Structural determinants for the membrane interaction of novel bioactive undecapeptides derived from gaegurin 5

Gaegurin 5 is a 24-residue, membrane-active antimicrobial peptide isolated from the skin of an Asian frog, Rana rugosa. We recently reported the antimicrobial activities of two novel undecapeptides derived from an inactive N-terminal fragment (residues 1-11) of gaegurin 5 (Won, et al. J. Biol. Chem. 2004, 279, 14784-14791). In the present work, the anticancer activities of the two antimicrobial undecapeptide analogues were additionally identified. The relationships between their structural properties and biological activities were assessed by characterizing the fundamental structural determinant for the basic membrane interaction. The circular dichroism and nuclear magnetic resonance results revealed that in a membrane-mimetic environment, the active peptides adopt a more stabilized helical conformation than that of the inactive fragment, and this conformation conferred an overall amphipathicity to the active peptides. Therefore, the most decisive factor responsible for the activity and selectivity could be the intramolecular amphipathic cooperativity, rather than the amphipathicity itself. Especially, the tryptophan residue of the active peptides seems to play a crucial role at the critical amphipathic interface that promotes and balances the amphipathic cooperativity by stabilizing both the hydrophilic and hydrophobic interactions with the membrane. Altogether, the present results suggest that the two novel undecapeptides are worthy of therapeutic development as new antibiotic and anticancer agents and provide structural information about their action mechanism.     

Design of potent, non-toxic anticancer peptides based on the structure of the antimicrobial peptide, temporin-1CEa

Recent advances in the search for novel anticancer agents have indicated that the positively charged antimicrobial peptides have emerged as promising agents offering several advantages over the conventional anticancer drugs. As a naturally occurring, cationic, α-helical antimicrobial peptide, temproin-1CEa has been proved to exhibit a potent anticancer effect and a moderate hemolytic activity. In order to reduce the hemolytic activity of temporin-1CEa and improve its anticancer potency towards a range of human breast cancer cells, in the present study, six analogs of temporin-1CEa were rationally designed and synthesized. The amphipathicity levels and α-helical structural patterns of peptides were reserved, while their cationic property and hydrophobicity were changed. The results of MTT and hemolysis assay indicated that the analog peptides displayed an improved anticancer activity and showed an overall optimized therapeutic index. The hydrophobicity of peptides was positively correlated with their hemolytic and antitumor activities. Moreover, the data suggest a strategy of increasing the cationicity while maintaining the moderate hydrophobicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells such as human erythrocytes. This work highlights the potential for rational design and synthesis of improved antimicrobial peptides that have the capability to be used therapeutically for treatment of cancers.     

Proteome of monocled cobra (Naja kaouthia) venom and potent anti breast cancer peptide from trypsin hydrolyzate of the venom protein

Anticancer peptide is one of the target in the development of new anticancer drug. Bioactive peptide can be originated from isolated free peptide or produced by hydrolysis of protein. Protein is the main component of Naja kaouthia venom, and due to the toxicity of the venom, it can be assessed as the source of anticancer peptides. This study aims to characterize the venom protein and to identify peptides from the snake venom of N. kaouthia as anticancer. Proteome analysis was employed trypsin hydrolysis of N. kaouthia venom protein completed with HRMS analysis protein database query. Preparative tryptic hydrolysis of the protein followed by reverse-phased fractionation and anti breast cancer activity testing were performed to identify the potent anticancer from the hydrolysate. Proteomic analysis by high-resolution mass spectrometry revealed that there are 20 enzymatic and non-enzymatic proteins in N. kaouthia venom. The 25% methanol peptide fraction had the most active anticancer activity against MCF-7 breast cancer cells and showed promising selectivity (selectivity index = 12.87). Amino acid sequences of eight peptides were identified as potentially providing anticancer compounds. Molecular docking analysis showed that WWSDHR and IWDTIEK peptides gave specific interactions and better binding affinity energy with values of −9.3 kcal/mol and −8.4 kcal/mol, respectively. This study revealed peptides from the snake venom of N. kaouthia became a potent source of new anticancer agents.     

