A mini review on the antimicrobial peptides isolated from the genus <Emphasis Type="Italic">Hylarana</Emphasis> (Amphibia: Anura) with a proposed nomenclature for amphibian skin peptides

Hylarana is a well established frog genus coming under the family Ranidae. An increasing number of antimicrobial peptides have been isolated and characterized from the skin of frogs of this genus. This review covers the antimicrobial peptides reported so far from the frogs of Hylarana genus and to propose a consistent system of nomenclature for amphibian skin peptides. Multiple sequence alignment of the skin peptides from Hylarana genus has grouped them into six peptide families, and three bioactive peptides. Existing nomenclature of amphibian antimicrobial peptides is species centered with no implication to the genus which can lead to disparities, when frogs with same species name belonging to different genus have to be named. As per the proposed system the peptide should have the parent peptide name (e.g. Brevinin-1) followed by two uppercase letter of the genus, if two genera begin with the same letter–first letter should be the same followed by an appropriate second letter (e.g. HU for Huia and HM for Humenerana). This is succeeded by species name in lower case-orthologous peptides from different species may be characterized by the initial letter of that species, when two species begin with the same initial letter, second letter should be used appropriately (e.g. HLat for Hylarana aurata and HLan for Hylarana aurantiaca). Paralogs belonging to the same peptide family are assigned by numbers.     

Isolation and Some Properties of the Antimicrobial Peptide (Pa-AMP) from the Seeds of Pokeweed (Phytolacca americana).

An antimicrobial peptide, designated Pa-AMP, was purified by gel filtration on Sephadex G-75 followed by S-Sepharose, Cosmosil-SP, and reverse-phase HPLC from the seeds of pokeweed (Phytolacca americana). Pa-AMP is a basic peptide having an isoelectric point of over 10 and its extinction coefficient at 280 nm of 1% aqueous solution was 7.7. Pa-AMP has a molecular mass of 4 kDa and 3.4 kDa on tricine SDS-PAGE under nonreducing and reducing conditions, respectively. The N-terminal amino acid of Pa-AMP was blocked. The concentrations of peptide required for 50% inhibition (IC₅₀) of the growth of plant pathogenic fungi, Gram-positive, and Gram-negative bacteria were 3 to 41 μg/ml. Differing from other peptides, Pa-AMP inhibited the growth of some Gram-negative bacteria.     

Studies on in vivo induction of HIV-1 envelope-specific cytotoxic T lymphocytes by synthetic peptides from the V3 loop region of HIV-1 IIIB gp120

We have previously reported the induction of MHC class I-restricted, CD8⁺ cytotoxic T lymphocytes (CTLs) specific to human immunodeficiency virus type 1 (HIV-1) in mice by a 15-amino acid peptide (R15K) from the V3 loop in gp120. We now present evidence showing that CTL activity induced by R15K was stable for 8–10 weeks after a single injection and that as little as 20 μg peptide was sufficient for efficient CTL induction in vivo. While induction of CTLs was efficient with R15K emulsified in either complete or incomplete Freund's adjuvant, only a low-level CTL response was observed in mice immunized with R15K in either alum or saline. We analyzed a series of carrier-free synthetic peptides ranging in length from 8 to 24 amino acids from the V3 loop region and observed that peptide R10I consisting of 10 amino acids from the middle portion of R15K was more efficient for CTL induction. Additionally, lymph node cells from mice immunized with 24 and 15 amino acid peptides (N24G and R15K, respectively) when restimulated in vitro with R10I exhibited greater HIV-1 env-specific CTL activity than when either of the longer peptides was used for restimulation. A peptide consisting of only 8 amino acids (R8K) was sufficient neither for inducing primary CTLs nor for in vitro restimulation of lymph node CTL precursors. These results establish that a carrier-free 10-amino acid synthetic peptide from the V3 loop region in HIV-1 gp120 has the optimal sequence for efficient induction of HIV env-specific CTLs in mice.     

