LP2, a stable lanthipeptide derived from cAng-(1-7), exerts myeloprotective action in mice.

OBJECTIVES: Linear unstable angiotensins stimulate hematopoiesis. Here we address: (1) Is cyclic angiotensin-(1-7) myeloprotective in mice? (2) Is cyclic angiotensin-(1-7) stable in rat? (3) Does LP2, a cyclic angiotensin-(1-7) with an N-terminal d-lysine, exert myeloprotective action in tumor-bearing mice? MATERIALS AND METHODS: Cyclic angiotensin-(1-7)'s capacity to restore levels of blood platelets and white blood cells was studied in gemcitabine-treated mice. The stability of cyclic angiotensin-(1-7) in rat was measured in blood samples taken after injection or infusion. The capacity of LP2 to restore total bone marrow cell levels in mice after treatment with 5-fluoruracil was measured. In addition, the capacity of LP2 to counter anemia in tumor-bearing mice treated with erlotinib was measured. RESULTS: Cyclic angiotensin-(1-7) dose-dependently restored blood platelet levels in gemcitabine-treated mice, whereas its capacity to restore levels of white blood cells was less. In vivo aminoterminal breakdown of cyclic angiotensin-(1-7) yielded cyclic angiotensin-(2-7) and cyclic angiotensin-(3-7). LP2 significantly (p < .0001 at 100 µg/kg/day) restored bone marrow cell counts in mice after treatment with 5-fluoruracil. LP2 also significantly (p < .05) countered anemia in tumor-bearing mice treated with erlotinib. CONCLUSIONS: LP2 exerts myeloprotective action with perspectives for continuation of its clinical development.

LP2, a cyclic angiotensin-(1-7) analog extended with an N-terminal D-lysine, impairs growth of patient-derived xenografts of colorectal carcinoma in mice.

LP2 is a 4, 7 D, L lanthionine-stabilized analog of angiotensin-(1-7), with an N-terminal D-lysine, resistant to breakdown by peptidases. It is a specific agonist of the angiotensin II type 2 receptor. Consistent with its high specificity and stability, LP2 has shown excellent safety and pharmacokinetics in a first-in-human clinical phase Ia trial. Here, based on strong rationales, we studied the capacity of LP2 to inhibit the growth of patient-derived xenografts of colorectal cancer in mice. Prior to efficacy studies, immunohistochemistry on an untreated tissue array demonstrated that the AT2R expression is reduced in human colorectal cancer and in stroma when compared to tumor adjacent tissue. Subsequent studies demonstrated that LP2 at a subcutaneously injected dose as low as 0.2 µg/kg/day inhibited patient-derived xenografts of colorectal carcinoma in mice. Kinome analyses and validation of elected kinase inhibition indicated that LP2-mediated AT2R stimulation inhibited PI3K/AKT/mTOR which resulted in apoptosis via CDKs. LP2 acted synergistically with 5-FU and the EGFR inhibitor erlotinib. Taken together, the extremely low dose of LP2 at which antitumor activity is exerted, the synergism with selected drugs and, together with its excellent specificity, safety and stability, warrant further evaluation of LP2's inhibitory potential of colorectal cancer.

GRPR-selective PET imaging of prostate cancer using [(18)F]-lanthionine-bombesin analogs.

The gastrin-releasing peptide receptor (GRPR) is overexpressed in a variety of human malignancies, including prostate cancer. Bombesin (BBN) is a 14 amino acids peptide that selectively binds to GRPR. In this study, we developed two novel Al(18)F-labeled lanthionine-stabilized BBN analogs, designated Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN, for positron emission tomography (PET) imaging of GRPR expression using xenograft prostate cancer models. (Methyl)lanthionine-stabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN analogs were conjugated with a NOTA chelator and radiolabeled with Al(18)F using the aluminum fluoride strategy. Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN was labeled with Al(18)F with good radiochemical yield and specific activity>30 GBq/µmol for both radiotracers. The logD values measured for Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN were -2.14 ± 0.14 and -2.34 ± 0.15, respectively. In athymic nude PC-3 xenografts, at 120 min post injection (p.i.), the uptake of Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN in prostate cancer (PC-3) mouse models was 0.82 ± 0.23% ID/g and 1.40 ± 0.81% ID/g, respectively. An excess of unlabeled ɛ-aminocaproic acid-BBN(7-14) (300-fold) was co-injected to assess GRPR binding specificity. Tumor uptake of Al(18)F-NOTA-4,7-lanthionine-BBN and Al(18)F-NOTA-2,6-lanthionine-BBN in PC-3 tumors was evaluated by microPET (µPET) imaging at 30, 60 and 120 min p.i. Blocking studies showed decreased uptake in PC-3 bearing mice. Stabilized 4,7-lanthionine-BBN and 2,6-lanthionine-BBN peptides were rapidly and successfully labeled with (18)F. Both tracers may have potential for GRPR-positive tumor imaging.