In vitro antibacterial and antimalarial activity of dehydrophenylalanine-containing undecapeptides alone and in combination with drugs

A set of three cationic undecapeptides, analogous to the previously reported peptide VS2 (KWΔFWKΔFVKΔFVK), was created by alanine substitution in order to probe the effect of hydrophobicity on peptide activity. The activities of these peptides were determined against Escherichia coli, Staphylococcus aureus and the malaria parasite Plasmodium falciparum. VA1, the closest analogue of VS2, showed five-fold augmented activity [minimum inhibitory concentration (MIC)=10 μM] against the Gram-positive bacterium S. aureus. The designed analogues were non-haemolytic and non-cytotoxic at their MICs and clinically relevant concentrations. By alanine substitution, it was also possible to probe the critical role of tryptophan residues in determining peptide potency. Circular dichroism studies of the peptides in a membrane-mimetic system showed a correlation between peptide helicity and antimicrobial activity. The peptides were also tested in combination with sublethal concentrations of antibiotic drugs (rifampicin and kanamycin) and the antimalarial drug chloroquine. In combination with these drugs, the effect of the peptides was synergistic or additive. These results provide insight into basic design principles for generating new clinically relevant lead peptides. It also provides an alternative strategy where a peptide and a non-peptide drug can be used in combination to battle increasingly drug-resistant microbes.     

Structural and charge requirements for antimicrobial and hemolytic activity in the peptide PKLLETFLSKWIG,corresponding to the hydrophobic region of the antimicrobial protein bovine seminalplasmin

Several analogs of the 13-residue antimicrobial and hemolytic peptide PKLLETFLSKWIG (SPF), which is the most hydrophobic region of the 47-residue antimicrobial protein seminalplasmin [Sitaram, N. & Nagaraj, R. (1990) J. Biol. Chem. 265, 10438-104423 have been synthesized. The antimicrobial and hemolytic properties of the peptides were investigated with a view to gain an insight into the structural and charge requirements for these activities of SPF. Peptides in which E was replaced by K exhibited considerably improved antimicrobial activity with no concomitant increase in hemolytic activity. A peptide in which the aromatic amino acids were replaced by leucine exhibited antimicrobial activity like those of the peptides which had aromatic amino acids. Interchange in the positions of E and K and total replacement of K by E resulted in complete loss of activity. The peptides having antimicrobial activities showed appreciable helical content in a hydrophobic environment, whereas inactive peptides did not. Thus, by suitable‘engineering’ the biological activity of a short 13-residue peptide can be altered to yield peptides specifically having only antimicrobial activity with increased potency. © Munksgaard 1995.     

HAZ,a novel peptide with broad-spectrum antibacterial activity

ObjectiveThe growing microbial resistance to antibiotics is a global public concern, which creates serious needs for newer antimicrobial agents. Antimicrobial peptides (AMPs) are increasingly exploited in drug development as therapeutic candidates. Here, we aimed to design and characterize a novel peptide with broad spectrum antimicrobial activity.MethodsHybridization and sequence modification approaches were used to design the novel peptide, named HAZ, aiming at optimizing the physicochemical parameters involved in antimicrobial activity. Peptide activities were assessed alone or combined with different selected antibiotics against various sensitive and drug-resistant bacterial strains. In addition, the hemolysis and the cytotoxic activities of HAZ peptide were evaluated on human red blood cells and epithelial adenocarcinoma cells (A549), respectively.ResultsHAZ peptide was sequentially modified to result in favored physicochemical parameters (helicity 95.24 %, hydrophobic ratio 47 %, and net charge of 8 + ). Functional assessment of HAZ revealed significant antimicrobial activity, with MIC values of 15 – 20 µM against tested bacterial strains. It also exhibited biofilm eradication activity at slightly higher concentrations. HAZ-antibiotics combinations exhibited a synergistic action mode that led to dramatic decrease in the MIC values for both HAZ peptide and the antibiotic. Such efficacy was accompanied with minimal hemolytic toxicity on human erythrocytes. Importantly, HAZ displayed promising anticancer activity against human lung cancer cells.ConclusionRationally-designed antimicrobial peptides offer promising alternatives to the current antibiotics for management of infectious diseases. HAZ peptide is a broad-spectrum AMP, and a promising candidate for antimicrobial and anticancer drug development.     