Preparation of Golgi Membrane Fractions Containing Lectin-binding Sites from Suspension-cultured Rice Cells

Bluegill-degrading bacteria were isolated from various environmental sources. Brevibacillus sp. BGM1 degraded bluegill efficiently at 50 °C, and its culture supernatant showed the highest peptide and amino acid concentrations as trichloroacetic acid (TCA) soluble fraction (ASF) (10.7 mg/ml) of all supernatants obtained with bluegill as a substrate. Strain BGM1 secreted a protease(s) into the medium, and the concentration of peptides and amino acids gradually increased. The fertile effect of the degraded bluegill products (DGP) on Brassica rapa was also investigated. The root hair density of B. rapa grown with DGP at a concentration of 30 μg peptides and amino acids/ml was about 1.7 times higher than when grown with the same concentration of undegraded bluegill. DGP was shown to increase root hair numbers and adventitious root formation. The results of this study suggest that a specific peptide(s) for promotion of root hair is produced from the order Perciformes with a protease(s) from BGM1.     

Fungicidal activity of the human peptide hepcidin 20 alone or in combination with other antifungals against Candida glabrata isolates

Candida glabrata infections are often difficult to eradicate due to the intrinsically low susceptibility to azoles of this species. In addition, C. glabrata has also been shown to be insensitive to several cationic peptides, which have been shown to be promising novel therapeutic candidates for the treatment of fungal infection. In this study, the in vitro fungicidal activity of the human cationic peptide hepcidin 20 (Hep-20) was evaluated against clinical isolates of C. glabrata with different levels of fluconazole susceptibility. Interestingly, all isolates were susceptible to Hep-20 (100–200 μg/ml) at pH 7.4, whereas the fungicidal effect of the peptide was higher (50 μg/ml) at acidic pH values. In addition, an increased antifungal activity was observed for Hep-20 with amphotericin B and a synergistic effect was demonstrated for the Hep-20/fluconazole and Hep-20/caspofungin combinations.     

Comparative aspects of natriuretic peptide physiology in non-mammalian vertebrates: a review

The natriuretic peptide system is a complex family of peptides and receptors that is primarily linked to the maintenance of osmotic and cardiovascular homeostasis. A natriuretic peptide system is present in each vertebrate class but there are varying degrees of complexity in the system. In agnathans and chondrichthyians, only one natriuretic peptide has been identified, while new data has revealed that multiple types of natriuretic peptides are present in bony fish. However, it seems in tetrapods that there has been a reduction in the number of natriuretic peptide genes, such that only three natriuretic peptides are present in mammals. The peptides act via a family of guanylyl cyclase receptors to generate the second messenger cGMP, which mediates a range of physiological effects at key targets such as the gills, kidney and the cardiovascular system. This review summarises the current knowledge of the natriuretic peptide system in non-mammalian vertebrates and discusses the physiological actions of the peptides.Abbreviations ANP atrial natriuretic peptide - AVT arginine vasotocin - BNP brain natriuretic peptide - cDNA complementary deoxyribonucleic acid - CNP C-type natriuretic peptide - cAMP adenosine 3, 5 cyclic monophosphate - cGMP guanosine 3, 5 cyclic monophosphate - GC guanylyl cyclase - GFR glomerular filtration rate - mRNA messenger ribonucleic acid - NPR natriuretic peptide receptor - NPs natriuretic peptides - sCP salmon cardiac peptide - VIP vasoactive intestinal peptide - VNP ventricular natriuretic peptide Communicated by I.D. Hume     

Inhibitory and anti‐inflammatory effects of two antimicrobial peptides moronecidin and temporin‐1Dra against Propionibacterium acnes in vitro and in vivo