To protect peptide pharmaceuticals against peptidases.

INTRODUCTION: The major hurdle in the application and delivery of peptide pharmaceuticals is their rapid in vivo breakdown. METHODS: We here combined two approaches to stabilize peptide pharmaceuticals, introduction of D-amino acids and cyclization, by applying an innovative enzymatic method. This method yields peptides with thioether bridges between a D-amino acid and an L-amino acid. On the basis of guidelines concerning the flanking residues of serines/threonines and cysteines, a peptide of interest is designed with serine/threonine and cysteine at appropriate positions to allow their effective participation in cyclization. In Lactococcus lactis the peptide of interest is directly or via a spacer genetically fused to a lantibiotic leader peptide which induces enzyme-catalysed synthesis of a thioether-bridged peptide. The peptide is translocated via a lantibiotic transporter, analysed by mass spectrometry and the leader peptide is removed. Because of its therapeutic relevance and terminal modifications we chose the decapeptide Luteïnizing Hormone Release Hormone (LHRH) as a test case for thioether bridge introduction. The N-terminal pyroglutamate protects against aminopeptidase activity; the amidated C-terminus, which occurs in 50% of all therapeutic peptides, precludes carboxypeptidase action and is essential for optimal receptor interaction. We had Lactococcus posttranslationally introduce a thioether bridge between residues 4 and 7 of the Leu7Cys-LHRH analog QHWSYGCRPG. The N-terminal glutamine of the thioether-bridged peptide could be converted in pyroglutamate. The introduction of the thioether bridge proved to be compatible with subsequent chemical and enzymatic amidation methods. In this way biologically produced thioether LHRH was compared with LHRH isomers obtained by base-assisted sulfur extrusion. RESULTS: Biologically produced thioether LHRH is the most stable thioether LHRH isomer with strongly enhanced proteolytic resistance compared to natural LHRH. DISCUSSION: The data convincingly demonstrate the broad perspective of stereo- and regiospecifically generating cyclized peptide pharmaceuticals with significantly enhanced therapeutic potential.

Biosynthesis, immunity, regulation, mode of action and engineering of the model lantibiotic nisin.

This review discusses the state-of-the-art in molecular research on the most prominent and widely applied lantibiotic, i.e., nisin. The developments in understanding its complex biosynthesis and mode of action are highlighted. Moreover, novel applications arising from engineering either nisin itself, or from the construction of totally novel dehydrated and/or lanthionine-containing peptides with desired bioactivities are described. Several challenges still exist in understanding the immunity system and the unique multiple reactions occurring on a single substrate molecule, carried out by the dehydratase NisB and the cyclization enzyme NisC. The recent elucidation of the 3-D structure of NisC forms the exciting beginning of further 3-D-structure determinations of the other biosynthetic enzymes, transporters and immunity proteins. Advances in achieving in vitro activities of lanthionine-forming enzymes will greatly enhance our understanding of the molecular characteristics of the biosynthesis process, opening up new avenues for developing unique and novel biocatalytic processes.

Enterocin P causes potassium ion efflux from Enterococcus faecium T136 cells.

Enterocin P is a bacteriocin produced by Enterococcus faecium P13. We studied the mechanism of its bactericidal action using enterocin-P-sensitive E. faecium T136 cells. The bacteriocin is incapable of dissipating the transmembrane pH gradient. On the other hand, depending on the buffer used, enterocin P dissipates the transmembrane potential. Enterocin P efficiently elicits efflux of potassium ions, but not of intracellularly accumulated anions like phosphate and glutamate. Taken together, these data demonstrate that enterocin P forms specific, potassium ion-conducting pores in the cytoplasmic membrane of target cells.

Comparison of the membrane interaction and permeabilization by the designed peptide Ac-MB21-NH2 and truncated dermaseptin S3.