Convergent evolution-guided design of antimicrobial peptides derived from influenza A virus hemagglutinin

Antimicrobial activity and solution structures of four 13-amino acid peptides derived from the fusion domain of viral hemagglutinin proteins are presented. The results show that carboxyl-terminal amidation is a key factor to switch a viral fusion domain-derived sequence into an antimicrobial peptide. Optimization of amphiphilic balance on the amidated analogue largely improves efficacy and enlarges antimicrobial spectra of these peptides. Our work indicates that viral fusion domains have potential to be engineered into potent antimicrobial peptides.     

Database screening and in vivo efficacy of antimicrobial peptides against methicillin-resistant Staphylococcus aureus USA300

Natural antimicrobial peptides (AMPs) are promising candidates for developing a generation of new antimicrobials to meet the challenge of antibiotic-resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). To facilitate the search for new candidates, we have utilised the Antimicrobial Peptide Database (APD), which contains natural AMPs from bacteria, fungi, plants and animals. This study demonstrates the identification of novel templates against MRSA by screening 30 peptides selected from the APD. These peptides are short (<25 residues), cysteine-free, cationic and represent candidates from different biological sources such as bacteria, insects, arachnids, tunicates, amphibians, fish and mammals. Six peptides, including ascaphin-8, database-screened antimicrobial peptide 1 (DASamP1), DASamP2, lycotoxin I, maculatin 1.3 and piscidin 1, were found to exert potent antimicrobial activity against an MRSA USA300 isolate. Although five of the six peptides showed broad-spectrum antibacterial activity, DASamP1 displayed killing of MRSA in vitro but not of Escherichia coli, Bacillus subtilis or Pseudomonas aeruginosa. In addition, DASamP1 suppressed early biofilm formation in a mouse model of catheter-associated MRSA infection. DASamP1 is a novel, short and potent peptide that will be a useful starting template for further developing novel anti-MRSA peptides.     

Discovery of two bombinin peptides with antimicrobial and anticancer activities from the skin secretion of Oriental fire‐bellied toad,Bombina orientalis

Amphibian skin secretions are known to contain numerous peptides with a large array of biological activities. Bombinins are a group of amphibian‐derived peptides with broad spectrum antimicrobial activities that have been only identified from the ancient toad species, Bombina. In this study, we described the identification and characterization of a novel bombinin precursor which encoded a bombinin‐like peptide (BLP‐7) and a novel bombinin H‐type peptide (named as Bombinin H‐BO) from the skin secretion of Oriental fire‐bellied toad, Bombina orientalis. The primary structures of both mature peptides were determined by combinations of molecular cloning of peptide precursor‐encoding cDNAs and mass spectrometry techniques. Secondary structure prediction revealed that both peptides had cationic amphipathic α‐helical structural features. The synthetic replicate of BLP‐7 displayed more potent antimicrobial activity than Bombinin H‐BO against Gram‐positive and Gram‐negative bacteria and yeast. Also, in vitro antitumour assay showed that both peptides possessed obvious antiproliferative activity on three human hepatoma cells (Hep G2/SK‐HEP‐1/Huh7) at the non‐toxic doses. These results indicate the peptide family of bombinins could be a potential source of drug candidates for anti‐infection and anticancer therapy.     