Proliferation of Propionibacterium acnes (P. acnes) is one of the main pathogenetic mechanisms of acne. Antimicrobial peptides with low‐drug resistance and nonresidual are potential anti‐acne agents. In this study, two antimicrobial peptides named temporin‐1Dra and moronecidin were synthesized and tested their antimicrobial activity against P. acnes in vitro and in vivo. These two peptides inhibited the growth of Escherichia coli, Staphylococcus aureus, Candida albicans, and P. acnes. The minimal inhibitory concentrations (MICs) of temporin‐1Dra and moronecidin to P. acnes were 30 and 10 μM, respectively. Both peptides exhibited strong resistance to heat and pH, but no obvious cytotoxicity to HaCaT cells. They also displayed persistent antimicrobial activities in the microbial challenge test. In the P. acnes‐induced inflammation mouse model, moronecidin significantly decreased the ear swelling thickness in a concentration‐dependent manner. At the 14th day after injection, 20 μg/day moronecidin reduced the ear swelling thickness to 46.15 ± 5.23% compared with the normal cream group. Tissue staining showed that moronecidin effectively reduced abscess and thickness of the dermis layer. Our results indicate that the antimicrobial peptide moronecidin could be developed as a potential natural anti‐acne agent in the cosmetics or pharmaceutical industries.     

In vitro activity of novel in silico‐developed antimicrobial peptides against a panel of bacterial pathogens

Antimicrobial‐peptide‐based therapies could represent a reliable alternative to overcome antibiotic resistance, as they offer potential advantages such as rapid microbicidal activity and multiple activities against a broad spectrum of bacterial pathogens. Three synthetic antimicrobial peptides (AMPs), AMP72, AMP126, and also AMP2041, designed by using ad hoc screening software developed in house, were synthesized and tested against nine reference strains. The peptides showed a partial β‐sheet structure in 10‐mM phosphate buffer. Low cytolytic activity towards both human cell lines (epithelial, endothelial, and fibroblast) and sheep erythrocytes was observed for all peptides. The antimicrobial activity was dose dependent with a minimum bactericidal concentration (MBC) ranging from 0.17 to 10.12 μM (0.4–18.5 µg/ml) for Gram‐negative and 0.94 to 20.65 μM (1.72‐46.5 µg/ml) for Gram‐positive bacteria. Interestingly, in high‐salt environment, the antibacterial activity was generally maintained for Gram‐negative bacteria. All peptides achieved complete bacterial killing in 20 min or less against Gram‐negative bacteria. A linear time‐dependent membrane permeabilization was observed for the tested peptides at 12.5 µg/ml. In a medium containing Mg²⁺ and Ca²⁺, the peptide combination with EDTA restores the antimicrobial activity particularly for AMP2041. Moreover, in combination with anti‐infective agents (quinolones or aminoglycosides) known to bind divalent cation, AMP126 and AMP2041 showed additive activity in comparison with colistin. Our results suggest the following: (i) there is excellent activity against Gram‐negative bacteria, (ii) there is low cytolytic activity, (iii) the presence of a chelating agent restores the antimicrobial activity in a medium containing Mg²⁺ and Ca²⁺, and (iv) the MBC value of the combination AMPs–conventional antibiotics was lower than the MBC of single agents alone. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Novel antibacterial peptides induced by probiotics in Hermetia illucens (Diptera: Stratiomyidae) larvae

There is a need to discover new therapeutic substances due to the emergence of deadly infectious diseases and various antibiotic resistance. We focused on the larvae that are utilized as a medical insect for the treatment of skin damage in Europe and America. This study was to investigate the pharmacological activities of novel antibacterial peptides isolated from Hermetia illucens larvae against the Klebsiella pneumoniae and Shigella dysenteriae. The larvae were immunized by probiotics (Lactobacillus casei) for 24 h. The hemolymph from the immunized larvae was fractionated through reverse‐phase chromatography. Peptides were purified using HPLC and the coomassie blue staining, and identified using Nano‐LC‐ESI‐MS/MS system. Antibacterial activities of the peptides were evaluated by turbidometric assay, liquid broth dilution assay, resazurin assay, and agar disk diffusion method. The minimum inhibitory concentrations (MICs) of the peptides were measured as 150 μg/mL through the turbidometric, liquid broth dilution, and resazurin assays. The peptides effectively inhibited their growth/proliferation as well as the survival rate of the tested bacteria. Furthermore, the immunized larvae exhibited overexpression of the peptides compared to non‐immunized larvae. These results demonstrate that the peptides induced by H. illucens exert strong antibacterial activity against Gram‐negative bacteria. The results suggest that the activation of the humoral immunity induced by immunization functioned to enhance the production of antibacterial peptides from the insect and their antibacterial properties. This study indicates the potential of the peptides produced from larvae as antibacterial peptide substance for the development of novel antibacterial drugs.     

SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC₅₀) value of each peptide was greater than 1000 μg mL⁻¹. The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 μg mL⁻¹ concentration showed a reduction of more than log₁₀ 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log₁₀ 4.8 to log₁₀ 2.5 and log₁₀ 3.1 respectively. The concentration of SLAM homologous peptide (25 μg mL⁻¹) to exert its anti-PPRV effect was much less than its CC₅₀ level (>1000 μg mL⁻¹). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.     

Establishment of an in Vitro Model of the Human Placental Barrier by Placenta Slice Culture and Ussing Chamber

Aggregation occurs through hydrophobic interactions when a polypeptide chain refolds in non-native states or when genetic variants of biologically active proteins assume inappropriate conformations, as observed in the case of dysfunctional serpins. Here, using the molecular chaperone BiP from bovine liver microsomes, we characterized the hydrophobic nature of the peptide segment which is considered to be a site required for aggregation among a non-inhibitory serpin ovalbumin in a heat-denatured state. Screening of the peptide scan for binding of BiP showed that BiP-binding sites are mostly buried in the folded ovalbumin. When ovalbumin was heat-denatured, the denatured protein was recognized by the antibody that reacts with the hydrophobic surface of the amino-terminal segment of ovalbumin. This antibody significantly suppressed the binding of BiP to denatured ovalbumin. BiP also bound the immobilized peptide in an ATP-dependent manner and the peptide stimulated the ATPase activity of BiP with a K _m of 165 μM and a V _max of 0.4 nmol/min per milligram. Measurement of surface plasmon resonance showed that the peptide had a K _d of 0.52 μM by BiP, lower than that for RCMLA (K _d=1.1 μM) and even lower than that of the peptide P10K, PLSRTLSVAAKK, (K _d=21 μM). These results demonstrate that the aggregation-prone site on heat-denatured ovalbumin has almost the same hydrophobic nature of interacting with the molecular chaperone BiP as the conventionally known peptides that bind to the Escherichia coli chaperone DnaK.     

Construction and synthesis of lactoferricin derivatives with enhanced antibacterial activity

A series of peptides derived from sequences from human, bovine, murine and caprine lactoferrin has been prepared and investigated for antibacterial effect. Among the four species investigated peptides based on the bovine sequence displayed significant activity. The bovine sequence, bovine lactoferricin, showed a MIC value of 30 μg/mL on E. coli and S. aureus, whereas the three other lactoferricins possessed MIC values above 200 μg/mL. Based on these findings, novel peptides with enhanced antibacterial activities, were prepared with sequences designed by molecular modelling and structure‐activity studies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