Ac-MB21-NH(2) (Ac-FASLLGKALKALAKQ-NH(2)) and dermaseptin S3(1-16)-NH(2) (ALWKNMLKGIGKLAGK-NH(2)) are cationic amphipathic peptides with antimicrobial activity against a broad spectrum of microorganisms including various fungi. The interaction of the peptides with liposomes was studied by exploiting the tryptophan fluorescence of F1W-Ac-MB21-NH(2) and dermaseptin S3(1-16)-NH(2). Spectral analysis and the use of quenchers indicate that the tryptophans of both peptides insert more deeply in anionic than in zwitterionic liposomes. Membrane insertion correlates with the formation of an alpha-helical peptide structure. Both peptides permeabilize liposomes composed of anionic, cylindric phospholipids more efficiently than liposomes formed of zwitterionic, conic (phospho)lipids.

Bacteriocins: mechanism of membrane insertion and pore formation.

Lactic acid bacteria produce several types of pore forming peptides. Class I bacteriocins are lantibiotics that contain (methyl)lanthionine residues that may form intramolecular thioether rings. These peptides generally have a broad spectrum of activity and form unstable pores. Class II bacteriocins are small, heat stable peptides mostly with a narrow spectrum of activity. Most bacteriocins interact with anionic lipids that are abundantly present in the membranes of gram-positive bacteria. 'Docking molecules' may enhance the conductivity and stability of lantibiotic pores, while 'receptors' in the target membrane may determine specificity of class II bacteriocins. Insertion into the membrane of many bacteriocins is proton motive force driven. Lantibiotics may form pores according to a 'wedge-like' model, while class II bacteriocins may enhance membrane permeability either by the formation of a 'barrel stave' pore or by a 'carpet' mechanism.

Complementary and overlapping selectivity of the two-peptide bacteriocins plantaricin EF and JK.

Plantaricin EF and JK are both two-peptide bacteriocins produced by Lactobacillus plantarum C11. The mechanism of plantaricin EF and JK action was studied on L. plantarum 965 cells. Both plantaricins form pores in the membranes of target cells and dissipate the transmembrane electrical potential (Deltapsi) and pH gradient (DeltapH). The plantaricin EF pores efficiently conduct small monovalent cations, but conductivity for anions is low or absent. Plantaricin JK pores show high conductivity for specific anions but low conductivity for cations. These data indicate that L. plantarum C11 produces bacteriocins with complementary ion selectivity, thereby ensuring efficient killing of target bacteria.

The lantibiotic nisin induces transmembrane movement of a fluorescent phospholipid.

Nisin is a pore-forming antimicrobial peptide. The capacity of nisin to induce transmembrane movement of a fluorescent phospholipid in lipid vesicles was investigated. Unilamellar phospholipid vesicles that contained a fluorescent phospholipid (1-acyl-2-(6-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)amino]caproyl)-sn-glycero-3-phosphocholine) in the inner leaflet of the bilayer were used. Nisin-induced movement of the fluorescent phospholipid from the inner leaflet to the outer leaflet of the membrane reached stable levels, which were dependent on the concentration of nisin added. The rate constant k of this nisin-induced transmembrane movement increased with the nisin concentration but was not dependent on temperature within the range of 5 to 30 degrees C. In contrast, the rate constant of movement of fluorescent phospholipid from vesicle to vesicle strongly depended on temperature. The data indicate that nisin transiently disturbs the phospholipid organization of the target membrane.

Plantaricin A is an amphiphilic alpha-helical bacteriocin-like pheromone which exerts antimicrobial and pheromone activities through different mechanisms.

Production of bacteriocins by lactic acid bacteria is in some cases regulated by a quorum sensing mechanism that involves a secreted bacteriocin-like peptide pheromone. In the case of Lactobacillus plantarum C11, this pheromone, the 26-mer plantaricin A (PlnA), has the interesting property of having both bacteriocin and pheromone activities. To gain insight into how PlnA functions as a pheromone and as a bacteriocin, the L- and D-enantiomers of an N-terminally truncated form of PlnA were synthesized (PlnA-22L and PlnA-22D; PlnA-22L has full biological activity). With circular dichroism, it was shown that the two peptides are unstructured in aqueous solution, but they adopt mirror-image amphiphilic helical structures in the presence of trifluoroethanol and membrane-mimicking entities such as micelles of dodecylphosphocholine and negatively charged Ole2GroPGro liposomes, but not in the presence of zwitterionic Ole2GroPCho liposomes. Thus, the negative charge on the membrane is important for structuring of the (positively charged) PlnA peptides. In terms of in vivo antimicrobial activity, PlnA-22L and PlnA-22D behaved almost identically. Likewise, the peptides dissipated the membrane potential and the transmembrane pH gradient in sensitive cells equally effectively. PlnA-22L induced bacteriocin production in L. plantarum C11 (i.e., displayed pheromone activity), the level of induction being clearly dose-dependent. PlnA-22D did not display pheromone activity, but, at high concentrations, was able to inhibit the pheromone activity of PlnA-22L. The results indicate that the antimicrobial activity of PlnA does not require chiral interactions and is mediated through the formation of a strongly amphiphilic alpha-helical structure. In contrast, PlnA's pheromone activity is dependent on a chiral interaction between the amphiphilic helix (PlnA-22L) and a receptor protein. One may speculate that PlnA is an evolutionary intermediate between a true bacteriocin and a pheromone.