Structure-function relationships and conformational properties of alpha-MSH(6-13) analogues with candidacidal activity

Alpha-melanocyte-stimulating hormone (alpha-MSH) is an endogenous linear tridecapeptide with potent anti-inflammatory effects. We firstly demonstrated that alpha-MSH and its C-terminal sequence Lys-Pro-Val [alpha-MSH(11-13)] have antimicrobial effects against two major and representative pathogens: Staphylococcus aureus and Candida albicans. Successively, in an attempt to improve the candidacidal activity of alpha-MSH and to better understand the peptide structure-antifungal activity relations, we have recently designed and synthesized novel peptide analogues. We focused on the sequence alpha-MSH(6-13), which contains the invariant melanocortin core sequence His-Phe-Arg-Trp (6-9) and also contains the sequence Lys-Pro-Val (11-13) important for antimicrobial activity. In that structure-activity study, we discovered several compounds that have greater candidacidal activity than alpha-MSH, among which the peptide [d-Nal-7,Phe-12]-alpha-MSH(6-13) was the most potent. Here, we report a detailed conformational analysis by spectroscopic and computational methods of three peptides, alpha-MSH(6-13) (1), [d-Nal-7,Phe-12]-alpha-MSH(6-13) (2) and [d-Nal-7,Asp-12]-alpha-MSH(6-13) (3). Peptides were chosen on the basis of their candidacidal activities and were studied in membrane mimetic environment (SDS micelles). Different turn structures were observed for the three peptides and a conformation-activity model was developed based on these results. This study offers a structural basis for the design of novel peptide and non-peptide analogues to be used as new antimicrobial agents.     

Design,synthesis, and antimicrobial activities of novel functional peptides against Gram‐positive and Gram‐negative bacteria

The extensive use of antibiotics in medicine results in the multidrug resistance of bacteria, making the development of new antimicrobial agents an urgent need. Antimicrobial peptides (AMPs) are considered as a new class of antibiotic with characteristics including an ability to kill target cells rapidly and a broad spectrum of activity. We have developed a potent antimicrobial peptide MAP‐0403 (MIC = 5 μM), but it exhibited a high hemolytic side‐effect (70.7%). To reduce its hemolytic effect and increase antimicrobial activity, three peptides derivatives of MAP‐0403 (J‐1, J‐2, and J‐3) were designed, synthesized by solid phase synthesis, purified by RP‐HPLC, and characterized by MALDI‐TOF MS. Structure–activity relationships of these peptides were studied by using circular dichroism and antimicrobial assays. The percentage of helical structure in J‐1, J‐2, and J‐3 was lower than that of MAP‐0403. The antimicrobial activity of J‐1 was the same as that of MAP‐0403 (MIC = 5 μM), J‐2 exhibited the highest activity (MIC = 2.5 μM), while J‐3 showed decreased activity (MIC = 10 μM). Compared to MAP‐0403, J‐2 showed significantly reduced hemolytic effect (3.4%), while J‐1 and J‐3 showed slightly decreased hemolytic effect (46.2%, 55.6%, respectively). Peptide J‐2 was discovered as a novel and potent antimicrobial agents.     

Marine peptides and related compounds in clinical trial

Advances in the sophisticated instruments for the isolation and characterization of marine natural products, and development in the biological assay systems, have resulted in the discovery of various compounds of biomedical application. Marine natural products have been a source of new leads for the treatment of many deadly diseases such as cancer, acquired immuno-deficiency syndrome (AIDS) etc. The compounds of marine origin are diverse in structural class from simple linear peptides to complex macrocyclic polyethers. Number of marine peptides have been isolated in recent years which exhibit potent biological activities, and many of the compounds showed promising anticancer activity. Didemnin was the first marine peptide that entered in human clinical trials in US for the treatment of cancer, and other anticancer peptides such as kahalalide F, hemiasterlin, dolastatins, cemadotin, soblidotin, didemnins and aplidine have entered in the clinical trials. Clinical status of anticancer marine derived peptides have been discussed and reviewed.     