The immunoregulatory effects of edeine analogues in mice

The edeines analogs were tested in several in vitro and in vivo assays using the mouse model, with edeine B (peptide W1) and cyclosporine A as reference compounds. The peptides displayed moderate, stimulatory effects on concanavalin A-induced (ConA-induced) splenocyte proliferation, whereas their effects on pokeweed mitogen-induced (PWM-induced) splenocyte proliferation were inhibitory. The peptides inhibited lipopolysacharide-induced (LPS-induced) tumor necrosis factor alpha production but had little effect on interleukin 6 production. In the model of the humoral immune response in vitro to sheep red blood cells, peptide 1 was distinctly stimulatory in the investigated concentrations (1-100 μg/ml), whereas peptides 3 and 4 only stimulated the number of antibody-forming cells at the highest concentration (100 μg/ml). In the model of the delayed type hypersensitivity in vivo to ovalbumin, the peptides were moderately suppressive (3 being the most active). The reference peptide W1 stimulated ConA-induced cell proliferation at 1–10 μg/ml but was inhibitory at 100 μg/ml. It also inhibited PWM-induced cell proliferation in a dose-dependent manner. This peptide had no effect on the humoral immune response in vitro or on cytokine production, but inhibited DTH reaction in vivo. The relationship between structure and activity, and a possible mode of action of the peptides, is discussed in this paper.     

Relationships between the Circadian System and Alzheimer's Disease-Like Symptoms in Drosophila

Circadian clocks coordinate physiological, neurological, and behavioral functions into circa 24 hour rhythms, and the molecular mechanisms underlying circadian clock oscillations are conserved from Drosophila to humans. Clock oscillations and clock-controlled rhythms are known to dampen during aging; additionally, genetic or environmental clock disruption leads to accelerated aging and increased susceptibility to age-related pathologies. Neurodegenerative diseases, such as Alzheimer''s disease (AD), are associated with a decay of circadian rhythms, but it is not clear whether circadian disruption accelerates neuronal and motor decline associated with these diseases. To address this question, we utilized transgenic Drosophila expressing various Amyloid-β (Aβ) peptides, which are prone to form aggregates characteristic of AD pathology in humans. We compared development of AD-like symptoms in adult flies expressing Aβ peptides in the wild type background and in flies with clocks disrupted via a null mutation in the clock gene period (per⁰¹). No significant differences were observed in longevity, climbing ability and brain neurodegeneration levels between control and clock-deficient flies, suggesting that loss of clock function does not exacerbate pathogenicity caused by human-derived Aβ peptides in flies. However, AD-like pathologies affected the circadian system in aging flies. We report that rest/activity rhythms were impaired in an age-dependent manner. Flies expressing the highly pathogenic arctic Aβ peptide showed a dramatic degradation of these rhythms in tune with their reduced longevity and impaired climbing ability. At the same time, the central pacemaker remained intact in these flies providing evidence that expression of Aβ peptides causes rhythm degradation downstream from the central clock mechanism.     

Tertiary structure-related activity of tick defensin (persulcatusin) in the taiga tick, <Emphasis Type="Italic">Ixodes persulcatus</Emphasis>

Defensins are small cysteine-rich cationic proteins found in both vertebrates and invertebrates constituting the front line of host innate immunity. To examine the importance of the tertiary structure of tick defensin in its antimicrobial activity, we synthesized two types of the peptides with tertiary structure or primary one on basis of the information of the sequence in the defensin originated from the taiga tick, Ixodes persulcatus. Chemically synthesized peptides were used to investigate the activity spectrum against Staphylococcus aureus, Borrelia garinii and flora-associated bacteria. Both synthetic peptides showed antimicrobial activity against S. aureus in short-time killing within 1 h, but they do not show the activity against B. garinii, Stenotrophomonas maltophila and Bacillus spp., which were frequently isolated from the midgut of I. persulcatus. The teriary structure brought more potent activity to S. aureus than primary one in short-time killing. We also examined its antimicrobial activity by evaluation of growth inhibition in the presence of the synthetic peptides. Minimum inhibitory concentration (MIC) was ranged from 1.2 to 5.0 μg/ml in tertiary peptide and from 10 to 40 μg/ml in primary peptide, when 10 strains of S. aureus were used. From the curve of cumulative inhibition rates, MIC50 (MIC which half of the strains showed) to S. aureus is about 1.2 μg/ml in the peptide with tertiary structure and about 10 μg/ml in the linear one. Corynebacterium renale is 10 times or more sensitive to tertiary peptide than primary one. In conclusion, the presence of 3 disulfide bridges, which stabilize the molecule and maintain the tertiary structure, is considered to have an effect on their antimicrobial activities against Gram-positive bacteria such as S. aureus.     

Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF)

To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5–8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in flies but for other bioactive peptides in various invertebrates. Arch. Insect Biochem. Physiol. 37:146–157, 1998. © 1998 Wiley-Liss, Inc.     

Structural and functional characterization of peptides derived from the carboxy‐terminal region of a defensin from the tick Ornithodoros savignyi

Tick defensins may serve as templates for the development of multifunctional peptides. The purpose of this study was to evaluate shorter peptides derived from tick defensin isoform 2 (OsDef2) in terms of their antibacterial, antioxidant, and cytotoxic activities. We compared the structural and functional properties of a synthetic peptide derived from the carboxy‐terminal of the parent peptide (Os) to that of an analogue in which the three cysteine residues were omitted (Os–C). Here, we report that both peptides were bactericidal (MBC values ranging from 0.94–15 µg/ml) to both Gram‐positive and Gram‐negative bacteria, whereas the parent peptide only exhibited Gram‐positive antibacterial activity. The Os peptide was found to be two‐fold more active than Os–C against three of the four tested bacteria but equally active against Staphylococcus aureus. Os showed rapid killing kinetics against both Escherichia coli and Bacillus subtilis, whereas Os–C took longer, suggesting different modes of action. Scanning electron microscopy showed that in contrast to melittin for which blebbing of bacterial surfaces was observed, cells exposed to either peptide appeared flattened and empty. Circular dichroism data indicated that in a membrane‐mimicking environment, the cysteine‐containing peptide has a higher α‐helical content. Both peptides were found to be non‐toxic to mammalian cells. Moreover, the peptides displayed potent antioxidant activity and were 12 times more active than melittin. Multifunctional peptides hold potential for a wide range of clinical applications and further investigation into their mode of antibacterial and antioxidant properties is therefore warranted. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Synergistic Effect of α-Hexachlorocy-clohexane with Pyrethrins

The more potent inhibitory activity against angiotensin-converting enzyme (ACE) was excised from a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) preparation of Bacillus stearothermophilus by heating at 120°C in 1 m AcOH–20mm HCI, as compared with GAPDH preparations of yeast and pig. Sufficient excision of B. stearothermophilus ACE inhibitors required a longer proteolysis time of 60 min. Two inhibitors were then purified by gel-permeation and reverse-phase chromatog-raphies. One of the B. stearothermophilus ACE inhibitors, BG-1, was the GAPDH peptide 68–77 (Gly-Lys-Glu-Ile-Ile-Val-Lys-Ala-Glu-Arg, IC₅₀: 32 μm). Another inhibitor, BG-2 (Gly-Lys-Met-Val-Lys-Val-Val-Ser-Trp-Tyr, IC₅₀: 6 μM), corresponded to GAPDH peptide 304–313. These sequences were quite different from those of vertebrate GAPDH peptides and the venom peptide family with ACE inhibitory activity. BG-2 was found to be a non-competitive type inhibitor, differing from many natural peptide inhibitors. Thus, B. stearothermophilus GAPDH seemed to be a good source of new type ACE inhibitors, in addition to the advantages due to its thermophilic property.     

Anticancer potency of small linear and cyclic tetrapeptides and pharmacokinetic investigations of peptide binding to human serum albumin

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a β^2,2‐amino acid with either two 2‐naphthyl‐methylene or two para‐CF₃‐benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC₅₀) 11–70 μM] compared to the linear tetrapeptide counterparts (IC₅₀ 18.7 to >413 μM). The most potent cyclic tetrapeptide c3 had a 16.5‐fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9‐fold) preference for Ramos cells (IC₅₀ 23 μM) compared to healthy human lung fibroblast cells (MRC‐5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22–28 μM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide‐based cancer drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.