Role of transmembrane pH gradient and membrane binding in nisin pore formation.

Nisin is a cationic antimicrobial peptide that belongs to the group of lantibiotics. It is thought to form oligomeric pores in the target membrane by a mechanism that requires the transmembrane electrical potential delta psi and that involves local pertubation of the lipid bilayer structure. Here we show that nisin does not form exclusively voltage-dependent pores: even in the absence of a delta psi, nisin is able to dissipate the transmembrane pH gradient (delta pH) in sensitive Lactococcus lactis cells and proteoliposomes. The rate of dissipation increases with the magnitude of the delta pH. Nisin forms pores only when the delta pH is inside alkaline. The efficiency of delta psi-induced pore formation is strongly affected by the external pH, whereas delta pH-induced pore formation is rather insensitive to the external pH. Nisin(1-12), an amino-terminal fragment of nisin, and (des-deltaAla5)-(nisin(1-32) amide have a strongly reduced capacity to dissipate the delta psi and delta pH in cytochrome c oxidase proteoliposomes and L. lactis cells. Both variants bind with reduced efficiency to liposomes containing negatively charged phospholipids, suggesting that both ring A and rings C to E play a role in membrane binding. Nisin(1-12) competes with nisin for membrane binding and antagonizes pore formation. These findings are consistent with the wedge model of nisin-induced pore formation.

Mechanism of lantibiotic-induced pore-formation.

Nisin and other lantibiotics have a bacteriocidal effect against Gram-positive bacteria, and also inhibit the outgrowth of bacterial spores. The bacteriocidal effect appears to be due to the formation of pores in the bacterial membrane. In the absence of anionic membrane phospholipids, the lantibiotic nisin acts as an anion selective carrier. In the presence of anionic phospholipids, nisin forms nonselective, transient, multi-state pores in cells, proteoliposomes, liposomes and black lipid membranes. Pore formation involves distinct steps. First, nisin associates tightly with the anionic membrane surface leading to a high local concentration. This results in a disturbance of the lipid dynamics near the phospholipid polar head group-water interface, and an immobilization of lipids. In the presence of a transmembrane electrical potential above the threshold level, the molecules reorient, presumably as an aggregate, from a surface-bound into a membrane-inserted configuration. Co-insertion of bound, anionic phospholipids results in bending of the lipid surface giving rise to a wedge-like, nonspecific, water-filled pore.

Selective internalization of choline-phospholipids in Plasmodium falciparum parasitized human erythrocytes.

We have incubated control and Plasmodium falciparum parasitized human erythrocytes with lipid vesicles containing radiolabeled long-chain phosphatidylcholine and sphingomyelin, in the presence of a nonspecific lipid transfer protein. Most of the radiolabeled phospholipids were, immediately thereafter, available for extracellular phospholipases, suggesting that uptake of vesicles as such did not occur. In time, the amount of phosphatidylcholine inserted in the outer leaflet of the host cell membrane of parasitized erythrocytes decreased, indicating that phosphatidylcholine was being internalized in parasitized erythrocytes. The exclusion of sphingomyelin from the internalization process suggests that the removal of phosphatidylcholine from the outer leaflet of the erythrocyte membrane is caused by transbilayer migration, rather than by endocytosis. The extent of phosphatidylcholine internalization indicates that part of it does not remain in the inner leaflet of the host cell membrane, but is taken up by the intraerythrocytic parasite. Individual phosphatidylcholine species, containing 16:0/18:1-, 16:0/18:2- and 16:0/20:4-fatty acids, showed similar extents of internalization, after being incorporated in parasitized erythrocytes by a phosphatidylcholine specific transfer protein.

Growth inhibition of Plasmodium falciparum in in vitro cultures by selective action of tryptophan-N-formylated gramicidin incorporated in lipid vesicles.