Lipid membrane-binding properties of tryptophan analogues of linear amphipathic beta-sheet cationic antimicrobial peptides using surface plasmon resonance

Using a surface plasmon resonance (SPR) system, we investigated the lipid membrane-binding properties of four analogues of the 18-residue linear amphipathic beta-sheet cationic antimicrobial peptide (KIGAKI)3-NH2, each of which contains a single isoleucine-to-tryptophan substitution. The results of the SPR study revealed significant differences in the binding characteristics of the peptides depending upon the position of tryptophan residues. These peptides showed higher binding affinity to membranes containing acidic phospholipids than zwitterionic phospholipids. The addition of dimethylsulfoxide to the running buffer was effective in maintaining the solubility of these peptide solutions and obtaining concentration-dependent sensorgrams for the kinetic analysis in this study. The kinetic binding data of SPR correlated closely with both the ability of the peptides to lyse liposomes with the same phospholipid composition and bactericidal activity. The results demonstrate that SPR may be a valuable tool to predict the membrane lytic properties of antimicrobial peptides.     

Structure–biological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin

The antimicrobial peptide, lactoferricin, is generated upon the gastric pepsin cleavage of lactoferrin and has many basic and hydrophobic amino acid residues essential for its biological activity. To investigate the structure-antimicrobial activity relationships, the basic amino acid-rich region of bovine lactoferricin (BLFC), RRWQWRMKKLG, was selected. Using chemically synthesized BLFC and its substituted peptides, the antimicrobial activities of the peptides were tested by determining the minimal inhibitory concentration (MIC) of Escherichia coli and Bacillus subtilis and the disruption of the outer cell membrane of E. coli, and the peptide's toxicities were assayed by hemolysis. The short peptide (B3) composed of only 11 residues had similar antimicrobial activities while losing most of the hemolytic activities as compared with the 25 residue-long ones (B1 and B2). The short peptides (B3, B5 and B7) with double arginines at the N-termini had more potent antimicrobial activity than those (B4 and B6) with lysine. However, no antimicrobial and hemolytic activities were found in B8, in which all basic amino acids were substituted with glutamic acid, and in B9, in which all hydrophobic amino acids were substituted with alanine. The circular dichroism (CD) spectra of the short peptides in 30 MM SDS were correlated with their antimicrobial activities. These results suggested that the 11-residue peptide of BLFC is involved in the interaction with bacterial phospholipid membranes and plays an important role in antimicrobial activity with little or no hemolytic activity. © Munksgaard 1996.     

Rational design of α-helical antimicrobial peptides to target Gram-negative pathogens, Acinetobacter baumannii and Pseudomonas aeruginosa: utilization of charge, 'specificity determinants,' total hydrophobicity, hydrophobe type and location as design parameters to improve the therapeutic ratio

The rapidly growing problem of increased resistance to classical antibiotics makes the development of new classes of antimicrobial agents with lower rates of resistance urgent. Amphipathic cationic α‐helical antimicrobial peptides have been proposed as a potential new class of antimicrobial agents. The goal of this study was to take a broad‐spectrum, 26‐residue, antimicrobial peptide in the all‐D conformation, peptide D1 (K13) with excellent biologic properties and address the question of whether a rational design approach could be used to enhance the biologic properties if the focus was on Gram‐negative pathogens only. To test this hypothesis, we used 11 and 6 diverse strains of Acinetobacter baumannii and Pseudomonas aeruginosa, respectively. We optimized the number and location of positively charged residues on the polar face, the number, location, and type of hydrophobe on the non‐polar face and varied the number of ‘specificity determinants’ in the center of the non‐polar face from 1 to 2 to develop four new antimicrobial peptides. We demonstrated not only improvements in antimicrobial activity, but also dramatic reductions in hemolytic activity and unprecedented improvements in therapeutic indices. Compared to our original starting peptide D1 (V13), peptide D16 had a 746‐fold improvement in hemolytic activity (i.e. decrease), maintained antimicrobial activity, and improved the therapeutic indices by 1305‐fold and 895‐fold against A. baumannii and P. aeruginosa, respectively. The resulting therapeutic indices for D16 were 3355 and 895 for A. baumannii and P. aeruginosa, respectively. D16 is an ideal candidate for commercialization as a clinical therapeutic to treat Gram‐negative bacterial infections.     

