continues.     

Therapeutic potential of the

Innate immune mechanisms respond rapidly to bacterial infection. A key cellular component of the innate immune response is the neutrophil, whose cytoplasmic granules contain a variety of antimicrobial proteins and peptides. Among these is the bactericidal/permeability-increasing protein (BPI), a cationic 55 kDa protein whose selective anti-infective action against Gram-negative bacteria is based on its high (nM) affinity for lipopolysaccharide (LPS, or “endotoxin”). Binding of BPI to Gram-negative bacteria results in growth inhibition, serves as an opsonin that enhances phagocytosis of bacteria and inhibits bacteria-induced inflammatory responses by blocking the interaction of LPS with host pro-inflammatory pathways. Expression of BPI appears to be developmentally regulated as human newborns apparently have lower neutrophil BPI levels than adults. BPI expression has also recently been demonstrated in human epithelial cells where it appears to be inducible by endogenous anti-inflammatory lipids (lipoxins). BPI’s potent anti-endotoxic activity against a broad range of Gram-negative bacterial pathogens is manifest in biological fluids and renders it an attractive template for pharmaceutical development. Indeed, rBPI₂₁, an active recombinant protein derived from human BPI, has proven safe in Phase I human trials, shown promise in Phase II trials and has recently completed a Phase III trial for severe meningococcaemia with apparent benefit. Identification and evaluation of additional disease entities characterised by Gram-negative bacteraemia and/or endotoxaemia as possible targets for BPI therapy continues.     

In-vitro activity of panipenem against clinical isolates in 2006

The antimicrobial activity of various antibiotics against clinical bacterial isolates recovered from patients with infectious diseases at the medical facilities in the Kanto region between March and September 2006 was evaluated. A total of 1030 clinical isolates were available for susceptibility tests: 420 aerobic Gram-positive organisms, 520 aerobic Gram-negative organisms, 30 anaerobic Gram-positive organisms and 60 anaerobic Gram-negative pathogens. Antimicrobial susceptibility data for Streptococcus pneumoniae and Haemophilus influenzae isolates from pediatric and adult patients were analyzed separately. Panipenem (PAPM), imipenem (IPM), meropenem (MEPM), biapenem (BIPM), doripenem (DRPM), cefozopran (CZOP), cefepime (CFPM), and sulbactam/cefoperazone (SBT/CPZ) were used as test antibiotics. PAPM, IPM and DRPM exhibited excellent in vitro antibacterial activities against methicillin-susceptible Staphylococcus, with all isolates exhibiting a MIC of < or =0.06 microg/mL. Against Streptococcus including penicillin-resistant S. pneumoniae, PAPM demonstrated the strongest antibacterial activity among the carbapenems with a MIC range of < or =0.06 to 0.12 microg/mL. Against Enterobacteriaceae, MEPM showed the strongest antibacterial activity, and PAPM had comparable activity to IPM. Against the extended-spectrum beta-lactamase producing Escherichia coli, Klebsiella species and Proteus species, the MICs for the cephems were high, however, those for the carbepenems were low. Against H. influenzae, PAPM had comparable activity to IPM. With respect to anaerobes, each of the carbapenems tested demonstrated almost the same strong antibacterial activity. In conclusion, 13 years has passed since PAPM was launched in 1993, PAPM still maintains potent antibacterial activity and is considered an effective antimicrobial agent for various types of infectious diseases.     

富含脯氨酸的抗菌肽研究进展

富含脯氨酸的抗菌肽是一族分离自动物的线性多肽 ,它们都具有抗菌活性 ,在天然免疫中具有重要的作用。这类抗菌肽按来源又分为两类 :来自哺乳动物的富含脯氨酸的抗菌肽和来自昆虫和其他脊椎动物的富含脯氨酸的抗菌肽。它们都富含脯氨酸 ,对革兰阴性菌起作用 ,其杀菌机制不同于大多抗菌肽的膜溶解作用。它们进入细菌胞质后 ,结合DnaK蛋白 ,抑制DnaK的生物功能 ,最终导致了细菌的死亡。这一作用机制对于药物的开发很有意义。除了抗菌作用外 ,富含脯氨酸的抗菌肽还具有其他重要的药理作用。对这方面作用研究比较多的是PR 39,它在诱导粘连蛋白聚糖在间充质细胞的表达 ,抑制嗜中性粒细胞NADPH氧化酶 ,抑制 1κBα和HIF 1α的降解等方面具有重要作用。这些发现表明PR 39在伤口修复、炎症、缺血再灌注损伤以及诱导血管生成等方面具有很好的应用前景。     

Antibacterial effect of four phenothiazines

Various types of phenothiazines were examined for antibacterial effect on 61 Gram-positive and Gram-negative bacterial strains in vitro. The investigated phenothiazines were two neuroleptic drugs, fluphenazine and chlorpromazine, and two antihistaminic drugs, alimemazine and promethazine. All four drugs have antibacterial effects in vitro, the phenothiazines being more potent against the Gram-positive microorganisms. The antibacterial potency of the drugs was measured as IC50: Fluphenazine 29 microM (15 micrograms/ml), alimemzaine 49 microM (37 micrograms/ml), promethazine 88 microM (28 micrograms/ml) and chlorpromazine 92 microM (29 micrograms/ml). The antibacterial potency of the drugs was linked neither to the neuroleptic nor the antihistaminic potency of the drugs, which is in agreement with results of earlier stereoisomeric investigations. Thus, the known phenothiazines may represent a pool of potentially new antimicrobial drugs. A therapeutic application of these results, however, requires additional in vitro an in vivo testing in an animal model. The bacterial model might be of value as a model system in the study of the interaction of neuropharmacological agents and other membrane active compounds on biological membranes.