We studied the differential effect of tryptophan-N-formylated gramicidin on uninfected and Plasmodium falciparum-infected erythrocytes. Trp-N-formylated gramicidin induces a much faster leakage of K+ from infected cells than from uninfected cell whereas, and at an even lower concentration, gramicidin A' causes a rapid K+ leakage from both uninfected and infected cells. We also studied the effect of Trp-N-formylated gramicidin and gramicidin A' incorporated in liposomes on the growth of Plasmodium falciparum in an in vitro culture. Incorporation of Trp-N-formylated gramicidin in the membranes of so-called 'stealth' vesicles strongly decreases the concentration needed to induce 50% inhibition of parasite growth. Moreover, no decrease in the K+ content of uninfected cells was observed when cells were exposed to liposome-incorporated Trp-N-formylated gramicidin at a concentration which causes full inhibition of parasite growth. These observations strongly suggest that Trp-N-formylated gramicidin incorporated in 'stealth' vesicles ends up specifically in the infected cell, thereby inhibiting the growth of the growth of the malaria parasite.

Selective elimination of malaria infected erythrocytes by a modified phospholipase A2 in vitro.

Pig pancreatic phospholipase A2 does not act on normal erythrocytes, but the membrane penetrating capacity is enhanced by the covalent attachment of one fatty acyl chain to Lys-116 of the enzyme. Taking advantage of the impaired packing of phospholipids in the membrane of Plasmodium infected erythrocytes it was demonstrated that a lauric acid derivative of phospholipase A2 is capable of exclusively attaching the infected erythrocytes in vitro, leaving the uninfected cells undisturbed. The chemically modified phospholipase A2 appeared to cause death of the parasite in cell cultures of infected erythrocytes.

Plasmodium knowlesi induces alterations in phosphatidylcholine and phosphatidylethanolamine molecular species composition of parasitized monkey erythrocytes.

Using high performance liquid chromatography and gas-liquid chromatography, we have characterized the phosphatidylcholine and phosphatidylethanolamine molecular species composition of trophozoite and schizont forms of Plasmodium knowlesi parasitized erythrocytes. Similarly, we determined these parameters in the erythrocyte membranes of trophozoite parasitized cells, unparasitized erythrocytes from infected monkeys before and after a chloroquine treatment and erythrocytes from monkeys that had never been infected. Plasma phosphatidylcholine molecular species composition was also studied. P. knowlesi parasitized erythrocytes presented higher amounts of 16:0/18:2-phosphatidylcholine than the various control cells, which appeared to be compensated for by a decrease in 18:0/20:4-, 16:0/20:3-, 16:0/18:1-, 18:0/18:2-, 18:0/20:3-, 16:0/16:0- and 16:0/18:0-phosphatidylcholines. In the case of phosphatidylethanolamine, the alterations were quantitatively of greater importance and consisted of an increase in, again, 16:0/18:2-phosphatidylethanolamine and a decrease in several species containing 20:4, namely 16:0/20:4-, 18:0/20:4- and 18:1/20:4-phosphatidylethanolamine; also the levels of alkoxy-phosphatidylethanolamines were markedly decreased. P. knowlesi development within monkey erythrocytes therefore appears to be associated with changes in phosphatidylcholine and phosphatidylethanolamine molecular species in the whole parasitized cell. These alterations are also exhibited by the host cell membrane, which provides the first experimental evidence that the parasite is able to manipulate the erythrocyte membrane lipid species composition. The consequences of these alterations on membrane physiology are discussed, as well as the implications that these data may have on the trafficking of phosphatidylcholine and phosphatidylethanolamine in the erythrocytes of P. knowlesi infected monkeys.

Phospholipid asymmetry in the plasma membrane of malaria infected erythrocytes.

The transbilayer distribution of glycerophospholipids in the plasma membrane of Plasmodium knowlesi infected erythrocytes was studied by using lysine-116-epsilon-N-palmitoyl amidinated pancreatic phospholipase A2. As a consequence of its superior membrane penetrating capacities, this modified enzyme rapidly degrades its substrates in the outer membrane leaflet of intact erythrocytes, a property that makes the enzyme an excellent tool to study the malaria parasitized red cell. The modified phospholipase A2 caused a nonlytic hydrolysis of up to 12-15% of the phosphatidylethanolamine and none of the phosphatidylserine in the red cell membrane, irrespective of whether the cells harboured trophozoite and schizont stages of parasites or no parasites at all. The absence of phosphatidylserine at the exterior surface of Plasmodium infected erythrocytes was confirmed by applying the prothrombinase assay on Plasmodium falciparum infected human erythrocytes. Consequently, the results from these and previous studies indicate that the plasma membrane of Plasmodium infected erythrocytes exhibit a normal transbilayer phospholipid asymmetry.

Phospholipid uptake by Plasmodium knowlesi infected erythrocytes.

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.