Antimicrobial activities of human beta-defensins against Bacillus species

Natural defences in the human body function to protect us from numerous environmental toxins and exposure to potential harmful biological agents. An important frontline defence is antimicrobial peptides. These peptides occur at environmental interfaces and serve to limit bacterial invasion. There has been little work comparing specific peptides as potential antimicrobial compounds. In this study, we evaluated the antimicrobial activity of peptides from the human beta-defensin (HBD) family against four species of Bacillus, chosen as models for Bacillus anthracis, a potential bioweapon. The impact of peptide concentration, sequence and protein binding was evaluated on their biological activity. The results indicated that HBD-3 was the most biologically active against Bacillus subtilis and Bacillus licheniformis, whilst HBD-2 was found to be most active against Bacillus cereus and Bacillus thuringiensis. Moreover, the antimicrobial activity of the peptides was directly related to peptide concentration and indirectly related to albumin concentration (i.e. protein binding).     

5-氟尿嘧啶衍生物的合成与抗肿瘤活性研究

目的寻找新型、高效的5-氟尿嘧啶衍生物类抗肿瘤化合物。方法设计、合成4个5-氟尿嘧啶衍生物,利用MTT方法测定这些化合物对4株肿瘤细胞的增殖抑制活性。结果 4个5-氟尿嘧啶衍生物均显示了较好的肿瘤细胞增殖抑制活性。结论 4个目标化合物对肿瘤细胞有较好的抑制活性,值得进一步研究。     

Anticancer activity of VmCT1 analogs against MCF‐7 cells

Antimicrobial peptides are considered promising drug candidates due to their broad range of activity. VmCT1 (Phe–Leu–Gly–Ala–Leu–Trp–Asn–Val–Ala–Lys–Ser–Val–Phe–NH₂) is an α‐helical antimicrobial peptide that was obtained from the Vaejovis mexicanus smithi scorpion venom. Some of its analogs showed to be as antimicrobial as the wild type, and they were designed for understanding the influence of physiochemical parameters on antimicrobial and hemolytic activity. Some cationic antimicrobial peptides exhibit anticancer activity so VmCT1 analogs were tested to verify the anticancer activity of this family of peptides. The analogs were synthesized, purified, characterized, and the conformational studies were performed. The anticancer activity was assessed against MCF‐7 mammary cancer cells. The results indicated that [Glu]⁷‐VmCT1‐NH₂, [Lys]³‐VmCT1‐NH₂, and [Lys]⁷‐VmCT1‐NH₂ analogs presented moderated helical tendency (0.23–0.61) and tendency of anticancer activity at 25 μmol/L in 24 hr of experiment; and [Trp]⁹‐VmCT1‐NH₂ analog that presented low helical tendency and moderated anticancer activity at 50 μmol/L. These results demonstrated that single substitutions on VmCT1 led to different physicochemical features and could assist on the understanding of anticancer activity of this peptide family.     

Synthesis and biological evaluation of novel peptides based on antimicrobial peptides as potential agents with antitumor and multidrug resistance‐reversing activities

Tumor chemotherapy, which plays an important role in the clinical treatment of metastatic cancer, is limited by low selectivity and drug resistance in clinical application. In our study, we selected antimicrobial peptide BP100 as a lead peptide, designed, and synthesized a series of novel antineoplastic peptides through solid‐phase synthesis. Among them, B4 and B8 showed excellent anticancer activity. As revealed by further investigations, these peptides could disrupt the cell membrane, trigger the cytochrome C release into cytoplasm, and ultimately lead to apoptosis. In addition, they also showed multidrug resistance‐reversing effects by performing effective antitumor activity against multidrug‐resistant cells. As a result, these peptides may possibly be regarded as a promising candidate for cancer treatment.